CN104726490A - Targeting carrier expressing human serum albumin and construction method thereof - Google Patents

Abstract

The invention discloses a targeting vector for expressing human serum albumin, which comprises a human serum albumin gene and two homologous sequences of chicken origin subjected to homologous recombination: 5' homology arm, 3' homology arm, It also contains the neo gene for drug screening and the reporter gene EGFP. The targeting vector expressing human serum albumin of the present invention can be used to prepare transgenic chickens. After the transgenic chickens are bred, human serum albumin will be specifically expressed in eggs (eggs), while other cells of the transgenic chickens It does not express human serum albumin, and is convenient for separation and purification, and the human serum albumin produced by transgenic chickens is more similar to normal serum albumin in humans after modification, which is more conducive to its biological function.

Description

Targeting vector expressing human serum albumin and its construction method

technical field

The present invention relates to a targeting vector capable of producing human serum albumin by using eggs of transgenic chickens, and relates to the independent design and cloning of a plurality of regulatory response elements suitable for the expression of human serum albumin in egg whites and the ability to recombine human serum albumin Based on the elements of the chicken ovalbumin sequence, and cloned by genetic engineering, the gene targeting vector ROSA26-shALBl that specifically expresses human serum albumin in the chicken oviduct tissue is constructed. The construction of this vector is obtained later and can express human serum albumin in egg white The core technology of protein transgenic chicken belongs to the technical field of genetic engineering.

Background technique

Human serum albumin is a single-chain protein composed of 585 amino acids with a molecular weight of 67kDa. Albumin is the protein with the highest content in plasma. Albumin has the functions of maintaining blood osmotic pressure, anti-shock, transportation and detoxification, and promoting liver cell repair and regeneration. It is the most widely used blood product in the world. Clinically, it is mainly used for the treatment of burns, hemorrhagic shock, edema, and hypoalbuminemia.

The traditional method of obtaining medical serum albumin is to extract it from the plasma of healthy people. However, due to the possibility of contamination of human proviruses in plasma products and the depletion of blood sources, this method is greatly limited and it is difficult to obtain sufficient blood products. At present, our country needs 120 tons of albumin every year, and the albumin that can be prepared through blood donation is only 1/3 of the required amount. Therefore, the supply of albumin is seriously in short supply.

At present, yeast and Escherichia coli are commonly used in recombinant biopharmaceuticals, but this method has many disadvantages, such as: large differences between microorganisms and human environment, low biological activity of expressed products, difficulty in completely removing adulterated microbial toxins, and complicated production processes. It is difficult to form large-scale production. Therefore, so far, no related products have been listed. Recently, international companies have also used transgenic technology to prepare transgenic animals to prepare human recombinant protein products. Compared with traditional microbial expression systems, animal bioreactors have the following characteristics: 1) The produced protein has high biological activity and can perform various recombinant proteins. Post-translational processing, very close to natural products; 2) large amount of expression, low cost; 3) easy purification, high quality products, avoiding the pollution of chemical and biological toxins in other production methods, safe and reliable; 4) easy industrialization: transfer The target gene can be inherited, so that a certain group has the same characteristics, and the group can be expanded through animal breeding for large-scale production. However, human genes are randomly inserted into the genome of animals. Although it has great advantages compared with microbial fermentation, there are also many uncontrollable factors and defects, and there is a lot of room for improvement: 1) The integration and expression of the target gene The efficiency is low; 2) It may cause gene mutation of the host cell; 3) The transgenic model does not match the expected results, or the expression of individual dysfunction; 4) The corresponding protein of the animal itself is still expressed, which is difficult to remove during purification, which is easy to cause Allergic reaction in humans. Therefore, transgenic animals are not yet ideal bioreactors.

In summary, finding a suitable method for expressing human serum albumin is an urgent problem to be solved.

Contents of the invention

Aiming at the above prior art, the present invention provides a targeting vector expressing human serum albumin and its construction method. The vector constructed by the present invention can integrate human serum albumin into the chicken genome and carry out gene knock-in with chicken ovalbumin as a target, transform the vector into the germ cells of chickens, and obtain transgenic chickens. Human serum albumin will be specifically expressed in eggs (eggs). The present invention genetically modifies chickens through gene recombination technology, changes the composition of its genome, and breeds transgenic chickens capable of expressing human serum albumin in chicken eggs (eggs), and ensures that the genetic modification changes can be inherited by offspring. Pass it on for a long time.

The present invention is achieved through the following technical solutions:

In order to meet the above-mentioned experimental design, the present invention recombines the regulatory response element suitable for the expression of human serum albumin in egg white and the gene of human serum albumin into the element of chicken ovalbumin sequence, and performs genetic engineering cloning to form human serum The gene targeting vector ROSA26-shALBl for the specific expression of albumin in chicken oviduct tissue, the specific technical scheme is as follows:

A targeting vector expressing human serum albumin, the construction map is shown in Figure 1, and the structure is: it contains the human serum albumin gene (the sequence is shown in SEQ ID NO.2, and the expressed protein sequence is shown in SEQ ID NO. 1), and two homologous sequences of the chicken source that undergo homologous recombination: 5'homologous arm, 3'homologous arm, and the 5'homologous arm is 1.7kb (shown in SEQ ID NO.3), The 3' homology arm is 5.2kb (shown in SEQ ID NO.4), the 5' end of the human serum albumin gene is connected with a kozak sequence, and the kozak sequence is GCCACC, and also includes a neo gene and a reporter gene for drug screening EGFP.

The method for constructing a targeting vector for expressing human serum albumin, the key of which is to construct a gene site-directed insertion that can accurately insert human serum albumin into the genome of PGC cells and successfully express human serum albumin The carrier, which is used to transfer the PGC cells with genetic material modification into embryos by microinjection technology, can obtain transgenic chickens. The oviduct cells and germ cells of the transgenic chicken can be developed from the modified PGC cells, express and produce human serum albumin during egg formation, and stably transmit the transgene modification in its offspring to produce offspring transgenic chickens. The construction method is as follows:

After searching through NCBI, after obtaining the target gene (gene of human serum albumin), design specific primers to amplify the 5' homology arm, 3' homology arm and human serum albumin gene (human serum albumin gene After amplification, a kozak sequence was added at the 5' end); then, the EGFP-ROSA-26 gene targeting vector (the vector is described in the reference: Canonical NF-kappaB activity, dispensable for B cell development, replacesBAFF-receptor signals and promotes B cell proliferation upon activation. Sasaki et al (Immunity.2006Jun.24(6):729-39.PubMed)) is used as the basis for site-directed insertion of human serum albumin gene integration: use pBluescript II SK (+) vector (commercialized vector) construct homology arm and human serum albumin gene fusion (first recombine the 5' homology arm into pBluescript II SK (+) vector, then recombine human serum albumin gene into pBluescript II SK (+) ) carrier, using specific primers to clone the fusion gene of the 5' homology arm and the human serum albumin gene), and then connecting the fusion gene to the EGFP-ROSA-26 gene targeting vector (using the infusion reagent, which is commercial product), and then connect the 3' homology arm to the EGFP-ROSA-26 gene targeting vector to obtain the targeting vector expressing human serum albumin; all sequences were obtained by PCR.

The specific primers are as follows:

The primers used to amplify the 5' homology arm are F1, F2:

F1: 5'-CCGCGGCCGCTCTATGGCGTCAAAGGTCA-3'; as shown in SEQ ID NO.5;

F2: 5'-CCCCCCGGGGAAAATGTAAGGATGG-3'; as shown in SEQ ID NO.6;

The primers used to amplify the human serum albumin gene are F3 and F4:

F3: 5'-CCCGGGGCCGAAATGAAGTGGGTAACCTTTAT-3'; as shown in SEQ ID NO.7;

F4: 5'-CCATCGATTTATAAGCCTAAGGCAGCTTGAC-3'; as shown in SEQ ID NO.8;

The primers used to amplify the 3' homology arm are F5, F6:

F5: 5'-TTCCACATCCACCGGTTGGTTTATTTAGAGTGGCA-3'; as shown in SEQ ID NO.9;

F6: 5'-GTTGGCGCCTACCGGACTGGCAGGGCTTATTTTCA-3'; as shown in SEQ ID NO.10;

Primers F7 and F8 are used to clone the 5' homology arm + human serum albumin gene connected to the pBluescript II SK (+) vector and add the homology arm required for infusion reagent connection:

F7: 5'-TCTTTCCAGTGGTTAGCTCTATGGCGTCAAAGGTC-3'; as shown in SEQ ID NO.11;

F8: 5'-ATACGAAGTTATTTATTATAAGCCTAAGGCAGCTT-3'; as shown in SEQ ID NO.12.

In the above method:

The 5' homology arm and human serum albumin fusion gene contains (5'-3' sequence) 5' homology arm (1.7kb), which is the upstream sequence of the chicken ovalbumin gene, including the ovalbumin promoter, 5 'Two enzyme cutting sites 5'NotI/3'XmaI are introduced at both ends of the homology arm;

Kozak (9bp) + human serum albumin cDNA sequence (1.8kb) contains a stop codon. The kozak sequence exists in a sequence of eukaryotic mRNA, which plays an important role in the initiation of translation and can enhance gene expression. Point introduction of 5'XmaI/3'ClaI;

The 3'homology arm (5.2kb) does not introduce a restriction site into the chicken ovalbumin sequence, and is connected by infusion reagent.

The 5' homology arm, the kozak9bp+ human serum albumin cDNA sequence and the 3' homology arm are PCR extension products, the kozak sequence is designed into the primer and connected to the 5' end of the human serum albumin cDNA sequence; the 5' homology arm The cDNA sequence of kozak9bp+ human serum albumin was first connected to pBluescript II SK (+) and then connected to the EGFP-ROSA-26 vector.

The application of the targeting vector expressing human serum albumin in the preparation of transgenic chickens: the above-mentioned targeting vector expressing human serum albumin is transformed into primordial germ cells of chickens, and after culturing and screening, microinjection technology is used to inject The successfully transformed primordial germ cells are injected into chicken embryos to cultivate transgenic chickens. After the transgenic chickens reproduce, the eggs (eggs) produced by them will specifically express human serum albumin, while other cells of the transgenic chickens do not. Expresses human serum albumin.

A method for obtaining a transgenic chicken expressing human serum albumin, the steps are: recombining the above-mentioned targeting vector expressing human serum albumin into chicken ovalbumin by means of homologous recombination (the schematic diagram is shown in Figure 3) Gene, and then the successfully transformed recombinant cells are injected into chicken embryos to cultivate transgenic chickens. After the transgenic chickens are bred, the eggs (eggs) produced by them will specifically express human serum albumin (take chicken oviduct as an example) Bioreaction Generator), while other cells of transgenic chickens do not express human serum albumin.

A method for preparing a bioreactor, the bioreactor is composed of a transgenic chicken oviduct, the method comprises using the targeting vector constructed above to express human serum albumin to prepare a transgenic chicken.

A method for producing human serum albumin from the oviduct of a transgenic chicken, the steps of which are: recombining the above-mentioned targeting vector expressing human serum albumin into the chicken ovalbumin gene by means of homologous recombination, and then recombining the successfully transformed recombination The cells are injected into chicken embryos, and transgenic chickens are bred, and the male and female individuals of the transgenic chickens are cultivated to sexual maturity and passed on (in order to increase the yield, human serum albumin in the egg white fraction of eggs produced by F0 or F1 female transgenic chickens can be analyzed Select high-yielding individuals as host animals for bioreactors or as breeders for bioreactors), and then separate and purify human serum albumin from eggs produced by female transgenic chickens.

The invention inserts the human serum albumin gene site-specifically into the chicken genome through a gene knock-in method, and uses the oviduct of the chicken as a bioreactor to express the target protein. The present invention selectively inserts the target fragment into the chicken ovalbumin gene through the method of homologous recombination, such an operation will not produce mutations in other parts of the host gene, and can well destroy its own ovalbumin expression, and more Importantly, chicken ovalbumin is only expressed in the oviduct, and the gene introduced by the present invention will not be expressed in other parts of the chicken body. Therefore, compared with other methods, the present invention uses transgenic chickens to produce human serum albumin has very obvious advantages: 1) chickens have the advantages of short generation interval, high production performance, easy feeding and management, and low cost. 2) By transferring the functional protein gene and expressing it in the chicken oviduct, using the chicken oviduct as a bioreactor, the egg can contain human serum albumin. Since there are only 8 kinds of proteins that make up the egg white, the transgenic protein is easy to separate from the components of the egg and collect. 3) Eggs have a natural sterile environment, and the expressed human serum albumin is not easily contaminated. 4) Compared with albumin produced by other transgenic methods, the protein prepared by using the chicken oviduct as a bioreactor can be glycosylated correctly, and the oligosaccharides attached to the nascent polypeptide in the chicken body are more similar to humans than those of mammals , so the human serum albumin produced by the transgenic chicken is more similar to the normal serum albumin in the human body after modification, which is more conducive to its biological function.

Description of drawings

Figure 1: Schematic diagram of gene targeting vector construction.

Figure 2: Agarose gel electrophoresis identification diagram of vector construction, it can be seen from Figure 2 that the size of the sequence fragment used to construct the vector of the present invention is consistent with the expectation.

Figure 3: Schematic diagram of homologous recombination.

detailed description

The present invention will be further described below in conjunction with embodiment.

The reagents and methods involved in the examples, unless otherwise specified, are conventional reagents and conventional methods in the art.

Example 1 Construction of a targeting vector expressing human serum albumin (a schematic diagram of the construction principle is shown in Figure 1)

(1) Extraction of Chicken Genomic DNA

1. Take 200 mg of chicken embryos at the 10th stage, cut them into pieces, add 0.5 ml of SENT (containing proteinase K 50 μg/ml and 10% SDS), transfer to a homogenizer for homogenization.

2. Transfer the homogenate to a 1.5ml centrifuge tube.

3. 60C water bath for 3 hours.

4. Add an equal volume of saturated phenol to the above sample treatment solution, and mix gently and thoroughly for 3 minutes.

5. Centrifuge at 5000g for 10min, and transfer the upper aqueous phase to another 1.5ml centrifuge tube.

6. Add an equal volume of saturated phenol, mix well, centrifuge at 5000g for 10min, and transfer the upper aqueous phase to another tube.

7. Add an equal volume of phenol/chloroform, mix gently, centrifuge at 5000g for 10min, and transfer the upper aqueous phase to another tube. If the aqueous phase is still not clear, this step can be repeated several times.

8. Add an equal volume of chloroform, mix gently, centrifuge at 5000g for 10min, and transfer the upper aqueous phase to another tube.

9. Add 1/10 volume of 3M sodium acetate (pH5.2) and 2.5 volumes of absolute ethanol, and gently invert to mix.

10. After flocs appear, centrifuge at 5000g for 5min, and discard the supernatant.

11. The precipitate was washed with 75% ethanol, centrifuged at 5000g for 3min, and the supernatant was discarded.

12. Volatile ethanol at room temperature, add 50% sterilized pure water to dissolve after the precipitate is nearly transparent.

(2) Extraction of RNA from human blood

1. Strict aseptic operation, take 2ml of fresh blood in 3.8% sodium citrate anticoagulant tube.

2. Centrifuge at 1,500 rpm for 5 minutes.

3. Carefully suck out the upper layer of plasma in the ultra-clean bench, and at the same time make up 2ml of whole blood volume with an equal amount of Hank's solution, and mix gently.

4. Take 4ml of lymphocyte separation solution in a 15ml sterilized transparent plastic centrifuge tube, carefully add 2ml of anticoagulated blood to make up the volume on the surface of the lymphocyte separation solution, and centrifuge at 3,000rpm for 10min at room temperature.

5. Carefully collect the cells on the interface, add to a centrifuge tube containing 4ml Hank’s solution, mix gently, and rinse the cells.

6. Centrifuge at 1,500rpm for 5min, repeat rinsing once, and the pellet is the desired lymphocytes.

7. Add Trizol, 1ml, to the treated lymphocytes, invert and mix until completely dissolved, and place at room temperature for 5 minutes.

8. Add 200 μl of chloroform, mix thoroughly by inversion for 1 min, form a uniform chylus, and centrifuge for 5 min.

9. Carefully transfer the supernatant to RNase-free 1.5ml eppendorf (after centrifugation and stratification, you can absorb about 350μl of the colorless liquid in the upper layer, and be careful not to absorb any intermediate layer).

10. Transfer the supernatant layer to a clean centrifuge tube, add an equal volume of isopropanol in an ice bath, mix by inverting, and incubate the mixed sample at -20°C for more than 20 minutes, 4°C, 12,000× centrifuge for 10 minutes. The RNA pellet usually forms a pellet that adheres to the wall or bottom of the tube.

11. Discard the supernatant, wash the RNA pellet once with 1 ml of 75% ethanol in an ice bath, wash the wall of the centrifuge tube upside down, and vortex the sample to suspend the pellet as much as possible, centrifuge at 12,000×g for 5 minutes at 4°C, and again Remove the supernatant and dry the pellet.

12. Add 20ul high-pressure DEPC water to dissolve.

(3) Synthesis of human cDNA

1. Heat-denature the extracted human mRNA at 65°C for 5 minutes, and place it on ice immediately.

2. Reaction solution configuration: as follows:

Reagents and dosage:

RNA solution: 2 μg; 5×RTBuffer: 4 μl; dNTP Mixture (each 10 mM): 2 μl; primer (mix): 1 μl; ReverTra Ace: 1 μl; Rnase free H ₂ O: up to 20 μl.

3. Reaction conditions:

5min at 65°C;

37℃15min;

98°C for 5 minutes;

5 minutes on ice;

(4) PCR amplification of 5' homology arm, 3' homology arm and human serum albumin gene

The PCR reaction system is shown in Table 1:

Table 1

5' homology arm 3' homology arm human serum albumin gene template 1ul 1ul 1ul Primer 1 1ul 1ul 1ul Primer 2 1ul 1ul 1ul High Fidelity Taq Enzyme 1ul 1ul 1ul mix reaction system 25ul 25ul 25ul pure water 21ul 21ul 21ul total capacity 50ul 50ul 50ul

The PCR reaction conditions are shown in Table 2:

Table 2

5' homology arm 3' homology arm human serum albumin gene Pre-denaturation at 94°C for 5 minutes √ √ √ Denatured for 30 seconds √ √ √ annealing 62°C 63°C 62°C Extend 1kb/min 2 minutes 5 minutes 2 minutes 72°C for 10 minutes √ √ √ 4°C √ √ √

"√" is the same condition adopted during the experiment.

(5) Carrier construction:

1. Complete the recovery and purification of PCR products according to the instructions of the commercial kit. Carry out the digestion and ligation reactions described below.

2. Enzyme digestion reaction (as shown in Table 3):

table 3

3. Follow the procedure of the Gel Recovery Kit to recover the digested product.

4. Carry out the ligation reaction at room temperature with DNA ligase, and ligate the DNA fragments to the carrier after reacting overnight (as shown in Table 4).

Table 4

5' homology arm 7ul human serum albumin gene 7ul pBluescript II SK (+) vector 3ul ligation buffer 2ul T4 DNA ligase 1ul Reaction conditions Overnight at room temperature

5. Transform the ligated vector into competent cells by heat shock transformation method to amplify the recombinant vector.

Experimental conditions: Add 20ul of the ligation product to 200ul competent cells, place on ice for 15 minutes, heat shock at 42°C for 1 minute, place on ice for 2 minutes, spread the bacteria on ampicillin-resistant agarose-LB solid culture basically. Incubate overnight. The grown single colonies were selected, amplified, and plasmids were extracted. (For the extraction method, please refer to the plasmid extraction kit of Kangwei Company).

6. Construction of ROSA26-shALB1 vector

6.1 Amplify the 3' homology arm F7F8 through primer F5F6 to amplify the 5' homology arm + human serum albumin gene sequence connected to the pBluescript II SK (+) vector, which should be explained that there are 15 identical There are 15 identical bases on the base F7F8. After amplification by PCR method, the PCR product is purified and recovered.

6.2 Carry out the ligation reaction according to the instructions of the infusion reagent.

6.3 Carry out the transformation in "5" of the connected vector, obtain the ROSA26-shALBl transgene vector with human serum albumin after amplification, and carry out PCR identification on the obtained recombinant vector, and the result shows that the target recombinant vector is obtained (as shown in Figure 2 shown).

Embodiment two Identify the expression of human serum albumin:

The gene targeting vector constructed in Example 1 containing homology arms and human serum albumin was named ROSA26-shALB1 vector and linearized with AsiSI. Resuspend ⁵ *106 DT-40 (commercialized cells) in 400ul electroporation buffer, add 20ug linearized ROSA26-shALBl gene recombination vector, and exponentially decay pulse (200V, with900–1,100uF) for electroporation transformation Into DT-40 cells, and after 24-48 hours of transfection, the expression of the light-emitting protein GFP carried by the vector itself was first observed with a fluorescence microscope. The expression of human serum albumin in DT-40 cells was identified by Western analysis. Vectors with high expression efficiency were selected for sequencing analysis, and the vectors with correct sequences were amplified in large quantities and then transferred into PGC cells.

Embodiment three

PGC cell culture: 5% FBS, 10 ng/ml LIF, 10 ng/ml mlbFGF, 10 ng/ml SCF, 0.1 mmol/L MEM non-essential amino acids, 0.1 mmol/L β-mercaptoethanol, 2 mmol /L L-glutamine, 100IU/ml penicillin and 100ug/ml streptomycin are the complete culture solution. Cells were inoculated into 6-well cell culture plates at a density of 1×10 ⁶ cells/well, placed in an incubator containing 5% CO ₂ at 37°C, and cultured on the feeder cell layer. During the culture period, half of the medium was changed every other day. Feeder cells are generally buffalo rat liver (BRL) cells rat liver cells or ouabain-resistant (STO) fibroblasts cells anti-ouabain fibroblasts.

Subculture and cryopreservation: Add cells and medium to a centrifuge tube and centrifuge at 300g for 5 minutes, resuspend the cells in a solution containing 10% (fetal bovine serum) FBS, 1% double antibodies (penicillin, streptomycin) and 10% The DMSO high-sugar DMEM medium was frozen at a density of 2.5*10 ⁴ /ml, frozen at -80°C, and placed in liquid nitrogen after 24 hours.

Example 4 Identification of PGC cell characteristics

RT-PCR Detection of the Expression of Stem Cell Pluripotency Marker Genes

(1) Extraction of total RNA:

1. Sample preparation: Blow off the cultured PGC clones from the feeder layer, digest them with 0.25% trypsin-0.05% EDTA into single cells, inoculate them on a culture plate covered with 0.1% gelatin for 30 minutes at a differential speed, and remove impurities feeder layer, the enriched PGCs were collected by centrifugation;

2. Add 1mL TRIzol to the cells, vigorously shake and beat repeatedly, let stand at room temperature for 5min, then transfer to 1.5ml EP tube;

3. Add chloroform according to the ratio of 1ml TRlzol to 200ul of chloroform, and shake vigorously for 30s to fully mix into milky white, and stand at room temperature for 5min;

4. Centrifuge at 12,000g at 2-8°C for 15min. After centrifugation, the tube is divided into three phases: the lower layer is a red phenol phase, the middle layer is a chloroform phase, and the upper layer is a colorless water phase;

5. RNA precipitation: Carefully pipette the upper aqueous phase into another new EP tube treated with OEPC, add an equal volume of isopropanol, place at room temperature for 10 minutes, and centrifuge at less than 12,000g for 30 minutes at 2-8°C to take the supernatant;

6. Discard the supernatant, wash the precipitate with 1.5ml of 75% ethanol, shake vigorously, centrifuge at 7500g for 10min at 2-8°C, use an automatic micropipette to absorb the ethanol as much as possible, and place it in an ultra-clean bench for a few minutes dry;

7. Determination of total RNA concentration: Detect the values of OD260 and OD280 with a UV spectrophotometer, and calculate the concentration of RNA and the ratio of OD260/OD280.

(2) Synthesis of cDNA:

1. Heat denaturation of RNA at 65°C for 5 minutes, then place it on ice immediately;

2. Reaction solution configuration: as follows:

RNA solution: 2μg; 5×RT Buffer: 4μl; dNTP Mixture (10mM each): 2μl; primer (mix): 1μl; ReverTra Ace: 1μl; Rnase free H2O: upto 20μl;

3. Reaction conditions:

5min at 65°C;

37℃15min;

98°C for 5 minutes;

5 minutes on ice.

The CVH and Dazl genes of cultured cells were identified by RT-PCR, and the cells specifically expressing PGC genes were selected for subsequent experiments. CVH (chicken homolog to vasa gene, cvh) is a specific molecular marker of primordial germ cells, and it is a gene specifically expressed in chicken primordial germ cells. The Dazl gene plays a key role in the early differentiation of germ cells. The deletion or mutation of this gene can cause the loss of germ cells and the inability to produce gametes.

Determination of telomerase activity in PGC cells. After the cells were washed with PBS buffer, the cells were collected, and the telomerase detection kit was used to operate according to the instructions, and a positive control and a negative control were set up.

Embodiment five

Genetic modification of PGC cells, after the assembled carrier was linearized with AsiSI endonuclease, it was transformed into PGC cells according to the method in Example 2, and a negative control was set up at the same time for electroporation, and the neo gene was contained on the ROSA26-shALBl carrier, As a positive selection gene; HSV-tk gene as a negative selection gene, linearized DNA into PGC cells, continue to culture in vitro, and double selection with neomycin (G418) and lethal nucleoside analogs (FIAU). No screening drug was added in the first few days of culture, and the medium containing screening drug should be replaced every 2 days thereafter. Because the randomly inserted DNA is usually integrated into the DNA of the recipient cell from the beginning to the end, the HSV-tk+ gene product can convert FIAU into a toxic substance and cause cell death. If homologous recombination occurs, since the HSV-tk gene cannot be integrated outside the homologous region, the neo gene can be retained within the homologous region, so that the recipient cells have a positive selection effect against G418, and have no transformation function but a negative effect on FIAU. Therefore, the surviving cells are considered to have undergone homologous recombination with the introduced gene.

Example 6 Identification of PGC Cell Modifications

After the cell selection after transfection, the surviving cells need to be further identified, and the identification of human serum albumin gene expression is carried out by PCR and Western blot, and Southern blot is used to identify whether human serum albumin is inserted into the PGC cell genome In , it was determined that the human serum albumin gene could be expressed.

Embodiment 7 The preparation of transgenic chicken

After the screened positive PGC-hAlb cells were digested, the cells were injected into StageX chicken embryos by microinjection. Place the injected chicken embryos in a 37°C rotary incubator until hatching is complete.

Embodiment eight identification of transgenic chicken

The hatched transgenic chickens were selected to collect blood, and the blood cells were separated. PCR and Southern blot were used to detect whether the human serum albumin gene was inserted into the genome and whether the transcription could be completed. Ovalbumin was extracted from transgenic chickens after laying eggs for Western blot detection to determine the expression of human serum albumin.

Claims (10)

1. express the targeting vector of human serum albumin for one kind, it is characterized in that: include human serum albumin gene, and carry out two homologous sequences of Ji Yuan of homologous recombination: 5 ' homology arm, 3 ' homology arm, the sequence of 5 ' homology arm is as shown in SEQ ID NO.3, the sequence of 3 ' homology arm is as shown in SEQ ID NO.4,5 ' end of human serum albumin gene is connected with kozak sequence, also includes the neo gene and reporter gene EGFP that carry out drug screening simultaneously.

2. the construction process of the targeting vector of expression human serum albumin according to claim 1, is characterized in that: design Auele Specific Primer, carries out the amplification of 5 ' homology arm, 3 ' homology arm and human serum albumin gene; Then, use pBluescriptII SK(+) vector construction homology arm and human serum albumin gene merge, then fusion gene is connected on EGFP-ROSA-26 gene targeting carrier, again 3 ' homology arm is connected on EGFP-ROSA-26 gene targeting carrier, the targeting vector of human serum albumin must be expressed.

3. the construction process of the targeting vector of expression human serum albumin according to claim 2, it is characterized in that: described pBluescript II SK(+) the concrete mode that merges of vector construction homology arm and human serum albumin gene is: first recombinate 5 ' homology arm pBluescript II SK(+) on carrier, recombinate human serum albumin gene pBluescript IISK(+ again) on carrier, utilize Auele Specific Primer to be got off by the fusion gene cloning of 5 ' homology arm and human serum albumin gene.

4. the construction process of the targeting vector of the expression human serum albumin according to Claims 2 or 3, is characterized in that: described Auele Specific Primer is as follows:

Primer for the 5 ' homology arm that increases is F1, F2:

F1:5 '-CCGCGGCCGCTCTATGGCGTCAAAGGTCA-3 '; As shown in SEQ ID NO.5;

F2:5 '-CCCCCCGGGGAAAATGTAAGGATGG-3 '; As shown in SEQ ID NO.6;

Primer for the human serum albumin gene that increases is F3, F4:

F3:5 '-CCCGGGGCCGAAATGAAGTGGGTAACCTTTAT-3 '; As shown in SEQ ID NO.7;

F4:5 '-CCATCGATTTATAAGCCTAAGGCAGCTTGAC-3 '; As shown in SEQ ID NO.8;

Primer for the 3 ' homology arm that increases is F5, F6:

F5:5 '-TTCCACATCCACCGGTTGGTTTATTTAGAGTGGCA-3 '; As shown in SEQ ID NO.9;

F6:5 '-GTTGGCGCCTACCGGACTGGCAGGGCTTATTTTCA-3 '; As shown in SEQ ID NO.10;

Primers F 7, F8 will be for being connected to pBluescriptIISK(+) 5 ' homology arm+human serum albumin gene on carrier clone and add infusion reagent connect needed for homology arm:

F7:5 '-TCTTTCCAGTGGTTAGCTCTATGGCGTCAAAGGTC-3 '; As shown in SEQ ID NO.11;

F8:5 '-ATACGAAGTTATTTATTATAAGCCTAAGGCAGCTT-3 '; As shown in SEQ ID NO.12.

5. the targeting vector of expression human serum albumin according to claim 1 is preparing the application in transgenic chicken.

6. application according to claim 5, it is characterized in that: during embody rule, the targeting vector of the expression human serum albumin of claim 1 is transformed the archeocyte of chicken, after cultivating screening, utilize microinjection technique, successful for conversion archeocyte is injected in Embryo Gallus domesticus, cultivates transgenic chicken.

7. one kind obtains the method for the transgenic chicken of expressing human serum albumin, it is characterized in that: by the method for homologous recombination, the targeting vector of expression human serum albumin according to claim 1 is recombined in the oralbumin gene of chicken, then successful for conversion reconstitution cell is injected in Embryo Gallus domesticus, cultivates transgenic chicken.

8. prepare a method for bio-reactor, it is characterized in that: described bio-reactor is made up of transgenic chicken oviduct, method comprises prepares transgenic chicken with the targeting vector of expression human serum albumin according to claim 1.

9. one kind produces the method for human serum albumin from transgenic chicken oviduct, it is characterized in that: by the method for homologous recombination, the targeting vector of expression human serum albumin according to claim 1 is recombined in the oralbumin gene of chicken, then successful for conversion reconstitution cell is injected in Embryo Gallus domesticus, cultivate transgenic chicken, cultivating transgenic chicken female male to sexual maturity goes down to posterity, then separation and purification human serum albumin from the egg that transgenic chicken female individuals is produced.

10. the method producing human serum albumin from transgenic chicken oviduct according to claim 9, it is characterized in that: cultivate transgenic chicken female male to sexual maturity and after going down to posterity, the content of human serum albumin in the egg white component that analysis F0 or F1 generation female transgenic chicken individuals produce, selects high yield individual as the host animal of bio-reactor or as bio-reactor kind chicken.