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CN114409800B - Method for preparing recombinant cystatin C - Google Patents

Method for preparing recombinant cystatin C Download PDF

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Publication number
CN114409800B
CN114409800B CN202111551776.9A CN202111551776A CN114409800B CN 114409800 B CN114409800 B CN 114409800B CN 202111551776 A CN202111551776 A CN 202111551776A CN 114409800 B CN114409800 B CN 114409800B
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protein
cys
cystatin
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fusion protein
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CN114409800A (en
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蒋析文
黄黉
黄珞
颜青青
况修丽
肖兰花
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Guangzhou Da'an Gene Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

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Abstract

The invention discloses a method for preparing recombinant cystatin C, in particular to a method for expressing Cys C protein by adopting genetic engineering escherichia coli recombinant fusion, wherein the expressed Cys C recombinant protein is fused with a specific tag sequence at the N end, so that the high-efficiency expression in the escherichia coli system is realized, the stability of the protein is high, and the protein has excellent immunological activity.

Description

Method for preparing recombinant cystatin C
Technical Field
The invention belongs to the field of biotechnology. More particularly, to a method for preparing recombinant cystatin C.
Background
Cystatin C (Cystatin C or Cys C for short) is also called cysteine protease inhibitor C, and is a cysteine protease inhibitor. It is an ideal endogenous marker for reflecting the change of Glomerular Filtration Rate (GFR), and cystatin C is currently used as a recognized kidney function detection index at home and abroad.
The traditional method for extracting natural cystatin from tissues such as placenta, urine and the like has the problems of low efficiency, low purity, high purification cost and the like, unknown potential risks exist, the purification steps are complex, the difficulty of obtaining high-purity protein is great, the cross between the polyclonal antiserum prepared by immunization with the natural protein and other components in human serum is serious, and great difficulty is brought to the optimization of the product performance. At present, the expression of human Cys C has been reported to be studied using E.coli expression systems, but since the expression of human Cys C in E.coli belongs to heterologous expression, it is reported to be in the form of inclusion bodies. The expression product in the form of inclusion bodies must undergo complex denaturation and renaturation treatments to restore their biological activity. The expression of yeast and CHO cell systems is reported, but the production cost is high, the expression quantity is low, and the industrial production of Cys C is not facilitated.
The preparation of cystatin C diagnostic kit requires a high-specificity and high-sensitivity anti-Cys C monoclonal antibody, and high-purity cystatin C protein must be obtained for obtaining the monoclonal antibody. Cys C is mainly extracted from urine of patients in the past, so that not only is the source limited, but also the cost is high and the yield is extremely low. Therefore, the skilled person is dedicated to develop a cystatin C protein preparation process with high expression efficiency and easy purification.
Disclosure of Invention
The invention provides a cystatin C protein preparation process with high expression efficiency and easy purification.
In a first aspect of the invention, there is provided a cystatin C fusion protein selected from the group consisting of:
(A) A polypeptide having the amino acid sequence shown in SEQ ID NO. 3;
(B) A polypeptide having 90% or more homology (preferably 95% or more homology; preferably 96% or more homology; most preferably 97% or more homology) to the amino acid sequence shown in SEQ ID NO. 3, and which retains the activity of the polypeptide shown in SEQ ID NO. 1;
(C) And (3) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues in any one of the amino acid sequences shown in SEQ ID NO. 3 and keeps the activity of the polypeptide shown in SEQ ID NO. 1.
In another preferred embodiment, the fusion protein is isolated.
In another preferred embodiment, the cystatin C fusion protein has an amino acid sequence shown in SEQ ID NO. 3.
In a second aspect of the invention there is provided an isolated codon optimised polynucleotide encoding the fusion protein of the first aspect of the invention.
In another preferred embodiment, the polynucleotide is selected from the group consisting of:
(a) A polynucleotide with a sequence shown as SEQ ID NO. 4;
(b) A polynucleotide having a nucleotide sequence having a homology of 95% (preferably 98%) or more with the sequence shown in SEQ ID NO. 4;
(c) A polynucleotide complementary to the polynucleotide of any one of (a) - (b).
In a third aspect of the invention there is provided an expression vector comprising a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the expression vector is a pET-32a (+) expression vector.
In a fourth aspect of the invention there is provided a host cell comprising an expression vector according to the third aspect of the invention or having integrated into its genome a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the host cell is a prokaryotic cell, preferably the host cell is E.coli, more preferably E.coli Rosetta (DE 3) strain.
In a fifth aspect of the invention, there is provided a method of preparing a fusion protein according to the first aspect of the invention, comprising the steps of:
culturing the cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the fusion protein of the first aspect of the invention; and isolating the fusion protein.
In a sixth aspect of the invention there is provided a kit comprising a fusion protein according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention or an expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows that Cys C protein was not soluble expressed in SB, TB, SOC medium at 37℃and 18 ℃.
FIG. 2 shows that the tagged Cys C protein is soluble expressed in large amounts in the supernatant in TB medium.
FIG. 3 shows an electrophoretogram after purification of a large number of expressed products.
FIG. 4 shows the stability of recombinant fusion Cys C proteins.
FIG. 5 shows that the recombinantly expressed Cys C protein binds well to antibodies.
Detailed Description
The invention adopts the recombinant fusion expression of the Cys C protein by the genetic engineering escherichia coli, and the expressed Cys C is fused with a specific tag sequence at the N end, so that the high-efficiency expression in the solubility of an escherichia coli system is realized, the stability of the protein is high, and the protein has immunological activity. The method for recombinant fusion expression of Cys C protein has the advantages of high yield, short production period, easy purification of expression products, low cost and the like, and can realize industrial production of Cys C protein.
Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
Fusion proteins and their preparation
In the present invention, "fusion protein", "recombinant protein", "protein of the invention" and "fusion protein of the invention" are used interchangeably and refer to a protein having the amino acid sequence shown in SEQ ID No. 3 or a derivative thereof. The proteins of the invention may be monomers or multimers (e.g., dimers) formed from monomers. Furthermore, it is understood that the term also includes active fragments and derivatives of fusion proteins.
As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the polynucleotides and polypeptides in the native state in living cells are not isolated or purified, the same polynucleotides or polypeptides are isolated or purified if they are separated from other substances that are present in the native state.
As used herein, an "isolated fusion protein" refers to a fusion protein that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. The skilled artisan can purify fusion proteins using standard protein purification techniques. Substantially pure proteins can produce a single main band on a non-reducing polyacrylamide gel.
The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.
As used herein, the term "primer" refers to the generic term for oligonucleotides that, when paired with a template, are capable of synthesizing a DNA strand complementary to the template from the primer under the action of a DNA polymerase. The primer may be natural RNA, DNA, or natural nucleotide in any form. The primer may even be a non-natural nucleotide such as LNA or ZNA, etc. The primer is "substantially" (or "essentially") complementary to a particular sequence on one strand of the template. The primer must be sufficiently complementary to one strand on the template to begin extension, but the sequence of the primer need not be perfectly complementary to the sequence of the template. For example, a primer that is complementary to the template at the 3 'end is added to the 5' end of a primer that is not complementary to the template, and such primer is still substantially complementary to the template. Primers that are not perfectly complementary may also form primer-template complexes with the template, so long as they are sufficiently long to bind to the template, thereby allowing amplification.
The full-length nucleotide sequence of the fusion protein of the present invention or a component thereof or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present invention. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors or fusion protein coding sequences of the invention, and methods for producing the proteins of the invention by recombinant techniques.
The polynucleotide sequences of the present invention may be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally, there are the following steps:
(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) A host cell cultured in a suitable medium;
(3) Separating and purifying the protein from the culture medium or the cells.
Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.
Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell may be a eukaryotic cell, such as a mammalian cell. Representative examples are: animal cells of CHO, NS0, COS7, or 293 cells, and the like; prokaryotic cells, such as E.coli, are also contemplated.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The proteins obtained in the above methods can be isolated and purified by various separation methods using their physical, chemical and other properties, if necessary. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
In a preferred embodiment of the present invention, the amino acid sequence and gene sequence of Cys C described in the present invention are as follows:
base sequence:
MAGPLRAPLLLLAILAVALAVSPAAGSSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCCFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA(SEQ ID NO:1)
base sequence optimized for synonymous codon preference of E.coli:
ATGGCAGGACCCCTAAGGGCTCCATTATTGCTTCTGGCGATTTTGGCGGTAGCGCTGGCGGTGAGCCCGGCAGCAGGCTCGAGCCCAGGTAAACCGCCTCGCTTGGTCGGTGGTCCGATGGACGCCAGCGTTGAGGAAGAAGGCGTGCGTCGTGCGCTCGACTTCGCAGTTGGTGAATACAACAAAGCTTCTAATGATATGTATCATGGCCGTGCGCTGCAGGTTGTTCGCGCACGTAAGCAAATCGTGGCCGGTGTGAATTACTTCCTGGATGTTGAGTTGGGCCGTACGACCTGTACCAAGACCCAACCGAACCTGGACAACTGCCCGTTTCACGATCAGCCGCACCTGAAACGCAAGGCGTTTTGCTCCTTCCAAATCTATGCTGTGCCGTGGCAGGGCACCATGACTCTGAGCAAGTCCACCTGCCAGGACGCG(SEQ ID NO:2)。
in a preferred embodiment of the present invention, the amino acid sequence and gene sequence of the recombinant Cys C of the present invention are as follows:
base sequence:
SSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCCFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDASSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCCFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA(SEQ ID NO:3)
base sequence optimized for synonymous codon preference of escherichia coli
TCAAGTCCCGGAAAACCACCTAGGCTAGTAGGCGGTCCAATGGATGCCTCTGTTGAGGAAGAGGGCGTGCGTCGCGCGTTGGACTTTGCAGTTGGTGAATATAACAAAGCTTCCAACGATATGTACCATAGCCGTGCGCTGCAAGTAATGAGAGCTCGCAAGCAGATTGTCGCGGGTGTAAATTACTTCTTGGACGTTGAGCTCGGTCGTACCACCTGTACCAAGACCCAGCCGAATCTGGACAACTGCCCGTTTCACGATCAACCGCACCTGAAACGATAGGCGTTCTGCTCGTTCCAAATCTATGCAGTTCCGTGGCAGGGCACCATGACTCTGAGCAAAAGCACGTGCCAGGACGCGTCGAGCCCAGGTAAACCGCCTCGCTTGGTCGGTGGTCCGATGGACGCCAGCGTTGAGGAAGAAGGCGTGCGTCGTGCGCTCGACTTCGCAGTTGGTGAATACAACAAAGCTTCTAATGATATGTATCATGGCCGTGCGCTGCAGGTTGTTCGCGCACGTAAGCAAATCGTGGCCGGTGTGAATTACTTCCTGGATGTTGAGTTGGGCCGTACGACCTGTACCAAGACCCAACCGAACCTGGACAACTGCCCGTTTCACGATCAGCCGCACCTGAAACGCAAGGCGTTTTGCTCCTTCCAAATCTATGCTGTGCCGTGGCAGGGCACCATGACTCTGAGCAAGTCCACCTGCCAGGACGCG(SEQ ID NO:4)。
Genetically engineered cells
The present invention provides a genetically engineered cell (host cell) which is a prokaryotic cell (preferably E.coli) and has an expression cassette for the fusion protein of the present invention integrated into the genome of the cell; or the cell contains an expression vector containing the expression cassette of the fusion protein of the invention.
In a preferred embodiment, the expression cassette of the fusion protein of the invention comprises the following elements operably linked 5 'to 3': a promoter, a start codon, an ORF sequence of the fusion protein and a stop codon.
In the present invention, the term "operably linked" means a configuration in which a regulatory sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the regulatory sequence directs the expression of the coding sequence.
Materials and methods
1. Biological material
The host E.coli Rosetta (DE 3) strain used in this experiment (purchased from Tian Gen Biotechnology (Beijing) Co., ltd.), an expression vector (purchased from Kirschner Biotechnology Co., ltd.), and a Cys C protein activity assay kit (Elisa kit, purchased from Darui Biotechnology Co., ltd.).
2. Vector construction
The synonymous codon preference optimization of the escherichia coli is carried out on the human Cys C gene and the recombinant fusion Cys C protein gene designed by the invention, the optimized codon sequence is obtained, after artificial synthesis, the expression vector is constructed and is respectively connected to the vector pET-28a (+) and the vector pET-32a (+) (the downstream of the insertion site in the vector is provided with a coding (His) multiplied by 6 tag gene).
3. Construction of host E.coli
And taking an expression vector, and transforming the expression vector into escherichia coli competent Rosetta (DE 3) to obtain host escherichia coli.
4. Expression and purification of the Gene of interest
And fermenting and culturing the obtained host escherichia coli to obtain thalli, crushing the thalli, centrifuging, and carrying out Ni-column affinity chromatography on a bacterial liquid supernatant to obtain the human myoglobin target protein.
The invention has the beneficial effects that:
(1) The recombinant Cys C of the present invention significantly enhances the solubility of the protein, and is more expressed in the supernatant.
(2) The recombinant Cys C not only greatly promotes the soluble expression efficiency of the protein, but also enhances the stability of the protein.
(3) The codon sequence obtained by optimization can be expressed in a large amount in escherichia coli, and the expression quantity of the recombinant Cys C is obviously improved.
(4) The recombinant Cys C expressed in the escherichia coli is mainly a soluble fusion protein, so the purification process is simple.
(5) The recombinant Cys C of the present invention expressed by prokaryotic recombination maintains excellent antigen performance.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Example 1
1) The gene of human Cys C provided by NCBI is taken as a reference, the amino acid sequence shown in SEQ ID NO. 1 is determined without a tag vector in combination with the experimental design requirement of the invention, and after the optimization of synonymous codon preference of escherichia coli (SEQ ID NO. 2), the linking vector is pET-28a (+) (C-terminal fusion expression (His) 6 tag in the vector) which is synthesized by Nanjing Jinshi biotechnology Co.
The amino acid sequence of the fusion Cys C protein with the tag is shown as SEQ ID NO. 3, and the first 26 amino acids of the N end of the SEQ ID NO. 1 are excised in the fusion Cys C protein, so that the soluble expression of the Cys C protein is better promoted. After optimization of the synonymous codon preference of E.coli (SEQ ID NO: 4), the ligation vector was pET-32a (+) (C-terminal fusion expression (His) 6 tag in the vector) and was synthesized by Nanjing Jinsri Biotechnology Co., ltd.
2) Recombinant plasmid introduction into host E.coli
Taking 1 mu L of expression plasmid, adding the expression plasmid into 30 mu L of escherichia coli competent Rosetta (DE 3) under ice bath condition, standing for 20min in ice bath, heat-shocking for 90s, standing on ice immediately for 2min, adding 400 mu L of SOC culture medium without antibiotics, and culturing at 37 ℃ and 220rpm for 50min in a shaking way.
mu.L of the bacterial liquid was uniformly spread on LB plates containing 100. Mu.g/mL of kana resistance, and incubated overnight at 37 ℃.
3) Expression process of target Gene
Picking the monoclonal in step 2), aseptically inoculating into TB medium containing 100 μg/mL kana resistance, shake culturing at 37deg.C 220rpm to OD 600 Between 0.6 and 0.8, IPTG was induced and incubated overnight at 37℃and 18℃with shaking, respectively.
Sampling and ultrasonication for SDS-PAGE identification:
the unlabeled Cys C protein predicted molecular weight 15.81KD, the results of fig. 1 show that Cys C protein was not found to be soluble expressed in SB, TB, SOC medium at 37 ℃ and 18 ℃;
the predicted molecular weight of the coupled tag fusion Cys C protein is 36.71KD, and as shown in FIG. 2, the coupled tag Cys C protein can be expressed in a large amount in the supernatant in a TB culture medium, and the expression amount accounts for more than 80% of the total protein of the thalli.
EXAMPLE 2 purification of a large amount of the product
Shake flask culture of 1.5L bacterial liquid, and centrifugal collection of bacterial body wet weight: 26g. About 4g of the cells were weighed, and 35ml of Lysis Buffer was added thereto to resuspend on ice. Centrifuging at 20000rpm at 4deg.C for 30min after ultrasonic crushing, collecting supernatant, and filtering with 0.22 μm needle filter to obtain filtered bacterial liquid.
Filtering, subjecting to Ni-column affinity chromatography, eluting with 50mM Tris-HCl,50mM NaCl,200mM imidazole pH7.0 to obtain target protein, eluting to obtain 7ml protein with concentration of 2.13mg/ml, and electrophoresing the target protein as shown in figure 3.
The expression content of the target protein is calculated to be 97mg/L, and the purity can reach 95 percent.
Example 3 protein stability assay
The obtained target proteins were separately packed in 2mL EP tubes, 1 mL/branch, and sealed with a sealing film.
3 of the 3 animals per batch, 1 animal was subjected to 4℃as a control, and the other 4 animals were subjected to 37℃for one week of acceleration.
Samples were taken at 3 and 7 days for identification, and stability of the proteins was tested by unclle (Unchained Lab company, inc. Of usa, unclle multifunctional protein stability analysis system).
The results of FIG. 4 show that the recombinant fusion Cys C protein has better stability after 3 days at 37℃and no significant difference from 4℃placement.
EXAMPLE 4 Elisa assay of immunological Activity
The purified fusion Cys C protein was taken and coated overnight at 4℃by adding 0.2. Mu.g/well to the ELISA plate.
After the coating is finished, the coating is washed for 3 times by a plate washer, and is added into a constant temperature incubator with the temperature of the skim milk powder of 37 ℃ for sealing for 1 hour.
Then, human Cys C protein polyclonal antibodies (available from Darui biotechnology Co., ltd., guangzhou) diluted with PBS at different multiples of 1K, 2K, 4K, 8K, and 16K were added, incubated for 2 hours at 37℃in a constant temperature incubator, and plates were washed 3 times after the completion.
Adding secondary antibody (purchased from biological engineering (Shanghai) Co., ltd.) and incubating at 37deg.C for 30min, washing 5 times, tapping dry solution, adding TMB color development solution, and developing at room temperature for 10min in dark place until light blue is visible.
Stop solution 50. Mu.L/well was added and OD was measured on a microplate reader at 450nm/630 nm.
As shown in FIG. 5, the recombinant Cys C protein binds well to the antibody and has excellent immunogenicity.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
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<120> method for preparing recombinant cystatin C
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 146
<212> PRT
<213> person (Homo sapiens)
<400> 1
Met Ala Gly Pro Leu Arg Ala Pro Leu Leu Leu Leu Ala Ile Leu Ala
1 5 10 15
Val Ala Leu Ala Val Ser Pro Ala Ala Gly Ser Ser Pro Gly Lys Pro
20 25 30
Pro Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly
35 40 45
Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser
50 55 60
Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg Lys
65 70 75 80
Gln Ile Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg
85 90 95
Thr Thr Cys Thr Lys Thr Gln Pro Asn Leu Asp Asn Cys Cys Phe His
100 105 110
Asp Gln Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr
115 120 125
Ala Val Pro Trp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys Gln
130 135 140
Asp Ala
145
<210> 2
<211> 438
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
atggcaggac ccctaagggc tccattattg cttctggcga ttttggcggt agcgctggcg 60
gtgagcccgg cagcaggctc gagcccaggt aaaccgcctc gcttggtcgg tggtccgatg 120
gacgccagcg ttgaggaaga aggcgtgcgt cgtgcgctcg acttcgcagt tggtgaatac 180
aacaaagctt ctaatgatat gtatcatggc cgtgcgctgc aggttgttcg cgcacgtaag 240
caaatcgtgg ccggtgtgaa ttacttcctg gatgttgagt tgggccgtac gacctgtacc 300
aagacccaac cgaacctgga caactgcccg tttcacgatc agccgcacct gaaacgcaag 360
gcgttttgct ccttccaaat ctatgctgtg ccgtggcagg gcaccatgac tctgagcaag 420
tccacctgcc aggacgcg 438
<210> 3
<211> 240
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 3
Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly Gly Pro Met Asp Ala
1 5 10 15
Ser Val Glu Glu Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val Gly
20 25 30
Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr His Ser Arg Ala Leu Gln
35 40 45
Val Val Arg Ala Arg Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu
50 55 60
Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Thr Gln Pro Asn Leu
65 70 75 80
Asp Asn Cys Cys Phe His Asp Gln Pro His Leu Lys Arg Lys Ala Phe
85 90 95
Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gln Gly Thr Met Thr Leu
100 105 110
Ser Lys Ser Thr Cys Gln Asp Ala Ser Ser Pro Gly Lys Pro Pro Arg
115 120 125
Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly Val Arg
130 135 140
Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser Asn Asp
145 150 155 160
Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg Lys Gln Ile
165 170 175
Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg Thr Thr
180 185 190
Cys Thr Lys Thr Gln Pro Asn Leu Asp Asn Cys Cys Phe His Asp Gln
195 200 205
Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr Ala Val
210 215 220
Pro Trp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys Gln Asp Ala
225 230 235 240
<210> 4
<211> 720
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
tcaagtcccg gaaaaccacc taggctagta ggcggtccaa tggatgcctc tgttgaggaa 60
gagggcgtgc gtcgcgcgtt ggactttgca gttggtgaat ataacaaagc ttccaacgat 120
atgtaccata gccgtgcgct gcaagtaatg agagctcgca agcagattgt cgcgggtgta 180
aattacttct tggacgttga gctcggtcgt accacctgta ccaagaccca gccgaatctg 240
gacaactgcc cgtttcacga tcaaccgcac ctgaaacgat aggcgttctg ctcgttccaa 300
atctatgcag ttccgtggca gggcaccatg actctgagca aaagcacgtg ccaggacgcg 360
tcgagcccag gtaaaccgcc tcgcttggtc ggtggtccga tggacgccag cgttgaggaa 420
gaaggcgtgc gtcgtgcgct cgacttcgca gttggtgaat acaacaaagc ttctaatgat 480
atgtatcatg gccgtgcgct gcaggttgtt cgcgcacgta agcaaatcgt ggccggtgtg 540
aattacttcc tggatgttga gttgggccgt acgacctgta ccaagaccca accgaacctg 600
gacaactgcc cgtttcacga tcagccgcac ctgaaacgca aggcgttttg ctccttccaa 660
atctatgctg tgccgtggca gggcaccatg actctgagca agtccacctg ccaggacgcg 720

Claims (10)

1. The cystatin C fusion protein is characterized in that the amino acid sequence of the cystatin C fusion protein is shown as SEQ ID NO. 3.
2. The cystatin C fusion protein of claim 1, wherein the cystatin C fusion protein is isolated.
3. An isolated codon optimized polynucleotide encoding the cystatin C fusion protein of claim 1.
4. A polynucleotide according to claim 3, wherein the sequence of the polynucleotide is as shown in SEQ ID No. 4.
5. An expression vector comprising the polynucleotide of claim 3.
6. A host cell comprising the expression vector of claim 5 or having integrated into its genome the polynucleotide of claim 3.
7. The host cell of claim 6, wherein the host cell is a prokaryotic cell.
8. The host cell of claim 7, wherein the host cell is e.
9. A method of preparing the cystatin C fusion protein according to claim 1, comprising the steps of:
culturing the cell of claim 6 under conditions suitable for expression, thereby expressing the cystatin C fusion protein of claim 1; and isolating the cystatin C fusion protein.
10. A kit comprising the cystatin C fusion protein of claim 1, the polynucleotide of claim 3 or the expression vector of claim 5 or the host cell of claim 6.
CN202111551776.9A 2021-12-17 2021-12-17 Method for preparing recombinant cystatin C Active CN114409800B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087622A (en) * 2015-07-08 2015-11-25 北京嘉万生物技术有限公司 Method for improving soluble expression quantity of human cystatin C protein in escherichia coli
CN109975552A (en) * 2017-12-28 2019-07-05 江苏众红生物工程创药研究院有限公司 A kind of recombination cystatin C albumen and its application in detection kit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087622A (en) * 2015-07-08 2015-11-25 北京嘉万生物技术有限公司 Method for improving soluble expression quantity of human cystatin C protein in escherichia coli
CN109975552A (en) * 2017-12-28 2019-07-05 江苏众红生物工程创药研究院有限公司 A kind of recombination cystatin C albumen and its application in detection kit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
cystatin C [Homo sapiens].GenBank: AAA52164.1.2016,序列及注释. *

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