CN115232834A - Gene editing system for construction of OCA-1A albinism model pig nuclear transfer donor cells and its application - Google Patents

Abstract

The invention discloses a gene editing system for constructing OCA-1A type albinism model pig nuclear transplantation donor cells and its application. The present invention provides a method for preparing recombinant cells: the DNA molecules shown in SEQ ID NO: 19 in the chromosomal DNA of pig cells are replaced with the DNA molecules shown in SEQ ID NO: 18 to obtain recombinant cells. Implementation method: the target sequence binding region is TYR-gRNA1 shown in nucleotides 3-22 in SEQ ID NO:16, and the target sequence binding region is shown in nucleotides 3-22 in SEQ ID NO:17. Pig cells were co-transfected with TYR-gRNA2 shown in SEQ ID NO: 18, TYR-E1mut-ss126 shown in SEQ ID NO: 18, and NCN protein. The invention has great application value for the research and development of OCA-1A albinism drugs and for revealing the pathogenesis of the disease.

Description

Gene editing of porcine nuclear transfer donor cells for the construction of OCA-1A albinism model system and its application

technical field

The invention belongs to the field of biotechnology, in particular to the field of gene editing technology, and more particularly, based on the CRISPR/Cas9 system and ssODN homologous recombination technology, a gene editing system for constructing OCA-1A albinism model pig nuclear transplantation donor cells and its application .

Background technique

Gene editing is a biotechnology that has made significant progress in recent years, including gene editing based on homologous recombination to nuclease-based editing technologies such as ZFN, TALEN, CRISPR/Cas9, among which CRISPR/Cas9 technology is currently the most advanced. gene editing technology. At present, gene editing technology is increasingly applied to the production of animal models.

Homologous recombination (HDR) is the exchange of DNA sequence information through sequence homology: that is, the repair template contains the desired insert, and the two ends of the repair template are recombination arms with sequence homology near the insertion site. In the past, double-stranded DNA (dsDNA) was commonly used as a repair template, but recent studies have revealed the superiority of single-stranded oligonucleotide deoxynucleotides (ssODN) as HDR donor templates. First, ssODN as a donor template has higher insertion site specificity than dsDNA template, and dsDNA template is prone to random insertion. Secondly, ssODN requires shorter homologous recombination arms than dsDNA templates. The design of recombination arms of 30-60 bases on one side can achieve efficient and stable HDR, which provides higher insertion efficiency than similar dsDNA templates. high. Third, dsDNA is easily incorporated by the NHEJ repair pathway, leading to duplication of homology arms or partial integration of the dsDNA template, while ssODN is not prone to this phenomenon. In addition, dsDNAs are harmful to cultured cells, and linear or plasmid dsDNAs have lower transfection efficiency and cause adverse cell reactions, and ssODN templates have more advantages in these aspects.

Albinism is a general term for a variety of hereditary diseases caused by reduced or lack of melanin synthesis, mainly manifested as hypopigmentation of the skin, hair and eyes. Mutations in a variety of genes can cause the symptoms of albinism. Albinism can be divided into two categories: non-syndromic albinism and syndromic albinism, according to the location of pigment loss and the presence or absence of other system abnormalities. Non-syndromic albinism can be subdivided into ocular albinism (OA) with only ocular pigment deficiency and oculocutaneous albinism (OCA) with pigment loss in skin, hair, and eyes (ie, with systemic symptoms).

OCA is a group of autosomal recessive genetic diseases with reduced or lack of melanin pigmentation in the eyes, skin and hair as the main clinical manifestations. More than 90% of patients with albinism have OCA, which is generally caused by mutations in genes related to melanin synthesis or transport. Light, iris translucency, nystagmus, hypopigmentation of the fundus and abnormalities of the visual fiber pathway. In recent years, OCAs have been classified according to the genes in which they are mutated: mutations in the tyrosinase gene (TYR) lead to OCA1, mutations in the OCA2 gene (previously named the P gene) lead to OCA2, and mutations in the tyrosinase-related protein-1 gene (TYRP1 ) mutations lead to OCA3, and mutations in the SLC45A2 gene (previously named MATP or AIM1) lead to OCA4.

OCA-1 type accounts for about 40-50% of OCA, and is caused by the loss of function of the tyrosinase encoded by TYR gene mutation. Studies have shown that, depending on whether tyrosinase activity is completely lost, OCA-1 can be divided into two types: OCA-1A and OCA-1B, which are indistinguishable at birth. OCA-1A patients have a complete loss of tyrosinase activity, the most severe type, with complete loss of skin and eye pigment, and a drop in visual acuity to 5%. The activity of tyrosinase in patients with OCA-1B is significantly decreased, but not completely absent. The skin, hair and eye pigment of patients can increase with age and can be tanned.

The research on the occurrence and development mechanism of OCA-1 type caused by TYR mutation and the development of corresponding drugs need to be carried out on the basis of animal models. The commonly used animal model is the mouse model. Pathological and other aspects are very different from human beings, and cannot truly simulate the normal physiological and pathological states of human beings. As a large animal, pig has been the main meat supply animal for humans for a long time. Its body size and physiological function are similar to those of humans. It is easy to be bred and raised on a large scale, and has lower requirements on ethics and animal protection. It is an ideal human disease. model animal.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a gene editing system for constructing OCA-1A albinism model pig nuclear transplantation donor cells and its application.

The present invention provides a method for preparing recombinant cells, which includes the following steps: replacing the DNA molecules shown in SEQ ID NO: 19 in the chromosomal DNA of pig cells with the DNA molecules shown in SEQ ID NO: 18 to obtain recombinant cells.

The implementation manner of replacing the DNA molecule shown in SEQ ID NO: 19 in the chromosomal DNA of pig cells with the DNA molecule shown in SEQ ID NO: 18 is as follows: TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein Co-transfection of pig cells.

The co-transfection specifically adopts the mode of electric shock transfection.

The specific parameter settings of electroporation transfection can be: 1450V, 10ms, 3pulse.

For the co-transfection, a mammalian nucleofection kit (Neon kit, Thermofisher) and a NeonTM transfection system electroporator may be used.

The invention also provides the application of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein in the preparation kit.

The invention also provides the application of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and PRONCN proteins in the preparation kit.

The invention also provides the application of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and specific plasmids in the preparation kit.

The present invention also provides a kit comprising TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein.

The present invention also provides a kit comprising TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and PRONCN protein.

The present invention also provides a kit comprising TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and a specific plasmid.

Any of the above kits also include pig cells.

The use of any one of the above kits is as follows (a) or (b) or (c): (a) preparation of recombinant cells; (b) preparation of albinism model pigs; (c) preparation of albinism cell models or albinism tissue models or Albinism Organ Model.

The ratios of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein were as follows: 0.8-1.2 μg TYR-gRNA1: 0.8-1.2 μg TYR-gRNA2: 1.8-2.2 μg TYR-E1mut-ss126: 3-5 μg NCN protein.

The ratios of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein were as follows: 1 μg TYR-gRNA1: 1 μg TYR-gRNA2: 2 μg TYR-E1mut-ss126: 4 μg NCN protein.

The ratios of pig cells, TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein were as follows: 100,000 pig cells: 0.8-1.2μg TYR-gRNA1: 0.8-1.2μg TYR-gRNA2: 1.8-2.2 μg TYR-E1mut-ss126: 3-5 μg NCN protein.

The ratio of porcine cells, TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein was as follows: 100,000 porcine cells: 1 μg TYR-gRNA1: 1 μg TYR-gRNA2: 2 μg TYR-E1mut-ss126: 4 μg NCN protein.

Any of the above TYR-gRNA1 is sgRNA, and its target sequence binding region is shown in nucleotides 3-22 in SEQ ID NO: 16.

Any of the above TYR-gRNA2s are sgRNAs, and the target sequence binding region thereof is shown in nucleotides 3-22 in SEQ ID NO: 17.

Any of the above TYR-E1mut-ss126 is a single-stranded DNA molecule shown in SEQ ID NO: 18.

Any of the above NCN proteins are Cas9 proteins or fusion proteins with Cas9 proteins.

Specifically, the NCN protein is shown in SEQ ID NO:3.

Specifically, the TYR-gRNA1 is shown in SEQ ID NO: 16.

Specifically, the TYR-gRNA2 is shown in SEQ ID NO: 17.

Specifically, the TYR-gRNA1 is shown in SEQ ID NO: 10.

Specifically, the TYR-gRNA2 is shown in SEQ ID NO: 11.

Any of the above-mentioned porcine cells are porcine fibroblasts.

Any of the above-mentioned porcine cells are porcine primary fibroblasts.

The preparation method of the NCN protein comprises the following steps:

(1) The plasmid pKG-GE4 was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria;

(2) using liquid medium to cultivate the recombinant bacteria at 30°C, then adding IPTG and performing induction culture at 25°C, and then collecting the bacterial cells;

(3) thalline fragmentation is carried out by the collected thalline, and crude protein solution is collected;

(4) using affinity chromatography to purify the fusion protein with the His ₆ tag from the crude protein solution;

(5) using enterokinase with His ₆ tag to digest the fusion protein with His ₆ tag, and then using Ni-NTA resin to remove the protein with His ₆ tag to obtain purified NCN protein;

Plasmid pKG-GE4 has the fusion gene shown in nucleotides 5209-9852 in SEQ ID NO:1.

The preparation method of the NCN protein specifically comprises the following steps:

(1) The plasmid pKG-GE4 was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

(2) the recombinant bacteria obtained in step (1) are inoculated into the liquid LB medium containing ampicillin, and shake cultured;

(3) Inoculate the bacterial liquid obtained in step (2) into liquid LB medium, shake and cultivate at 30°C and 230rpm until OD _600nm value=1.0, then add IPTG to make the concentration in the system 0.5mM, then 25°C , 230rpm shaking culture for 12 hours, then centrifuged to collect bacterial cells;

(4) take the thalline obtained in step (3), wash with PBS buffer;

(5) taking the thalline obtained in step (4), adding a crude extraction buffer and suspending the thalline, then crushing the thalline, then collecting the supernatant by centrifugation, filtering with a 0.22 μm pore size filter, and collecting the filtrate;

(6) using affinity chromatography to purify the fusion protein with His ₆ tag (fusion protein shown in SEQ ID NO: 2) from the filtrate obtained in step (5);

(7) Take the post-column solution collected in step (6), concentrate using an ultrafiltration tube, and then dilute with 25mM Tris-HCl (pH8.0);

(8) adding the recombinant bovine enterokinase with His ₆ tag to the solution obtained in step (7), and cleaving with enzyme;

(9) mixing the solution obtained in step (8) with Ni-NTA resin, incubating, and then centrifuging to collect the supernatant;

(10) Take the supernatant obtained in step (9), concentrate using an ultrafiltration tube, and then add it to the enzyme storage solution, which is the NCN protein solution.

The specific method for purifying the fusion protein with His ₆ tag from the filtrate obtained in step (5) by affinity chromatography is as follows:

First equilibrate the Ni-NTA agarose column with 5 column volumes of equilibration solution (flow rate of 1ml/min); then load 50ml of the filtrate obtained in step (5) (flow rate of 0.5-1ml/min); then use 5 columns The column was washed with a volume of equilibration solution (flow rate of 1 ml/min); then the column was washed with 5 column volumes of buffer (flow rate of 1 ml/min) to remove impurities; Elute at a flow rate of -1 ml/min and collect the post-column solution (90-100 ml).

Any of the above PRONCN proteins include the following elements in sequence from upstream to downstream: a signal peptide, a molecular chaperone protein, a protein tag, a protease cleavage site, a nuclear localization signal, a Cas9 protein, and a nuclear localization signal.

The function of the signal peptide is to promote protein secretion and expression. The signal peptide can be selected from Escherichia coli alkaline phosphatase (phoA) signal peptide, Staphylococcus aureus protein A signal peptide, Escherichia coli outer membrane protein (ompa) signal peptide or signal peptide of any other prokaryotic gene, preferably base phoA signal peptide. The alkaline phosphatase signal peptide is used to guide the secretion and expression of the target protein into the bacterial periplasmic cavity, so as to be separated from the bacterial intracellular protein, and the target protein secreted into the bacterial periplasmic cavity is soluble and can be expressed in the bacterial periplasmic cavity. signal peptidase cleavage.

The function of the molecular chaperone protein is to increase the solubility of the protein. The molecular chaperone can be any protein that aids in the formation of disulfide bonds, preferably thioredoxin (TrxA protein). Thioreoxin, which can act as a molecular chaperone to help co-expressed target proteins (such as Cas9 proteins) form disulfide bonds, improve protein stability, correct folding, and increase the solubility and activity of target proteins.

The function of the protein tag is for protein purification. The tag can be a His tag (His-Tag, His ₆ protein tag), GST tag, Flag tag, HA tag, c-Myc tag or any other protein tag, more preferably a His tag. The His tag can be combined with the Ni column, and the target protein can be purified by one-step Ni column affinity chromatography, which can greatly simplify the purification process of the target protein.

The function of the protease cleavage site is to excise the non-functional segment after purification to release the Cas9 protein in its native form. The protease may be selected from Enterokinase, Factor Xa, Thrombin, TEV protease, HRV 3C protease, WELQut protease or any other endoprotease, More preferred is enterokinase. EK is an enterokinase cleavage site, which facilitates the use of enterokinase to excise the fused TrxA-His segment to obtain the natural form of the Cas9 protein. In this application, after the fusion protein is digested with the commercial enterokinase with His tag, the TrxA-His segment and the enterokinase with the His tag can be removed by one affinity chromatography to obtain the natural form of Cas9 protein, which avoids multiple purification and dialysis Damage and loss of target protein.

The nuclear localization signal may be any nuclear localization signal, preferably the SV40 nuclear localization signal and/or the nucleoplasmin nuclear localization signal. NLS is a nuclear localization signal, and an NLS site is designed at the N-terminal and C-terminal of Cas9, so that Cas9 can enter the nucleus more efficiently for gene editing.

The Cas9 protein can be saCas9 or spCas9, preferably spCas9 protein.

The PRONCN protein is specifically shown in SEQ ID NO:2.

Any of the above-mentioned specific plasmids sequentially include the following elements from upstream to downstream: promoter, operator, ribosome binding site, gene encoding PRONCN protein, and terminator.

Specifically, the promoter can be a T7 promoter. The T7 promoter is a strong promoter for prokaryotic expression, which can efficiently drive the expression of foreign genes.

The operon can be specifically the Lac operon. The Lac operon is a regulatory element for lactose-induced expression. After the bacteria grow to a certain number, IPTG can be used to induce the expression of the target protein at low temperature, which can avoid the effect of premature expression of the target protein on the growth of the host bacteria, and induce expression at low temperature. Also significantly improves the solubility of the expressed protein of interest.

The ribosome binding site is a ribosome binding site during protein translation, and is necessary for protein translation.

The terminator can specifically be a T7 terminator. The T7 terminator can effectively terminate gene transcription at the end of the target gene, avoiding the transcription and translation of other downstream sequences other than the target gene.

For the codons of spCas9 protein, this application has optimized the codons to fully adapt to the codon preference of the E. coli high-efficiency expression strain E.coli BL21(DE3) selected in this application, thereby improving the expression level of Cas9 protein .

The T7 promoter is shown as nucleotides 5121-5139 in SEQ ID NO:1.

The Lac operon is shown as nucleotides 5140-5164 in SEQ ID NO:1.

The ribosome binding site is shown as nucleotides 5178-5201 in SEQ ID NO:1.

The coding sequence of the alkaline phosphatase signal peptide is shown in nucleotides 5209-5271 in SEQ ID NO:1.

The coding sequence of TrxA protein is shown in nucleotides 5272-5598 in SEQ ID NO:1.

The coding sequence of His-Tag is shown in nucleotides 5620-5637 in SEQ ID NO:1.

The coding sequence of the enterokinase cleavage site is shown in nucleotides 5638-5652 in SEQ ID NO:1.

The coding sequence for the nuclear localization signal is shown in SEQ ID NO:1 at nucleotides 5656-5670.

The coding sequence of spCas9 protein is shown in nucleotides 5701-9801 in SEQ ID NO:1.

The coding sequence for the nuclear localization signal is shown at nucleotides 9802-9849 in SEQ ID NO:1.

The T7 terminator is nucleotides 9902-9949 in SEQ ID NO:1.

Specifically, the specific plasmid is plasmid pKG-GE4.

Plasmid pKG-GE4 has a DNA molecule represented by nucleotides 5121-9949 in SEQ ID NO:1.

Specifically, any of the above-mentioned plasmid pKG-GE4 is shown in SEQ ID NO: 1.

The present invention also protects the recombinant cells prepared by any of the above methods.

The invention also protects the application of the recombinant cells in the preparation of albinism model pigs.

The recombinant cells are used as donor cells for nuclear transplantation to carry out somatic cell cloning, and a cloned pig can be obtained, that is, an albino model pig.

The present invention also protects the porcine tissue of the model pig prepared by using the recombinant cell, that is, the albinism tissue model.

The present invention also protects the pig organ of the model pig prepared by using the recombinant cell, that is, the albinism organ model.

The present invention also protects the pig cells of the model pig prepared by using the recombinant cells, that is, the albinism cell model.

The present invention also protects the application of the recombinant cells, the albinism tissue model, the albinism organ model, the albinism cell model or the albinism model pig, as follows (d1) or (d2) or (d3) or ( d4):

(d1) Screening drugs for the treatment of albinism;

(d2) Evaluating the efficacy of albinism drugs;

(d3) Efficacy evaluation of gene therapy and/or cell therapy for albinism;

(d4) To study the pathogenesis of albinism.

Any of the pigs mentioned above may specifically be Congjiangxiang pigs.

Any of the above-mentioned albinisms may be OCA-1 albinism.

Any of the albinisms described above may be OCA-1A albinism.

OCA-1A albinism is caused by the following mutations in the TYR gene: R77L mutation of the TYR gene.

Pig TYR gene information: encodes tyrosinase; located on chromosome 9; GeneID is 407745, Sus scrofa. The amino acid sequence encoded by the porcine TYR gene is shown in SEQ ID NO:8. The porcine TYR gene has the DNA segment shown in SEQ ID NO:9.

Compared with the prior art, the present invention at least has the following beneficial effects:

(1) The research object (pig) of the present invention has better applicability than other animals (mice, primates).

Rodents such as rats and mice are very different from humans in terms of body size, organ size, physiology, and pathology, and cannot truly simulate the normal physiological and pathological states of humans. Studies have shown that more than 95% of drugs proven to be effective in rats and mice are ineffective in human clinical trials. As far as large animals are concerned, primates are the closest relatives to humans, but they are small in size, late in sexual maturity (mating at the age of 6-7), and are singleton animals. very high. In addition, primate cloning is inefficient, difficult and costly.

As a model animal, pigs do not have the above shortcomings. Pigs are the closest animals to humans except primates. Their body size, body weight, organ size, etc. are similar to those of humans. Pathogenesis and other aspects are very similar to humans. At the same time, pigs have early sexual maturity (4-6 months), high fecundity, and multiple litter, which can form a larger group within 2-3 years. In addition, the cloning technology of pigs is very mature, and the cost of cloning and feeding is much lower than that of primates. Pigs are therefore very suitable animals as models of human disease.

(2) The vector constructed by the invention uses a strong promoter T7-lac that can efficiently express the target protein to express the target protein, and uses the signal peptide of bacterial periplasmic protein alkaline phosphatase (phoA) to guide the secretion of the target protein It is expressed in the periplasmic cavity of bacteria, so as to be separated from bacterial intracellular proteins, and the target protein secreted into the periplasmic cavity of bacteria is soluble expression. At the same time, the thioredox protein TrxA and Cas9 protein are also used for fusion expression. TrxA can help the co-expressed target protein to form disulfide bonds, improve the stability of the protein, the correctness of folding, and increase the solubility and activity of the target protein. In order to facilitate the purification of the target protein, a His tag is designed, and the target protein can be purified by one-step Ni column affinity chromatography, which greatly simplifies the purification process of the target protein. At the same time, an enterokinase cleavage site was designed after the His tag to facilitate the excision of the fused TrxA-His polypeptide fragment to obtain the natural form of Cas9 protein. After the fusion protein is digested with His-tagged enterokinase, the TrxA-His polypeptide fragment and His-tagged enterokinase can be removed by one affinity chromatography to obtain the natural Cas9 protein, which avoids multiple purification and dialysis of the target protein. damage and loss. At the same time, the present invention also designs an NLS site at the N-terminus and C-terminus of Cas9, so that Cas9 can more effectively enter the nucleus for gene editing. In addition, the present invention selects E. coli BL21 (DE3) strain as the target protein expression strain, which can efficiently express the exogenous gene cloned in the expression vector (eg pET-32a) containing the phage T7 promoter. At the same time, for the codon of Cas9 protein, the present invention carries out codon optimization to make it completely adapt to the codon preference of the expression strain, thereby improving the expression level of the target protein. In addition, the present invention uses IPTG to induce the expression of the target protein at low temperature after the bacteria grows to a certain number, which can avoid the influence of the premature expression of the target protein on the growth of the host bacteria, and the induced expression at low temperature also significantly improves the expressed target protein. the solubility. After the above optimization design and experimental implementation, the activity of the obtained Cas9 protein has been significantly improved compared with the commercial Cas9 protein.

(3) The Cas9 high-efficiency protein constructed and expressed in the present invention is used in combination with gRNA transcribed in vitro to perform gene editing, and the optimal dosage ratio of Cas9 and gRNA is optimized, and the synthesized ssODN is used as Donor DNA to finally obtain the target site. The mutated single-cell clone ratio is as high as 18.75%, which is much higher than the conventional point mutation efficiency (<5%).

(4) The cloned pigs containing the target site mutation can be directly obtained by using the target gene point mutation single-cell clone obtained by the present invention to carry out somatic cell nuclear transplantation animal cloning, and the mutation can be stably inherited.

The method of microinjecting gene editing materials into fertilized eggs and then carrying out embryo transfer in the production of mouse models has a very low probability of directly obtaining point mutation offspring (less than 1%), which requires cross breeding of offspring. This is less suitable for modeling large animals (such as pigs) with longer gestation periods. Therefore, the present invention adopts the method of in vitro editing of primary cells and ssODN homologous recombination and screening positive editing single cell clones, which are technically difficult and challenging, and then directly obtains the corresponding disease model pigs through somatic cell nuclear transplantation animal cloning technology in the later stage. Greatly shorten the model pig production cycle and save manpower, material resources and financial resources.

The invention adopts CRISPR/Cas9 technology combined with ssODN homologous recombination technology to carry out site-directed mutation of TYR gene, simulates the natural pathogenic genetic characteristics of OCA-1A albinism, and obtains a single-cell clone with point mutation of TYR gene, which is used for the later passage of somatic cell nucleus. The transplanted animal cloning technology has laid the foundation for cultivating OCA-1A albinism model pigs. The model pig will provide a powerful experimental tool for studying the pathogenesis of OCA-1A albinism and drug development.

The invention lays a solid foundation for obtaining OCA-1A albinism model pigs with TYR gene point mutation through gene editing, and will help to study and reveal the pathogenesis of OCA-1A albinism caused by TYR gene point mutation, and can also be used for Researches on drug screening, drug efficacy testing, gene therapy and cell therapy can provide effective experimental data for further clinical application, and then provide a powerful experimental method for the successful treatment of human OCA-1A albinism. The invention has great application value for the research and development of OCA-1A albinism drugs and for revealing the pathogenesis of the disease.

Description of drawings

Figure 1 is a schematic diagram of the structure of plasmid pET-32a.

Figure 2 is a schematic diagram of the structure of plasmid pKG-GE4.

FIG. 3 is an electropherogram showing the optimized dosage ratio of gRNA and NCN protein in Example 3. FIG.

FIG. 4 is an electropherogram comparing the gene editing efficiency of NCN protein and commercial Cas9 protein in Example 3. FIG.

FIG. 5 is an electropherogram of PCR amplification with different primer pairs using the genome extracted from the ear tissue of pig named 1 as a template in Example 4. FIG.

FIG. 6 is an electrophoresis image of PCR amplification using the genomic DNAs of 18 pigs as templates and primer pairs consisting of TYR-E1-JDF70 and TYR-E1-JDR511 in Example 4, respectively.

FIG. 7 is an electropherogram comparing the editing efficiency of different targets in Example 4. FIG.

FIG. 8 is an electropherogram in Example 5. FIG.

Figure 9 shows the results of forward and reverse sequencing of the single-cell clone numbered 12 while aligning it with the wild-type sequence of the target site.

Figure 10 shows the results of forward and reverse sequencing of the single-cell clone numbered 8 while aligning it with the wild-type sequence of the target site.

Figure 11 shows the results of forward and reverse sequencing of the single-cell clone numbered 46 while aligning it with the wild-type sequence of the target site.

Figure 12 shows the results of forward and reverse sequencing of the single-cell clone numbered 20 while aligning it with the wild-type sequence of the target site.

Figure 13 shows the results of forward and reverse sequencing of single-cell clone numbered 9 while aligning it with the wild-type sequence of the target site.

Figure 14 shows the results of forward and reverse sequencing of the single-cell clone numbered 2 while aligning it with the wild-type sequence of the target site.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The recombinant plasmids constructed in the examples have all been verified by sequencing. The commercial Cas9-A protein is a commercially available Cas9 protein with good effect. The commercial Cas9-B protein is a commercially available Cas9 protein with good effect. Complete medium (% is volume ratio): 15% fetal bovine serum (Gibco)+83% DMEM medium (Gibco)+1% Penicillin-Streptomycin (Gibco)+1% HEPES (Solarbio). Cell culture conditions: constant temperature incubator at 37°C, 5% CO ₂ , 5% O ₂ .

The porcine primary fibroblasts used in the examples were all prepared from the primary Congjiangxiang pig ear tissue. The method of preparing primary porcine fibroblasts: ① Take 0.5 g of pig ear tissue, remove hair and bone tissue, then soak in 75% alcohol for 30-40s, and then use PBS containing 5% (volume ratio) Penicillin-Streptomycin (Gibco) The buffer was washed 5 times, and then washed once with PBS buffer; ② The tissue was cut into pieces with scissors, digested with 5 mL of 0.1% collagenase solution (Sigma), digested at 37 °C for 1 h, and then centrifuged at 500 g for 5 min, and the supernatant was discarded; ③ The pellet was discarded. Resuspend with 1mL of complete culture medium, then spread it into a 10cm-diameter cell culture dish containing 10mL of complete culture medium and sealed with 0.2% gelatin (VWR), and culture until the cells cover about 60% of the bottom of the dish; ④Complete After step ③, cells were digested with trypsin and collected, and then resuspended in complete culture medium. used for subsequent electroporation experiments.

The plasmid pKG-GE3 is a circular plasmid, as shown in SEQ ID NO: 2 in the patent application 202010084343.6. In SEQ ID NO: 2 in patent application 202010084343.6, nucleotides 395-680 constitute the CMV enhancer, nucleotides 682-890 constitute the EF1a promoter, and nucleotides 986-1006 encode nuclear localization signals (NLS), nucleotides 1016-1036 encode the nuclear localization signal (NLS), nucleotides 1037-5161 encode the Cas9 protein, and nucleotides 5162-5209 encode the nuclear localization signal (NLS), 5219 -5266 nucleotides encode nuclear localization signal (NLS), 5276-5332 nucleotides encode self-cleaving polypeptide P2A (the amino acid sequence of self-cleaving polypeptide P2A is "ATNFSLLKQAGDVEENPGP", and the position of self-cleaving breakage is The C-terminal starts between the first amino acid residue and the second amino acid residue), the 5333-6046 nucleotides encode the EGFP protein, and the 6056-6109 nucleotides encode the self-cleaving polypeptide T2A (self-cleaving polypeptide T2A The amino acid sequence is "EGRGSLLTCGDVEENPGP", and the position of self-cleavage is between the first amino acid residue and the second amino acid residue from the C-terminus), and the 6110-6703 nucleotides encode Puromycin protein (referred to as Puro protein). ), the 6722-7310 nucleotides constitute the WPRE sequence element, the 7382-7615 nucleotides constitute the 3'LTR sequence element, and the 7647-7871 nucleotides constitute the bGH poly(A) signal sequence element. In SEQ ID NO: 2 in patent application 202010084343.6, nucleotides 911-6706 form a fusion gene and express a fusion protein. Due to the presence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide T2A, the fusion protein spontaneously formed the following three proteins: a protein with Cas9 protein, a protein with EGFP protein, and a protein with Puro protein.

The pKG-U6 gRNA vector, the plasmid pKG-U6 gRNA, is a circular plasmid, as shown in SEQ ID NO: 3 in the patent application 202010084343.6. In SEQ ID NO: 3 in patent application 202010084343.6, the 2280-2539 nucleotides constitute the hU6 promoter, and the 2558-2637 nucleotides are used for transcription to form the gRNA backbone. When in use, a DNA molecule of about 20 bp (the target sequence binding region used for transcription to form gRNA) is inserted into the plasmid pKG-U6 gRNA to form a recombinant plasmid, and the recombinant plasmid is transcribed in cells to obtain gRNA.

Example 1. Construction of prokaryotic Cas9 high-efficiency expression vector

The schematic diagram of the structure of plasmid pET-32a is shown in Figure 1.

The plasmid pKG-GE4 was obtained by transforming the plasmid pET-32a as the starting plasmid. Plasmid pET32a-T7lac-phoA:SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter (referred to as plasmid pKG-GE4), as shown in SEQ ID NO: 1, is a circular plasmid, and the schematic diagram is shown in Figure 2.

In SEQ ID NO: 1, the 5121-5139 nucleotides constitute the T7 promoter, the 5140-5164 nucleotides encode the Lac operator, and the 5178-5201 nucleotides constitute the ribosome binding site Point (RBS), nucleotides 5209-5271 encode the alkaline phosphatase signal peptide (phoA signal peptide), nucleotides 5272-5598 encode the TrxA protein, and nucleotides 5620-5637 encode His-Tag , nucleotides 5638-5652 encode enterokinase cleavage site (EK cleavage site), nucleotides 5656-5670 encode nuclear localization signals, nucleotides 5701-9801 encode spCas9 protein, and Nucleotides 9802-9849 encode the nuclear localization signal, and nucleotides 9902-9949 constitute the T7 terminator. The nucleotides encoding the spCas9 protein have been codon-optimized for the E. coli BL21(DE3) strain.

The main modifications of the plasmid pKG-GE4 are as follows: ① The coding region of the TrxA protein is retained, and the TrxA protein can help the expressed target protein to form disulfide bonds and increase the solubility and activity of the target protein; Add alkali before the coding region of the TrxA protein The coding sequence of sex phosphatase signal peptide, the alkaline phosphatase signal peptide can guide the expressed target protein to be secreted into the periplasmic cavity of bacteria and can be digested by prokaryotic periplasmic signal peptidase; ② in the coding sequence of TrxA protein After that, the coding sequence of His-Tag is added, and His-Tag can be used for the enrichment of the expressed target protein; ③ Add the enterokinase cleavage site DDDDK (Asp-Asp-Asp-Asp-Lys) downstream of the coding sequence of His-Tag ), the purified protein will remove His-Tag and the upstream fused TrxA protein under the action of enterokinase; Both the upstream and downstream of the gene increase the nuclear localization signal coding sequence to increase the nuclear localization ability of the later purified Cas9 protein.

The fusion gene in plasmid pKG-GE4 is shown in nucleotides 5209-9852 in SEQ ID NO: 1, encoding the fusion protein shown in SEQ ID NO: 2 (fusion protein TrxA-His-EK-NLS-spCas9-NLS , referred to as PRONCN protein). Due to the existence of the alkaline phosphatase signal peptide and enterokinase cleavage site, the fusion protein is digested by enterokinase to form the protein shown in SEQ ID NO: 3, and the protein shown in SEQ ID NO: 3 is named as NCN protein.

Example 2. Preparation and purification of NCN protein

1. Induced expression

1. The plasmid pKG-GE4 was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

2. Inoculate the recombinant bacteria obtained in step 1 into a liquid LB medium containing 100 μg/ml ampicillin, and culture with shaking at 37° C. and 200 rpm overnight.

3. Inoculate the bacterial liquid obtained in step 2 into the liquid LB medium, shake and cultivate at 30°C and 230 rpm until the OD _600nm value = 1.0, and then add isopropyl thiogalactoside (IPTG) to the concentration in the system. It was 0.5 mM, followed by shaking culture at 25°C and 230 rpm for 12 hours, followed by centrifugation at 4°C and 10,000 g for 15 minutes, and the cells were collected.

4. Take the bacteria obtained in step 3 and wash with PBS buffer.

2. Purification of fusion protein TrxA-His-EK-NLS-spCas9-NLS

1. Take the cells obtained in step 1, add the crude extraction buffer and suspend the cells, then use a homogenizer to break the cells (1000par for three cycles), then centrifuge at 4°C and 15000g for 30min, collect the supernatant, and the supernatant The liquid was filtered through a 0.22 μm pore size filter, and the filtrate was collected. In this step, 10 ml of crude extraction buffer is mixed with each gram of wet weight of the bacterial cells.

Crude extraction buffer: containing 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM Imidazole, 1 mM PMSF, the balance being ddH ₂ O.

2. Purify the fusion protein by affinity chromatography.

First equilibrate the Ni-NTA agarose column with 5 column volumes of equilibration solution (flow rate of 1ml/min); then load 50ml of the filtrate obtained in step 1 (flow rate of 0.5-1ml/min); then use 5 column volumes of Wash the column with equilibration solution (flow rate of 1ml/min); then wash the column with 5 column volumes of buffer (flow rate of 1ml/min) to remove impurities; then use 10 column volumes of eluent at 0.5-1ml eluted at a flow rate of /min, and the post-column solution (90-100 ml) was collected.

Ni-NTA agarose column: GenScript, L00250/L00250-C, packing 10ml.

Equilibrium solution: containing 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM Imidazole, the balance being ddH ₂ O.

Buffer: 20 mM Tris-HCl (pH 8.0), 0.5 M _NaCl , 50 mM Imidazole, balance ddH2O.

Eluent: 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM Imidazole, balance ddH ₂ O.

3. Enzyme cleavage of fusion protein TrxA-His-EK-NLS-spCas9-NLS and purification of NCN protein

1. Take 15ml of the post-column solution collected in step 2, use an Amicon ultrafiltration tube (Sigma, UFC9100, 15ml capacity) to concentrate it to 200μl, and then dilute it to 1ml with 25mM Tris-HCl (pH8.0). Using 6 ultrafiltration tubes, a total of 6 ml was obtained.

2. Add the recombinant bovine enterokinase with the His ₆ tag of commercial origin (Sangon Bio, C620031, recombinant bovine enterokinase light chain, with the His ₆ tag, Recombinant Bovine Enterokinase Light Chain, His) to the solution obtained in step 1 ( about 6 ml), digested at 25°C for 16 hours. Add 2 units of enterokinase per 50 μg of protein.

3. Take the solution from step 2 (about 6ml), mix it with 480μl Ni-NTA resin (GenScript, L00250/L00250-C), rotate and mix at room temperature for 15min, then centrifuge at 7000g for 3min, collect the supernatant (4-5.5ml).

4. Take the supernatant obtained in step 3, use Amicon ultrafiltration tube (Sigma, UFC9100, capacity of 15ml) to concentrate it to 200μl, then add it to the enzyme stock solution, adjust the protein concentration to 5mg/ml, which is NCN protein solution.

After sequencing, the 15 N-terminal amino acid residues of the protein in the NCN protein solution are shown in positions 1 to 15 of SEQ ID NO: 3, that is, the NCN protein.

The NCN proteins used in subsequent examples were all provided by NCN protein solutions.

Enzyme stock solution (pH 7.4): containing 10 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, the balance being ddH ₂ O.

Example 3. Properties of NCN protein

The 2 gRNA targets selected to target the TTN gene are as follows:

TTN-gRNA1:AGAGCACAGTCAGCCTGGCG;

TTN-gRNA2: CTTCCAGAATTGGATCTCCG.

The primers used to identify the target fragment containing the gRNA in the TTN gene are as follows:

TTN-F55: TACGGAATTGGGGAGCCAGCGGA;

TTN-R560: CAAAGTTAACTCTCTGTGTCT.

1. Preparation of gRNA

1. Preparation of TTN-T7-gRNA1 transcription template and TTN-T7-gRNA2 transcription template

The TTN-T7-gRNA1 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO:4.

The TTN-T7-gRNA2 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO:5.

2. In vitro transcription to obtain gRNA

The TTN-T7-gRNA1 transcription template was taken and transcribed in vitro with Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified with MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain TTN-gRNA1. TTN-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO:6.

The TTN-T7-gRNA2 transcription template was taken and transcribed in vitro with Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified with MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain TTN-gRNA2. TTN-gRNA2 is a single-stranded RNA, as shown in SEQ ID NO:7.

2. Optimization of the dosage ratio of gRNA and NCN protein

1. Co-transfection of porcine primary fibroblasts

Group 1: TTN-gRNA1, TTN-gRNA2 and NCN proteins were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 0.5 μg TTN-gRNA1: 0.5 μg TTN-gRNA2: 4 μg NCN protein.

The second group: TTN-gRNA1, TTN-gRNA2 and NCN proteins were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 0.75 μg TTN-gRNA1: 0.75 μg TTN-gRNA2: 4 μg NCN protein.

The third group: TTN-gRNA1, TTN-gRNA2 and NCN proteins were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2: 4 μg NCN protein.

The fourth group: TTN-gRNA1, TTN-gRNA2 and NCN proteins were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1.25 μg TTN-gRNA1: 1.25 μg TTN-gRNA2: 4 μg NCN protein.

Group 5: TTN-gRNA1 and TTN-gRNA2 were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2.

Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).

2. After completing step 1, use the complete culture medium to culture for 12-18 hours, and then replace with a new complete culture medium for culture. The total incubation time after electroporation was 48 hours.

3. After completing step 2, trypsinize and collect cells, extract genomic DNA, perform PCR amplification with primer pair consisting of TTN-F55 and TTN-R560, and then perform 1% agarose gel electrophoresis.

The electropherogram is shown in Figure 3. The 505bp band is the wild-type band (WT), and the 254bp or so (the 505bp theoretical deletion of the wild-type band is 251bp) is the deletion mutant band (MT).

Gene deletion mutation efficiency=(MT gray level/MT band bp number)/(WT gray level/WT band bp number+MT gray level/MT band bp number)×100%. The efficiency of gene deletion mutation of the first group was 19.9%, the efficiency of gene deletion mutation of the second group was 39.9%, the efficiency of gene deletion mutation of the third group was 79.9%, and the efficiency of gene deletion mutation of the fourth group was 44.3%. No mutation occurred in the fifth group.

The results showed that the gene editing efficiency was the highest when the mass ratio of the two gRNAs to NCN protein was 1:1:4, and the actual dosage was 1μg:1μg:4μg. Therefore, the most suitable amount of the two gRNAs and NCN protein was determined to be 1 μg: 1 μg: 4 μg.

3. Comparison of gene editing efficiency between NCN protein and commercial Cas9 protein

1. Co-transfection of porcine primary fibroblasts

Cas9-A group: TTN-gRNA1, TTN-gRNA2 and commercial Cas9-A protein were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2: 4 μg Cas9-A protein.

pKG-GE4 group: TTN-gRNA1, TTN-gRNA2 and NCN proteins were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2: 4 μg NCN protein.

Cas9-B group: TTN-gRNA1, TTN-gRNA2 and commercial Cas9-B protein were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2: 4 μg Cas9-B protein.

Control group: TTN-gRNA1 and TTN-gRNA2 were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2.

Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).

2. After completing step 1, use the complete culture medium to culture for 12-18 hours, and then replace with a new complete culture medium for culture. The total incubation time after electroporation was 48 hours.

3. After completing step 2, trypsinize and collect cells, extract genomic DNA, perform PCR amplification with primer pair consisting of TTN-F55 and TTN-R560, and then perform 1% agarose gel electrophoresis.

The electropherogram is shown in Figure 4. The gene deletion mutation efficiency using commercial Cas9-A protein was 28.5%, the gene deletion mutation efficiency using NCN protein was 85.6%, and the gene deletion mutation efficiency using commercial Cas9-B protein was 16.6%.

The results show that, compared with the commercial Cas9 protein, the NCN protein prepared by the present invention significantly improves the gene editing efficiency.

Example 4. Screening of high-efficiency gRNA targets of TYR gene

Pig TYR gene information: encodes tyrosinase; located on chromosome 9; GeneID is 407745, Sus scrofa. The amino acid sequence encoded by the porcine TYR gene is shown in SEQ ID NO:8. In the genomic DNA, the porcine TYR gene has 5 exons. The human OCA-1A-related TYR gene point mutation is the R77L mutation. The R77L mutation corresponds to exon 1 of the porcine TYR gene. In the porcine genomic DNA, the partial sequence of the TYR gene (including the first exon and its upstream partial nucleotides) is shown in SEQ ID NO: 9.

1. Conservation analysis of TYR gene preset mutation sites and adjacent genome sequences

18 newborn Congjiangxiang pigs, including 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and 8 males (named A, B, C, D, E, F, G, H).

TYR-E1-JDF48:AGAATGCTCCTGGCTGCTTT;

TYR-E1-JDR511: CCTGTGGGGATGACGAAGTC;

TYR-E1-JDF70:ACTGCCTGCTCCTGGACTTTC;

TYR-E1-JDR508: GTGGGGATGACGAAGTCTGG.

The genome was extracted from the ear tissue of pig named 1 as a template, PCR amplification was performed using different primer pairs, and then 1% agarose gel electrophoresis was performed. The electropherogram is shown in Figure 5. In Figure 5: Group 1: using primer pair composed of TYR-E1-JDF48 and TYR-E1-JDR511; Group 2: using primer pair composed of TYR-E1-JDF48 and TYR-E1-JDR508; Group 3: using TYR- A primer pair consisting of E1-JDF70 and TYR-E1-JDR511; group 4: a primer pair consisting of TYR-E1-JDF70 and TYR-E1-JDR508. The results showed that the primer pair composed of TYR-E1-JDF70 and TYR-E1-JDR511 was preferably used to amplify the target fragment.

The genomic DNAs of 18 pigs were used as templates, and primer pairs consisting of TYR-E1-JDF70 and TYR-E1-JDR511 were used for PCR amplification, followed by 1% agarose gel electrophoresis. The electropherogram is shown in Figure 6. The PCR amplification products were recovered and sequenced, and the sequencing results were compared and analyzed with the TYR gene sequence in the public database. Conserved regions shared among 18 pigs were selected for the design of gRNA targets.

2. Screening targets

Several targets were initially screened by screening NGG (avoiding possible mutation sites), and 4 targets were further screened through preliminary experiments.

The 4 targets are as follows:

TYR-E1-gRNA1:ACCTCAGTTCCCCTTCACCG;

TYR-E1-gRNA2: GTCCTGTTGTAAAAGACGGA;

TYR-E1-gRNA3:AGACTCCCGTTCATCCACCC;

TYR-E1-gRNA4: GGTCCTGTTGTAAAAGACGG.

3. Preparation of recombinant plasmids

The plasmid pKG-U6gRNA was taken, digested with the restriction enzyme BbsI, and the vector backbone (a linear large fragment of about 3 kb) was recovered.

TYR-E1-gRNA1-S and TYR-E1-gRNA1-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (TYR-E1-gRNA1). The plasmid pKG-U6 gRNA (TYR-E1-gRNA1) expresses the sgRNA _TYR-E1-gRNA1 shown in SEQ ID NO: 10. sgRNA _TYR-E1-gRNA1 (SEQ ID NO: 10):

ACCUCAGUUCCCCUUCACCGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

TYR-E1-gRNA2-S and TYR-E1-gRNA2-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain the plasmid pKG-U6 gRNA (TYR-E1-gRNA2). The plasmid pKG-U6 gRNA (TYR-E1-gRNA2) expresses the sgRNA _TYR-E1-gRNA2 shown in SEQ ID NO: 11. sgRNA _TYR-E1-gRNA2 (SEQ ID NO: 11):

GUCCUGUUGUAAAAGACGGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

TYR-E1-gRNA3-S and TYR-E1-gRNA3-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with cohesive ends and the vector backbone were ligated to obtain plasmid pKG-U6gRNA (TYR-E1-gRNA3). The plasmid pKG-U6 gRNA (TYR-E1-gRNA3) expresses the sgRNA _TYR-E1-gRNA3 shown in SEQ ID NO:12. sgRNA _TYR-E1-gRNA3 (SEQ ID NO: 12):

AGACUCCCGUUCAUCCACCCguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

TYR-E1-gRNA4-S and TYR-E1-gRNA4-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule with cohesive ends and the vector backbone were ligated to obtain the plasmid pKG-U6gRNA (TYR-E1-gRNA4). The plasmid pKG-U6 gRNA (TYR-E1-gRNA4) expresses the sgRNA _TYR-E1-gRNA4 shown in SEQ ID NO:13. sgRNA _TYR-E1-gRNA4 (SEQ ID NO: 13):

GGUCCUGUUGUAAAAGACGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

TYR-E1-gRNA1-S: caccgACCTCAGTTCCCCTTCACCG;

TYR-E1-gRNA1-A: aaacCGGTGAAGGGGAACTGAGGTc;

TYR-E1-gRNA2-S: caccGTCCTGTTGTAAAAGACGGA;

TYR-E1-gRNA2-A:aaacTCCGTCTTTTACAACAGGAC;

TYR-E1-gRNA3-S: caccgAGACTCCCGTTCATCCACCC;

TYR-E1-gRNA3-A: aaacGGGTGGATGAACGGGAGTCTc;

TYR-E1-gRNA4-S: caccGGTCCTGTTGTAAAAGACGG;

TYR-E1-gRNA4-A: aaacCCGTCTTTTACAACAGGACC.

TYR-E1-gRNA1-S, TYR-E1-gRNA1-A, TYR-E1-gRNA2-S, TYR-E1-gRNA2-A, TYR-E1-gRNA3-S, TYR-E1-gRNA3-A, TYR- Both E1-gRNA4-S and TYR-E1-gRNA4-A are single-stranded DNA molecules.

4. Comparison of editing efficiency of different targets

1. Co-transfection

The first group: the plasmid pKG-U6gRNA (TYR-E1-gRNA1) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.92 μg plasmid pKG-U6gRNA (TYR-E1-gRNA1): 1.08 μg plasmid pKG-GE3.

The second group: the plasmid pKG-U6gRNA (TYR-E1-gRNA2) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.92 μg plasmid pKG-U6gRNA (TYR-E1-gRNA2): 1.08 μg plasmid pKG-GE3.

The third group: the plasmid pKG-U6gRNA (TYR-E1-gRNA3) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.92 μg plasmid pKG-U6gRNA (TYR-E1-gRNA3): 1.08 μg plasmid pKG-GE3.

The fourth group: the plasmid pKG-U6gRNA (TYR-E1-gRNA4) and the plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.92 μg plasmid pKG-U6gRNA (TYR-E1-gRNA4): 1.08 μg plasmid pKG-GE3.

The fifth group: porcine primary fibroblasts, electroporated with the same electroporation parameters without plasmids.

Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).

2. After completing step 1, use the complete culture medium to culture for 12-18 hours, and then replace with a new complete culture medium for culture. The total incubation time after electroporation was 48 hours.

3. After completing step 2, trypsinize and collect the cells, lyse the cells, extract genomic DNA, perform PCR amplification with primer pairs consisting of TYR-E1-JDF70 and TYR-E1-JDR511, and then perform 1% agarose gelation. gel electrophoresis. Detection of cell target gene mutation, electrophoresis chart shown in Figure 7.

The target product was recovered by cutting the gel and sent to a sequencing company for sequencing, and then the sequencing results were analyzed using the web version of the Synthego ICE tool to analyze the sequencing peak map to obtain the gene editing efficiency of different targets. The gene editing efficiencies of the first to fourth groups were 40%, 30%, 14%, and 18%, respectively, and no gene editing occurred in the fifth group. The results showed that TYR-E1-gRNA1 and TYR-E1-gRNA2 had higher editing efficiency.

Example 5. Preparation of single-cell clones with precise mutation of TYR gene using the method of somatic cell cloning

Two high-efficiency gRNA targets (TYR-E1-gRNA1 and TYR-E1-gRNA2) screened in Example 4 were selected.

1. Preparation of gRNA

1. Preparation of TYR-T7-gRNA1 transcription template and TYR-T7-gRNA5 transcription template

The TYR-T7-gRNA1 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO:14.

The TYR-T7-gRNA2 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO:15.

2. In vitro transcription to obtain gRNA

The TYR-T7-gRNA1 transcription template was taken and transcribed in vitro with Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified with MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain TYR-gRNA1. TYR-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO:16.

TYR-gRNA1 (SEQ ID NO: 16):

GGACCUCAGUUCCCCUUCACCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGGUGCUUUU.

The TYR-T7-gRNA2 transcription template was taken and transcribed in vitro with Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified with MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain TYR-gRNA2. TYR-gRNA2 is a single-stranded RNA, as shown in SEQ ID NO:17.

TYR-gRNA2 (SEQ ID NO: 17):

GGGUCCUGUUGUAAAAGACGGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGGUGCUUUU.

2. Synthesis of single-stranded Donor DNA mutated at the target site of the TYR gene

The single-stranded DNA corresponding to the R77L mutation was synthesized as Donor DNA. In addition to the target site mutation, the single-stranded DNA also contained the TYR-E1-gRNA1 and TYR-E1-gRNA2 target PAM or the 3'-end sequence synonymous mutation adjacent to the PAM. The single-stranded Donor DNA was named TYR-E1mut-ss126.

TYR-E1mut-ss126 is shown in SEQ ID NO:18.

TYR-E1mut-ss126 (SEQ ID NO: 18):

ggacatcattctgtccaaggcacccctgggacctcagttccccttcaccggAgtggatgaacTggagtcttggccctcAgtcttttacaacaggacctgccagtgctttggcaacttcatgggatt.

3. Transfection of porcine primary fibroblasts

1. Co-transfect porcine primary fibroblasts with TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein. Ratio: about 100,000 primary porcine fibroblasts: 1 μg TYR-gRNA1: 1 μg TYR-gRNA2: 2 μg TYR-E1mut-ss126: 4 μg NCN protein. Co-transfection was performed by electroporation, using a mammalian nucleofection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).

2. After completing step 1, use the complete culture medium to culture for 16-18 hours, and then replace with a new complete culture medium for culture. The total incubation time after electroporation was 48 hours.

3. After completing step 2, trypsinize and collect cells, then wash with complete culture medium, then resuspend with complete culture medium, and then pick individual clones and transfer them to 96-well plates (1 cell per well). , each well was filled with 100 μl of complete medium), and cultured for 2 weeks (replace with new complete medium every 2-3 days).

4. After completing step 3, trypsinize and collect the cells (about 2/3 of the cells obtained in each well are seeded into a 6-well plate with complete culture medium, and the remaining 1/3 is collected in a 1.5 mL centrifuge tube. ).

5. Take the 6-well plate from step 4, culture until the cells reach 80% confluence, digest with trypsin, collect the cells, and freeze the cells with cell freezing solution (90% complete medium + 10% DMSO, by volume). live.

6. Take the centrifuge tube of step 4, take the cells, perform cell lysis and extract genomic DNA, carry out PCR amplification with primer pairs consisting of TYR-E1-JDF70 and TYR-E1-JDR511, and then perform electrophoresis. Pig primary fibroblasts were used as wild-type controls (WT). The electropherogram is shown in Figure 8. The lane numbers in Figure 8 correspond to the cell numbers in Table 1.

7. After completing step 6, the PCR amplification product is recovered and sequenced.

There is only one sequencing result of porcine primary fibroblasts, and its genotype is homozygous wild type. If a single-cell clone has two sequencing results, one is consistent with the sequencing results of porcine primary fibroblasts, and the other is mutated compared with the sequencing results of porcine primary fibroblasts (mutations include one or more nucleotide deletions, insertions or substitutions), the genotype of the single-cell clone is heterozygous; if the sequencing results of a single-cell clone are two, compared with the sequencing results of porcine primary fibroblasts Mutation (mutation includes deletion, insertion or substitution of one or more nucleotides), the genotype of the single-cell clone is a biallelic different mutant; if the sequencing result of a single-cell clone is one, and the Compared with the sequencing results of porcine primary fibroblasts, there is a mutation (mutation includes deletion, insertion or substitution of one or more nucleotides), and the genotype of the single-cell clone is the same biallelic mutant; if a certain The sequencing result of the single-cell clone is one, which is consistent with the sequencing result of primary porcine fibroblasts, and the genotype of the single-cell clone is homozygous wild type.

The results are shown in Table 1. The genotypes of the single-cell clones numbered 5, 12, 18, 25, 30, 44, and 45 were homozygous wild-type. Single cells numbered 3, 8, 9, 11, 16, 17, 19, 23, 26, 27, 28, 29, 32, 33, 34, 36, 37, 38, 39, 41, 42, 47, 48 The genotype of the clone is heterozygous. The genotypes of the single-cell clones numbered 4, 7, 15, 21, 22, 24, 31, 35, and 46 were biallelic different mutants. The genotypes of the single-cell clones numbered 1, 2, 6, 10, 13, 14, 20, 40, and 43 were biallelic identical mutants. Among them, the single-cell clone numbered 9 is a heterozygous type of target site mutation (that is, one of the two homologous chromosomes has completed the replacement of single-stranded Donor DNA). The single-cell clones numbered 7, 15, 21, 24, 31, and 35 were biallelic different mutants of target site mutation (ie, one of the two homologous chromosomes completed the replacement of single-stranded Donor DNA). The single-cell clones numbered 2 and 40 are biallelic identical mutants of the target site mutation (that is, the single-stranded Donor DNA has been replaced on both homologous chromosomes). The rate of obtaining TYR gene exon 1 gene-edited single-cell clones was 85.42%, and single-cell clones with target site mutations (ie, single-cell clones numbered 2, 7, 9, 15, 21, 24, 31, 35, and 40) were obtained. cell clones) was 18.75%.

Exemplary sequencing alignment results are shown in Figures 9-14. Figure 9 shows the alignment results of forward and reverse sequencing of the single-cell clone numbered 12 with the target site wild-type sequence, which is homozygous wild-type. Figure 10 is the alignment result of the single-cell clone numbered 8 by reverse sequencing and the wild-type sequence of the target site, which is a heterozygous type. Figure 11 shows the alignment results of forward sequencing and reverse sequencing of the single-cell clone numbered 46 with the wild-type sequence of the target site, which is a biallelic different mutant. Figure 12 shows the alignment results of forward sequencing and reverse sequencing of the single-cell clone numbered 20 with the wild-type sequence of the target site, which is a biallelic identical mutant. Figure 13 shows the alignment results of forward sequencing and reverse sequencing of the single-cell clone numbered 9 with the wild-type sequence of the target site, which is a heterozygous type of the target site mutation. Figure 14 shows the alignment results of forward sequencing and reverse sequencing of the single-cell clone numbered 2 with the wild-type sequence of the target site, which is a biallelic identical mutant of the target site point mutation.

Table 1 Genotyping results of single-cell clones with TYR gene point mutation

Note: The target site mutation refers to the completion of the replacement of single-stranded Donor DNA; that is, the DNA molecule shown in SEQ ID NO: 19 in the chromosomal DNA is replaced by the DNA molecule shown in SEQ ID NO: 18.

The single-cell clones numbered 2 and 40 are biallelic identical mutants of the target site mutation (that is, the single-stranded Donor DNA has been replaced on both homologous chromosomes). Biallelic identical mutant cells mutated at the target site can be used for subsequent cloned pig production. The cells are used as donor cells for nuclear transplantation for somatic cell cloning, and cloned pigs can be obtained, which are model pigs of albinism.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the present invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> Nanjing Qizhen Genetic Engineering Co., Ltd.

<120> Gene Editing System for Construction of OCA-1A Albinism Model Pig Nuclear Transplantation Donor Cells and Its Application

<130> GNCYX212009

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9974

<212> DNA

<213> Artificial Sequence

<400> 1

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 540

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600

gagtattcaa catttccgtg tcgcccttat tcccttttttt gcggcatttt gccttcctgt 660

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620

aggatcttct tgagatcctt ttttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400

gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460

tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520

cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700

catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760

tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820

ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880

tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940

ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000

aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060

gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120

tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180

acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240

cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300

cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360

gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420

cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480

gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540

tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600

atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720

gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780

tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840

cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900

tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960

atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020

atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080

tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140

cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200

aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260

ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320

tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380

tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440

gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500

gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560

gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620

ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680

taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740

ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800

atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860

tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920

gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980

gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040

aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100

cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160

ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gaaacaaagc 5220

actattgcac tggcactctt accgttactg tttacccctg tgacaaaagc catgagcgat 5280

aaaattattc acctgactga cgacagtttt gacacggatg tactcaaagc ggacggggcg 5340

atcctcgtcg atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg 5400

gatgaaatcg ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcaa 5460

aaccctggca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa 5520

aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc 5580

ctcgacgcta acctggccgg ttctggttct ggccatatgc accatcatca tcatcatgac 5640

gatgacgata agatgcccaa aaagaaacga aaggtgggta tccacggagt cccagcagcc 5700

gacaaaaaat atagcatcgg cctggacatc ggtaccaaca gcgttggctg ggcagtgatc 5760

actgatgaat acaaagttcc atccaaaaaa tttaaagtac tgggcaacac cgaccgtcac 5820

tctatcaaaa aaaacctgat tggtgctctg ctgtttgaca gcggcgaaac tgctgaggct 5880

acccgtctga aacgtacggc tcgccgtcgc tacactcgtc gtaaaaaccg catctgttat 5940

ctgcaggaaa ttttctctaa cgaaatggca aaagttgatg atagcttctt tcatcgtctg 6000

gaagagagct tcctggtgga agaagataaa aaacacgaac gtcacccgat tttcggtaac 6060

attgtggatg aggttgccta ccacgagaaa tatccgacca tctaccatct gcgtaaaaaa 6120

ctggttgata gcactgacaa agcggatctg cgtctgatct acctggctct ggcacacatg 6180

atcaaattcc gtggtcactt cctgatcgaa ggtgatctga accctgataa ctccgacgtg 6240

gacaaactgt tcattcagct ggttcagacc tataaccagc tgttcgaaga aaacccgatc 6300

aacgcgtccg gtgtagacgc taaggcaatt ctgtctgcgc gtctgtctaa gtctcgtcgt 6360

ctggaaaacc tgattgcgca actgccaggt gaaaagaaaa acggcctgtt cggcaatctg 6420

atcgccctgt ccctgggtct gactccgaac tttaaatcca actttgacct ggcggaagat 6480

gccaagctgc agctgagcaa agatacctat gacgatgacc tggataacct gctggcacag 6540

atcggtgatc agtatgccga tctgttcctg gccgcgaaaa acctgtctga tgcgattctg 6600

ctgtctgata tcctgcgcgt taacactgaa attactaaag cgccgctgag cgcatccatg 6660

attaaacgtt acgatgaaca ccaccaggat ctgaccctgc tgaaagcgct ggtgcgtcag 6720

cagctgccgg aaaaatacaa ggagatcttc ttcgaccaga gcaaaaacgg ttacgcgggc 6780

tacattgatg gtggtgcatc tcaggaggaa ttctacaaat tcattaaacc gatcctggaa 6840

aaaatggatg gtactgaaga gctgctggtt aaactgaatc gtgaagatct gctgcgcaaa 6900

cagcgtacct tcgataacgg ttccatcccg catcagattc atctgggcga actgcacgct 6960

atcctgcgcc gtcaggaaga cttttatccg ttcctgaaag acaaccgtga gaaaattgaa 7020

aaaatcctga ccttccgtat tccgtactat gtaggtccgc tggcgcgtgg taactcccgt 7080

ttcgcttgga tgacccgcaa aagcgaagaa accatcaccc cgtggaattt cgaagaagtc 7140

gttgacaaag gcgcgtccgc gcagtctttc atcgaacgca tgacgaactt cgacaaaaac 7200

ctgccgaacg agaaagtgct gccgaaacac tctctgctgt acgagtactt cactgtgtac 7260

aacgaactga ccaaagtgaa atacgtcacc gaaggtatgc gtaaaccggc attcctgtcc 7320

ggtgagcaaa aaaaagcaat cgtggatctg ctgttcaaaa ccaaccgtaa agtaaccgtg 7380

aaacagctga aggaagacta tttcaagaaa atcgaatgtt ttgattctgt tgaaatctcc 7440

ggcgtggaag atcgcttcaa tgcgtccctg ggtacgtatc acgacctgct gaaaattatc 7500

aaagacaaag attttctgga caacgaggaa aacgaagaca tcctggagga tattgtactg 7560

accctgaccc tgttcgaaga ccgtgagatg atcgaagaac gcctgaaaac ctacgcccac 7620

ctgttcgatg acaaggtaat gaagcagctg aaacgtcgtc gttataccgg ctggggtcgt 7680

ctgtcccgta aactgatcaa tggcatccgt gataaacagt ctggcaaaac catcctggac 7740

ttcctgaaat ccgacggttt cgcgaatcgt aacttcatgc aactgattca tgacgattct 7800

ctgactttca aagaagacat ccagaaagca caggtttccg gccagggtga ctctctgcac 7860

gagcacattg ccaatctggc tggttctccg gctattaaaa agggtattct gcagactgtg 7920

aaagtagttg atgagctggt caaagtaatg ggccgtcaca agccggaaaa cattgtgatc 7980

gaaatggcac gtgaaaacca gacgacccag aaaggtcaga aaaactctcg tgaacgcatg 8040

aaacgtatcg aagaaggcat caaagaactg ggctctcaga tcctgaagga acaccctgta 8100

gaaaataccc agctgcagaa cgaaaagctg tatctgtatt acctgcagaa cggccgcgat 8160

atgtatgtgg accaggaact ggatatcaac cgcctgtccg attacgatgt agatcacatc 8220

gtgccgcaaa gcttcctgaa agacgacagc attgacaaca aagtactgac ccgttctgat 8280

aagaaccgtg gcaaatccga taacgtcccg tctgaagaag ttgttaaaaa aatgaaaaac 8340

tattggcgtc agctgctgaa cgcgaaactg atcacccagc gtaagttcga caatctgact 8400

aaagctgagc gcggtggtct gtccgaactg gataaagcgg gttttatcaa acgccagctg 8460

gttgaaaccc gtcagatcac gaagcacgtt gcgcagattc tggactctcg tatgaacacc 8520

aaatacgacg aaaacgacaa actgatccgc gaggttaagg ttatcaccct gaaaagcaaa 8580

ctggtatccg attttcgtaa agactttcag ttctacaaag tgcgcgaaat taacaactat 8640

caccacgctc acgatgcata tctgaatgca gttgttggca cggcgctgat caaaaagtat 8700

ccgaaactgg aatctgaatt cgtatacggc gattacaaag tgtatgacgt tcgtaagatg 8760

atcgcaaaat ccgagcagga aattggtaag gcgacggcga aatacttctt ttattccaat 8820

attatgaact ttttcaaaac cgaaatcacc ctggcgaatg gtgaaattcg taaacgcccg 8880

ctgatcgaaa ccaacggtga aactggtgaa atcgtttggg acaaaggccg cgacttcgcg 8940

accgtgcgta aagttctgtc tatgccgcaa gtgaacatcg tcaagaagac cgaagtacaa 9000

accggcggtt ttagcaaaga gagcattctg ccaaaacgta actccgacaa actgatcgcg 9060

cgcaagaaag actgggatcc gaaaaaatac ggtggtttcg attctccaac cgttgcttat 9120

tccgttctgg tggtagccaa agttgagaaa ggtaaaagca aaaaactgaa atccgtaaag 9180

gaactgctgg gtattactat catggagcgt agctccttcg aaaaaaaccc gatcgatttt 9240

ctggaagcga aaggctataa agaagtcaaa aaggacctga tcatcaaact gccaaaatac 9300

agcctgttcg agctggaaaa cggccgtaaa cgtatgctgg catctgcggg cgaactgcag 9360

aaaggcaacg agctggctct gccgtccaaa tacgtgaact ttctgtacct ggcctctcac 9420

tacgaaaaac tgaaaggttc cccggaagac aacgaacaga aacagctgtt cgtagagcag 9480

cacaaacact acctggacga gatcatcgaa cagatttctg aattttctaa acgtgtgatt 9540

ctggctgatg cgaatctgga taaagttctg tctgcctata acaagcatcg tgacaaaccg 9600

atccgcgaac aggctgagaa catcatccac ctgttcactc tgactaacct gggcgcgcca 9660

gcggctttca agtactttga taccaccatt gaccgcaagc gttacacctc cactaaagaa 9720

gtgctggacg cgactctgat ccaccagtcc atcaccggtc tgtacgagac ccgtatcgat 9780

ctgagccagc tgggcggtga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 9840

aagaaaaagt gacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 9900

actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 9960

aactatatcc ggat 9974

<210> 2

<211> 1547

<212> PRT

<213> Artificial Sequence

<400> 2

Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr

1 5 10 15

Pro Val Thr Lys Ala Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp

20 25 30

Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp

35 40 45

Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu

50 55 60

Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu

65 70 75 80

Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly

85 90 95

Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys

100 105 110

Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn

115 120 125

Leu Ala Gly Ser Gly Ser Gly His Met His His His His His His Asp

130 135 140

Asp Asp Asp Lys Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly

145 150 155 160

Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr

165 170 175

Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser

180 185 190

Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys

195 200 205

Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala

210 215 220

Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn

225 230 235 240

Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val

245 250 255

Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu

260 265 270

Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu

275 280 285

Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys

290 295 300

Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala

305 310 315 320

Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp

325 330 335

Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val

340 345 350

Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly

355 360 365

Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg

370 375 380

Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu

385 390 395 400

Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys

405 410 415

Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp

420 425 430

Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln

435 440 445

Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu

450 455 460

Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu

465 470 475 480

Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr

485 490 495

Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu

500 505 510

Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly

515 520 525

Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu

530 535 540

Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp

545 550 555 560

Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln

565 570 575

Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe

580 585 590

Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr

595 600 605

Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg

610 615 620

Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn

625 630 635 640

Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu

645 650 655

Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro

660 665 670

Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr

675 680 685

Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser

690 695 700

Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg

705 710 715 720

Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu

725 730 735

Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala

740 745 750

Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp

755 760 765

Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu

770 775 780

Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys

785 790 795 800

Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg

805 810 815

Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly

820 825 830

Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser

835 840 845

Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser

850 855 860

Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly

865 870 875 880

Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile

885 890 895

Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys

900 905 910

Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg

915 920 925

Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met

930 935 940

Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys

945 950 955 960

Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu

965 970 975

Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp

980 985 990

Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser

995 1000 1005

Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp

1010 1015 1020

Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys

1025 1030 1035 1040

Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1045 1050 1055

Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser

1060 1065 1070

Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg

1075 1080 1085

Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr

1090 1095 1100

Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr

1105 1110 1115 1120

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr

1125 1130 1135

Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu

1140 1145 1150

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu

1155 1160 1165

Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1170 1175 1180

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1185 1190 1195 1200

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1205 1210 1215

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1220 1225 1230

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys

1235 1240 1245

Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln

1250 1255 1260

Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp

1265 1270 1275 1280

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1285 1290 1295

Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val

1300 1305 1310

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly

1315 1320 1325

Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe

1330 1335 1340

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1345 1350 1355 1360

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1365 1370 1375

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1380 1385 1390

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu

1395 1400 1405

Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln

1410 1415 1420

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1425 1430 1435 1440

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1445 1450 1455

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1460 1465 1470

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys

1475 1480 1485

Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu

1490 1495 1500

Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1505 1510 1515 1520

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala

1525 1530 1535

Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1540 1545

<210> 3

<211> 1399

<212> PRT

<213> Artificial Sequence

<400> 3

Met Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala

1 5 10 15

Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly

20 25 30

Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys

35 40 45

Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly

50 55 60

Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys

65 70 75 80

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

85 90 95

Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe

100 105 110

Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His

115 120 125

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

130 135 140

Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser

145 150 155 160

Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met

165 170 175

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

180 185 190

Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn

195 200 205

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

210 215 220

Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu

225 230 235 240

Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu

245 250 255

Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp

260 265 270

Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp

275 280 285

Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu

290 295 300

Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile

305 310 315 320

Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met

325 330 335

Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala

340 345 350

Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp

355 360 365

Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln

370 375 380

Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly

385 390 395 400

Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys

405 410 415

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly

420 425 430

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

435 440 445

Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

450 455 460

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

465 470 475 480

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val

485 490 495

Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn

500 505 510

Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu

515 520 525

Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

530 535 540

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys

545 550 555 560

Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val

565 570 575

Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser

580 585 590

Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr

595 600 605

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

610 615 620

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

625 630 635 640

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

645 650 655

Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr

660 665 670

Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys

675 680 685

Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala

690 695 700

Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

705 710 715 720

Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His

725 730 735

Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

740 745 750

Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg

755 760 765

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

770 775 780

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

785 790 795 800

Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val

805 810 815

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

820 825 830

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

835 840 845

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp

850 855 860

Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly

865 870 875 880

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

885 890 895

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

900 905 910

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys

915 920 925

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

930 935 940

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu

945 950 955 960

Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys

965 970 975

Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

980 985 990

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

995 1000 1005

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

1010 1015 1020

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser

1025 1030 1035 1040

Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn

1045 1050 1055

Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1060 1065 1070

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val

1075 1080 1085

Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met

1090 1095 1100

Pro Gln Val Asn Ile Val Lys Lys Lys Thr Glu Val Gln Thr Gly Gly Phe

1105 1110 1115 1120

Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1125 1130 1135

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1140 1145 1150

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys

1155 1160 1165

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1170 1175 1180

Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys

1185 1190 1195 1200

Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1205 1210 1215

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1220 1225 1230

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1235 1240 1245

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1250 1255 1260

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr

1265 1270 1275 1280

Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile

1285 1290 1295

Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1300 1305 1310

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe

1315 1320 1325

Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr

1330 1335 1340

Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala

1345 1350 1355 1360

Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1365 1370 1375

Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala

1380 1385 1390

Gly Gln Ala Lys Lys Lys Lys Lys

1395

<210> 4

<211> 225

<212> DNA

<213> Artificial Sequence

<400> 4

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggagagc acagtcagcc tggcggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 5

<211> 225

<212> DNA

<213> Artificial Sequence

<400> 5

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggcttcc agaattggat ctccggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 6

<211> 102

<212> RNA

<213> Artificial Sequence

<400> 6

ggagagcaca gucagccugg cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 7

<211> 102

<212> RNA

<213> Artificial Sequence

<400> 7

ggcuuccaga auuggaucuc cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 8

<211> 531

<212> PRT

<213> Sus scrofa

<400> 8

Met Leu Leu Ala Ala Leu Tyr Cys Leu Leu Trp Thr Phe Gln Thr Ser

1 5 10 15

Ala Gly His Phe Pro Arg Ala Cys Ala Ser Ser Lys Asn Leu Met Glu

20 25 30

Lys Glu Cys Cys Pro Pro Trp Gly Gly Asp Gly Ser Pro Cys Gly Gln

35 40 45

Leu Ser Gly Arg Gly Ser Cys Gln Asp Ile Ile Leu Ser Lys Ala Pro

50 55 60

Leu Gly Pro Gln Phe Pro Phe Thr Gly Val Asp Glu Arg Glu Ser Trp

65 70 75 80

Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Phe Gly Asn Phe Met

85 90 95

Gly Phe Asn Cys Gly Ser Cys Lys Phe Gly Phe Gln Gly Pro Asn Cys

100 105 110

Thr Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Val

115 120 125

Pro Glu Lys Asn Lys Phe Leu Ala Tyr Leu Thr Leu Ala Lys His Thr

130 135 140

Thr Ser Pro Asp Phe Val Ile Pro Thr Gly Thr Tyr Gly Gln Met Asn

145 150 155 160

Asn Gly Ser Thr Pro Met Phe Ser Asp Ile Asn Ile Tyr Asp Leu Phe

165 170 175

Val Trp Met His Tyr Tyr Val Ser Arg Asp Thr Leu Leu Gly Gly Ser

180 185 190

Glu Ile Trp Arg Asp Ile Asp Phe Ala His Glu Ala Pro Gly Phe Leu

195 200 205

Pro Trp His Arg Leu Phe Leu Leu Val Trp Glu Gln Glu Ile Gln Lys

210 215 220

Leu Thr Gly Asp Glu Asn Phe Thr Ile Pro Tyr Trp Asp Trp Arg Asp

225 230 235 240

Ala Glu Asn Cys Asp Ile Cys Thr Asp Glu Tyr Met Gly Gly Arg Asn

245 250 255

Pro Ala Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp

260 265 270

Gln Ile Ile Cys Ser Arg Leu Glu Glu Tyr Asn Ser Arg Gln Ala Leu

275 280 285

Cys Asn Gly Thr Pro Glu Gly Pro Leu Leu Arg Asn Pro Gly Asn His

290 295 300

Asp Lys Ser Arg Thr Pro Arg Leu Pro Ser Ser Ala Asp Val Glu Phe

305 310 315 320

Cys Leu Ser Leu Thr Gln Tyr Glu Ser Gly Ser Met Asp Lys Ser Ala

325 330 335

Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr

340 345 350

Gly Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile

355 360 365

Tyr Met Asn Gly Thr Met Ser Gln Val Gln Gly Ser Ala Asn Asp Pro

370 375 380

Ile Phe Leu Leu His His Ala Phe Val Asp Ser Ile Phe Glu Gln Trp

385 390 395 400

Leu Arg Lys His His Pro Leu Leu Glu Val Tyr Pro Glu Ala Asn Ala

405 410 415

Pro Ile Gly His Asn Arg Glu Ser Tyr Met Val Pro Phe Ile Pro Leu

420 425 430

Tyr Arg Asn Gly Asp Phe Phe Ile Ser Ser Arg Asp Leu Gly Tyr Asp

435 440 445

Tyr Ser Tyr Leu Gln Asp Ser Glu Pro Asp Phe Phe Gln Asp Tyr Ile

450 455 460

Lys Pro Tyr Leu Glu Gln Ala Ser Arg Ile Trp Pro Trp Leu Leu Gly

465 470 475 480

Ala Ala Val Val Gly Ser Val Leu Thr Ala Val Leu Gly Gly Ile Thr

485 490 495

Arg Arg Leu Cys Cys Arg Arg Lys Arg Lys Arg Leu Pro Glu Glu Lys

500 505 510

Gln Pro Leu Leu Met Glu Lys Glu Asp Tyr Asp Ser Leu Leu Tyr Gln

515 520 525

Ser His Leu

530

<210> 9

<211> 869

<212> DNA

<213> Sus scrofa

<400> 9

tgtggctcaa ttaacaagct caaacagacc ttgtgagaac tagaggaaga atgctcctgg 60

ctgctttgta ctgcctgctc tggactttcc agacttccgc cggacacttc cctcgagcct 120

gtgcctcctc caagaacctg atggagaagg aatgctgccc accctgggga ggtgatggga 180

gtccctgtgg ccagctctca ggcaggggtt cctgtcagga catcattctg tccaaggcac 240

ccctgggacc tcagttcccc ttcaccgggg tggatgaacg ggagtcttgg ccctccgtct 300

tttacaacag gacctgccag tgctttggca acttcatggg atttaactgt ggaagttgta 360

agtttggctt tcagggaccc aactgcacag agaggcgact tttggtcaga agaaacatct 420

ttgatttgag tgtgccagag aagaacaaat ttcttgccta cctcacttta gcgaaacata 480

ctaccagccc agacttcgtc atccccacag gcacctatgg ccaaatgaat aatggatcaa 540

cacccatgtt tagtgacatc aacatttatg acctctttgt ctggatgcat tattacgtgt 600

ctagggacac actacttggg ggctctgaaa tctggagaga cattgatttt gctcatgaag 660

caccaggttt cctgccttgg catcgactct tcttgctggt atgggaacaa gaaatccaga 720

agctgacagg ggatgagaac ttcactattc catactggga ttggcgagat gcagaaaatt 780

gtgacatttg cacagatgag tacatgggag gacgcaaccc tgcaaaccct aatctactca 840

gcccagcatc cttcttctcc tcttggcag 869

<210> 10

<211> 100

<212> RNA

<213> Artificial Sequence

<400> 10

accucaguuc cccuucaccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 11

<211> 100

<212> RNA

<213> Artificial Sequence

<400> 11

guccuguugu aaaagacgga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 12

<211> 100

<212> RNA

<213> Artificial Sequence

<400> 12

agacucccgu ucauccaccc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 13

<211> 100

<212> RNA

<213> Artificial Sequence

<400> 13

gguccuguug uaaaagacgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

<210> 14

<211> 225

<212> DNA

<213> Artificial Sequence

<400> 14

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

ataggacctc agttcccctt caccggtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 15

<211> 225

<212> DNA

<213> Artificial Sequence

<400> 15

ggcttgtcgg actcttcgct attacgccag ctggcgaagg gggatgtgct gcaaggcgat 60

taagttgggt aacgccaggg ttttcccagt cacgacgtta ggaaattaat acgactcact 120

atagggtcct gttgtaaaag acggagtttt agagctagaa atagcaagtt aaaataaggc 180

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttt 225

<210> 16

<211> 102

<212> RNA

<213> Artificial Sequence

<400> 16

ggaccucagu uccccuucac cgguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 17

<211> 102

<212> RNA

<213> Artificial Sequence

<400> 17

ggguccuguu guaaaagacg gaguuuuaga gcuagaaaua gcaaguuaaa auaaggcuag 60

uccguuauca acuugaaaaa guggcaccga gucggugcuu uu 102

<210> 18

<211> 126

<212> DNA

<213> Artificial Sequence

<400> 18

ggacatcatt ctgtccaagg cacccctggg acctcagttc cccttcaccg gagtggatga 60

actggagtct tggccctcag tcttttacaa caggacctgc cagtgctttg gcaacttcat 120

gggatt 126

<210> 19

<211> 126

<212> DNA

<213> Sus scrofa

<400> 19

ggacatcatt ctgtccaagg cacccctggg acctcagttc cccttcaccg gggtggatga 60

acgggagtct tggccctccg tcttttacaa caggacctgc cagtgctttg gcaacttcat 120

gggatt 126

Claims (15)

1. A method for preparing recombinant cells, comprising the steps of: substituting the DNA molecules shown in SEQ ID NO: 19 in the chromosomal DNA of pig cells with the DNA molecules shown in SEQ ID NO: 18 to obtain recombinant cells. 2 . The method according to claim 1 , wherein the method of replacing the DNA molecule shown in SEQ ID NO: 19 in the chromosomal DNA of the pig cell with the DNA molecule shown in SEQ ID NO: 18 is: TYR -gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein are co-transfected into pig cells; the TYR-gRNA1 is sgRNA, and its target sequence binding region is shown in nucleotides 3-22 in SEQ ID NO: 16 ; Described TYR-gRNA2 is sgRNA, and its target sequence binding region is shown in 3-22 nucleotides in SEQ ID NO: 17; Described TYR-E1mut-ss126 is the single strand shown in SEQ ID NO: 18 DNA molecule; the NCN protein is Cas9 protein or a fusion protein with Cas9 protein. 3. The method according to claim 2, characterized in that: the ratios of porcine cells, TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein are successively: 100,000 porcine cells: 0.8-1.2 μg TYR-gRNA1: 0.8-1.2 μg TYR-gRNA2: 1.8-2.2 μg TYR-E1mut-ss126: 3-5 μg NCN protein. 4. The application of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein in the preparation kit; TYR-gRNA1 is TYR-gRNA1 described in claim 2; TYR-gRNA2 is TYR-gRNA2 described in claim 2; TYR-E1mut-ss126 is TYR-E1mut-ss126 described in claim 2; NCN The protein is the NCN protein described in claim 2; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing albinism model pigs; (c) preparing albinism cell model or albinism tissue model or albinism organ model . 5. The application of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and PRONCN protein in the preparation kit; TYR-gRNA1 is TYR-gRNA1 described in claim 2; TYR-gRNA2 is TYR-gRNA2 described in claim 2; TYR-E1mut-ss126 is TYR-E1mut-ss126 described in claim 2; The PRONCN protein sequentially includes the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease cleavage site, nuclear localization signal, Cas9 protein, nuclear localization signal; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing albinism model pigs; (c) preparing albinism cell model or albinism tissue model or albinism organ model . 6. The application of TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and specific plasmids in the preparation kit; TYR-gRNA1 is TYR-gRNA1 described in claim 2; TYR-gRNA2 is TYR-gRNA2 described in claim 2; TYR-E1mut-ss126 is TYR-E1mut-ss126 described in claim 2; The specific plasmid sequentially includes the following elements from upstream to downstream: promoter, operon, ribosome binding site, encoding gene of PRONCN protein, terminator; described PRONCN protein sequentially includes the following elements from upstream to downstream: signal peptide, Molecular chaperone protein, protein tag, protease cleavage site, nuclear localization signal, Cas9 protein, nuclear localization signal; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing albinism model pigs; (c) preparing albinism cell model or albinism tissue model or albinism organ model . 7. A kit comprising TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and NCN protein; TYR-gRNA1 is TYR-gRNA1 described in claim 2; TYR-gRNA2 is TYR-gRNA2 described in claim 2; TYR-E1mut-ss126 is TYR-E1mut-ss126 described in claim 2; NCN The protein is the NCN protein described in claim 2; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing albinism model pigs; (c) preparing albinism cell model or albinism tissue model or albinism organ model . 8. A kit comprising TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and PRONCN protein; TYR-gRNA1 is TYR-gRNA1 described in claim 2; TYR-gRNA2 is TYR-gRNA2 described in claim 2; TYR-E1mut-ss126 is TYR-E1mut-ss126 described in claim 2; The PRONCN protein sequentially includes the following elements from upstream to downstream: signal peptide, molecular chaperone protein, protein tag, protease cleavage site, nuclear localization signal, Cas9 protein, nuclear localization signal; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing albinism model pigs; (c) preparing albinism cell model or albinism tissue model or albinism organ model . 9. A kit comprising TYR-gRNA1, TYR-gRNA2, TYR-E1mut-ss126 and a specific plasmid; TYR-gRNA1 is TYR-gRNA1 described in claim 2; TYR-gRNA2 is TYR-gRNA2 described in claim 2; TYR-E1mut-ss126 is TYR-E1mut-ss126 described in claim 2; The specific plasmid sequentially includes the following elements from upstream to downstream: promoter, operon, ribosome binding site, encoding gene of PRONCN protein, terminator; described PRONCN protein sequentially includes the following elements from upstream to downstream: signal peptide, Molecular chaperone protein, protein tag, protease cleavage site, nuclear localization signal, Cas9 protein, nuclear localization signal; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant cells; (b) preparing albinism model pigs; (c) preparing albinism cell model or albinism tissue model or albinism organ model . 10. The method according to any one of claims 2 to 3 or the application according to claim 4 or the kit according to claim 7, wherein the NCN protein is as shown in SEQ ID NO: 3 Show. 11. The method or application or kit of claim 10, wherein: The preparation method of the NCN protein comprises the following steps: (1) The plasmid pKG-GE4 was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria; (2) using liquid medium to cultivate the recombinant bacteria at 30°C, then adding IPTG and inducing culture at 25°C, then collecting the bacterial cells; (3) thalline fragmentation is carried out by the collected thalline, and crude protein solution is collected; (4) using affinity chromatography to purify the fusion protein with the His ₆ tag from the crude protein solution; (5) using enterokinase with His ₆ tag to digest the fusion protein with His ₆ tag, and then using Ni-NTA resin to remove the protein with His ₆ tag to obtain purified NCN protein; Plasmid pKG-GE4 has the fusion gene shown in nucleotides 5209-9852 in SEQ ID NO:1. 12. The recombinant cell prepared by the method according to any one of claims 1 to 3. 13. The application of the recombinant cell of claim 12 in the preparation of albinism model pigs. 14. The pig tissue, pig organ or pig cell of albinism model pig prepared by using the recombinant cell of claim 12. 15. The application of the recombinant cell according to claim 12, the pig tissue according to claim 14, the pig organ according to claim 14, the pig cell according to claim 14 or the albinism model pig prepared by using the recombinant cell according to claim 12 , as (d1) or (d2) or (d3) or (d4) as follows: (d1) Screening drugs for the treatment of albinism; (d2) Evaluating the efficacy of albinism drugs; (d3) Efficacy evaluation of gene therapy and/or cell therapy for albinism; (d4) To study the pathogenesis of albinism.

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