CN112725380A - High-efficiency expression system for stably expressing foreign protein CHO cell, screening method thereof and application of high-efficiency expression system in PCV2Cap protein - Google Patents
High-efficiency expression system for stably expressing foreign protein CHO cell, screening method thereof and application of high-efficiency expression system in PCV2Cap protein Download PDFInfo
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Abstract
The invention discloses a CHO cell high-efficiency expression system, wherein the CHO cell contains a eukaryotic expression vector of CAG promoter gene-human interferon-alpha 2 signal peptide gene-Luc gene. The invention also discloses a screening method of the CHO cell high-efficiency expression system, which comprises the following steps: and screening a promoter and a signal peptide. The invention also discloses an application of the CHO cell high-efficiency expression system in PCV2Cap protein expression, wherein the application is to replace PCV2Cap protein genes with Luc genes in the eukaryotic expression vector. When the CHO cell high-efficiency expression system is used, the Luc gene is directly replaced by the target protein gene, so that the stable and high-efficiency secretory expression of the target protein in the CHO cell can be realized, and the application of the system in the expression of PCV2Cap protein is successful.
Description
Technical Field
The invention relates to a CHO cell high-efficiency expression system for stably expressing foreign protein, a screening method thereof and application in PCV2Cap protein, belonging to the field of biotechnology.
Background
The most common use in mammalian cell expression systems is the Chinese Hamster Ovary (CHO) expression system. The advantages of CHO cells widely used as host cells for the expression of foreign proteins are as follows: (1) the recombinant protein has high-efficiency posttranslational modification capacity, and the recombinant protein produced by CHO cells is more similar to natural protein molecules; (2) CHO cells have been extensively studied to demonstrate a safe host cell for the expression of foreign proteins, and thus they may be more easily qualified and approved for marketing therapeutic proteins from regulatory agencies such as the FDA; (3) the major drawback of using other mammalian cells for protein production is low productivity, but it can be overcome by gene amplification in CHO cells; (4) the CHO cells are easier to perform suspension cell culture in serum-free, and can perform high-density high-level expression of foreign proteins in a specific culture medium by serum-free suspension culture and domestication, thereby providing convenience for industrial production.
In order to further improve the secretory expression quantity of the target protein in the CHO cells, a promoter and a signal peptide in a transfection plasmid are key, so that the selection of a proper promoter and a proper signal peptide suitable for the expression of the CHO cells is a key factor for realizing the high-efficiency expression of the CHO cells, but the selection of the promoter and the signal peptide in the prior art is mainly combined with the target protein for one experiment, and has the advantages of small flux, long time and high cost. Therefore, it is necessary to develop a suitable screening method.
Porcine Circovirus Type 2 (PCV 2) is one of the major transmitting pathogens causing Porcine Circovirus-Associated Disease (PCVAD), and causes huge economic losses each year for the development of the piglet breeding and breeding industry in China and even in the world. Currently, vaccination is the primary measure for the control and prevention of PCVAD. The capsid protein (Cap) has been shown to be the most immunogenic protein of PCV2, and is closely related to infection and immunization with PCV 2. Thus, Cap protein is a potential target protein for developing PCV2 vaccine.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a CHO cell high-efficiency expression system and a screening method thereof; and secondly, the method is applied to PCV2Cap protein expression.
Therefore, in one aspect, the invention provides a CHO cell high-efficiency expression system, which is characterized in that the CHO cell contains a eukaryotic expression vector of CAG promoter gene-human interferon-alpha 2 signal peptide gene-Luc gene.
Preferably, the CAG promoter gene is shown as SEQ ID NO. 7; the human interferon-alpha 2 signal peptide gene is shown in SEQ ID NO. 4.
Preferably, the Luc gene is shown as SEQ ID NO. 8.
Preferably, the CHO cell of the invention is a CHO-K1 cell; the eukaryotic expression vector is pcDNA3.1.
In another aspect, the present invention further provides a screening method of the CHO cell high-efficiency expression system, wherein the method comprises the following steps: 1) screening of promoters: cloning the promoter gene and the Luc gene into a eukaryotic expression vector to obtain a recombinant eukaryotic expression vector containing the promoter gene-Luc gene; transfecting the recombinant eukaryotic expression vector into a CHO cell, culturing the CHO cell, detecting the expression condition of the Luc protein by using a chemiluminescence instrument and a wertern blot, and screening a promoter with high Luc expression quantity for screening subsequent signal peptides; 2) screening of signal peptides: cloning the signal peptide gene into the optimal recombinant eukaryotic expression vector containing the promoter gene-Luc gene screened in 1) to obtain the recombinant eukaryotic expression vector containing the promoter gene-signal peptide gene-Luc gene, wherein the sequence of the genes in the recombinant eukaryotic expression vector is the promoter gene-signal peptide gene-Luc gene; by culturing CHO cells, detecting the expression condition of Luc protein by using a chemiluminescence apparatus and a wertern blot, and screening signal peptide with high Luc expression quantity, a CHO cell high-efficiency expression system is obtained.
Preferably, the eukaryotic expression vector is pcDNA3.1, and the CHO cell is CHO-K1 cell.
On the other hand, the invention also provides application of the CHO cell high-efficiency expression system in PCV2Cap protein expression, wherein the application is to replace Luc gene with PCV2-Cap protein gene to obtain eukaryotic expression vector of the CHO cell containing CAG promoter-human interferon-alpha 2 signal peptide-PCV 2-Cap protein gene.
Preferably, the gene sequence of the PCV2-Cap protein is shown in SEQ ID NO. 6.
Preferably, the arrangement sequence of the nucleotides in the eukaryotic expression vector is SEQ ID NO.7-SEQ ID NO.4-SEQ ID NO. 6.
Preferably, the CHO cell line provided by the invention is a CHO-K1 cell line; the eukaryotic expression vector is pcDNA3.1.
When the CHO cell high-efficiency expression system is used, the Luc gene is directly replaced by the target protein gene, so that the stable and high-efficiency secretory expression of the target protein in the CHO cell can be realized, and a proper promoter and a proper signal peptide can be conveniently screened by using the screening method.
In order to obtain a stable transfer CHO cell strain capable of efficiently expressing PCV2Cap protein, the invention obtains the stable transfer CHO cell strain capable of stably and efficiently expressing PCV2Cap protein by applying the system and the method. Firstly, Luciferase (Luciferase, Luc) is used as a reporter gene, different promoters and signal peptides are screened to influence the expression of the Luciferase in CHO-K1 cells, on the basis, a eukaryotic expression vector capable of expressing PCV2Cap protein is constructed, and after CHO-K1 cells are transfected, cell strains capable of stably expressing Cap protein are screened. The concentration of the recombinant Cap protein after nickel column purification of the cell strain obtained by screening is 0.6mg/L under the condition of adherent culture in a laboratory; under the condition of shake flask culture, the yield can reach more than 1.0 g/L. And is easy to purify, thereby being easy for large-scale industrial production. The purified and recombined Cap protein is used for immunizing BALB/c mice, and the detection of antibody level, lymphocyte proliferation experiment and the like proves that the Cap protein has good immunogenicity, and can be used for preparing PCV2 genetic engineering subunit vaccines.
Drawings
FIG. 1 is a schematic diagram of the construction of pcDNA3.1-Luc recombinant vector.
FIG. 2 is a schematic diagram of the construction of pcDNA3.1-CAG-Luc recombinant vector.
FIG. 3 is the identification of reporter gene Luc protein expression quantity by the promoter, wherein A is the Western Blot detection result of Luc protein, B is the gray scale analysis statistical result of protein Luc expression quantity, and C is the chemiluminescence detection result of Luc.
FIG. 4pcDNA3.1-CAG-SPXSchematic representation of Luc recombinant vector construction.
FIG. 5 shows the effect of signal peptide on Luc reporter gene expression, where A is the chemiluminescence detection result of Luc, and B is the Western Blot detection result of Luc protein.
FIG. 6 is a schematic diagram of the construction of pcDNA3.1-CAG-SP4-Cap recombinant vector.
FIG. 7 shows the Western Blot identification result of the expression of the recombinant protein Cap of different cell clones, wherein A is the Western Blot identification of the expression of the Cap protein, and B is the gray scale analysis statistical analysis of the Cap protein Western Blot.
FIG. 8 shows the result of measurement of serum-specific antibody titer after mice are immunized with recombinant Cap protein.
FIG. 9 shows the result of detecting the spleen lymphocyte proliferation index of mice immunized with recombinant Cap protein.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only some of the embodiments of the invention, and not all of them.
The chemical reagents related to the invention are all made in China. The formulations of the reagents mentioned are as follows:
PBS：8gNaCl，0.2g KCl，1.44gNa2HPO4，0.24g KH2PO4soluble in ddH2O is constant volume to 1L; antigen coating buffer: 1.59g Na2CO3，2.93g NaHCO3，ddH2O is constant volume to 1L; ELISA washing solution: 0.05% Tween-20 in PBS; ELISA blocking solution: 5% skim milk powder, 5% BSA in PBS;ELISA antibody dilutions: 2% skimmed milk powder, 1% BSA in PBS; ELISA stop solution: 2M H2SO4。
EXAMPLE 1 establishment of CHO cell high-efficiency expression System
The vector commercialized pAAV-CAG-RFP and pNF kappa B-TA-Luc plasmids are used as templates, primers are designed, and a CAG promoter gene and a Luc reporter gene are respectively amplified by a PCR technology, wherein a PCR amplification primer reaction system is shown in Table 1.
TABLE 1 PCR primer design
The amplified CAG and Luc genes were digested with restriction enzymes BglII and XbaI, and ligated into pcDNA3.1 vector using molecular biology method. The pcDNA3.1-CAG-Luc and pcDNA3.1-Luc (note: the original vector is CMV promoter) recombinant vectors were obtained, and the vector maps are shown in FIG. 1 and FIG. 2.
The recombinant plasmids pcDNA3.1-CAG-Luc and pcDNA3.1-Luc which respectively carry the CAG promoters and the CMV promoters and are obtained by passing through CaCl2TG1 competent cells were transformed and positive clones were selected. The plasmids were extracted and cultured at a density of about 4.5X 10 by transfection with FuGENE6 reagent (Promega Co., Ltd.)6And in the CHO-K1 cells, the cells are collected for protein expression identification after lysis after 72h of transfection. The expression level of the Luc protein is detected by a chemiluminescence apparatus, the anti-Luc (Beijing Boaosen biotechnology, Inc., the product number is bsm-33318M) is used as a primary antibody, the expression condition of the Luc protein is detected by a Western Blot method, a promoter with high expression level is screened for later use, and the result proves that the effect of the CAG promoter is superior to that of a CMV promoter (P promoter)<0.05 or P<0.001) as shown in fig. 3.
Signal peptides of the following 5 genes were selected: SP1 (human serum albumin); SP2 (azacyclic protein precursor protein); SP3 (mouse signal peptide IgG κ); SP4 (human interferon-. alpha.2) and SP5 (human interleukin-2). Selecting proper enzyme cutting sites according to the sequences of the vector and the gene, and chemically synthesizing the following signal peptide sequences with XbaI restriction enzyme cutting sites and XhoI restriction enzyme cutting sites at two ends respectively: XbaI-SP 1-XhoI; XbaI-SP 2-XhoI; ③ XbaI-SP3-XhoI, fourthly XbaI-SP4-XhoI and fifthly XbaI-SP5-XhoI, and the five sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
The synthesized sequences (i), ii, iii, iv and v) are double digested with restriction enzymes XbaI and XhoI, respectively, and then ligated into pcDNA3.1-CAG-Luc vector by molecular biology method. Obtaining pcDNA3.1-CAG-SPXThe Luc recombinant vector has a vector map shown in FIG. 4.
The recombinant plasmid pcDNA3.1-CAG-SP obtained above is usedXLuc through CaCl2The method is transformed into TG1 competent cells, and positive clones are screened out. The plasmid was extracted and transfected into the plasmid by FuGENE6 (Promega) reagent at a density of about 4.5X 106And in the CHO-K1 cells, cell culture supernatant is collected after transfection for 72h, and meanwhile, the cells are lysed to collect lysate for protein expression identification. The expression level of the Luc protein is detected by a chemiluminescence apparatus, meanwhile, anti-Luc is used as a primary antibody, the expression condition of the Luc protein is detected by a Western Blot method, and a signal peptide with high expression quantity is screened for later use, and the result proves that the target protein secreted in a cell culture solution and the target protein expressed in the cell are high in the cell of the recombinant plasmid with the SP4 signal peptide, and is shown in figure 5.
Through screening of different promoters and signal peptides, the expression quantity of the target protein of the recombinant vector carrying the CAG promoter and the SP4 signal peptide is proved to be the highest, and a system for efficiently expressing the foreign protein by the CHO cell is established.
Example 2 establishment of CHO cell line stably expressing porcine circovirus type 2Cap protein
The gene sequence of the Shandong strain PCV2Cap (accession number: KY656098.1) is searched in an NCBI database, the nuclear localization signal peptide (1-123bp) is deleted, and the codon optimization based on a CHO cell expression system is carried out on the remaining 579bp nucleic acid sequence. Selecting proper enzyme cutting sites according to the sequences of the vector and the gene, and chemically synthesizing the following fusion genes: sixthly, XhoI-Cap-6 XHis-TGA-EcoRI, wherein TGA is a stop codon. The sequence is shown as SEQ ID NO. 6.
The sequence is digested twice by XhoI and EcoRI and is connected into pcDNA3.1-CAG-SP4 vector by molecular biology method. The pcDNA3.1-CAG-SP4-Cap recombinant vector was obtained, and the vector map is shown in FIG. 6.
The recombinant plasmid pcDNA3.1-CAG-SP4-Cap obtained above was passed through CaCl2The method is transformed into TG1 competent cells, and positive clones are screened out. Plasmids were extracted and transfected by the FuGENE6 reagent at a density of about 4.5X 106After 24 hours of transfection, the CHO-K1 cells were cultured by changing the medium with the complete medium with the final concentration of 700. mu.g/mL G418; the cell status was observed under the microscope every day, and every 48h the medium was replaced with complete medium with a final concentration of 700. mu.g/mL G418. After all the cells in the control group are completely dead, the G418 concentration is reduced to 350 mug/mL, the culture is continued, when the monoclonal cells are observed in the positive control group under a microscope, the cells in the experimental group are conveyed to a 60mm culture dish to be continuously cultured, and the monoclonal cell strains are screened out in a 96-well culture plate by a limiting dilution method.
After the screened monoclonal cell strain is subjected to amplification culture, collecting culture supernatant and cell lysate to perform SDS-PAG E electrophoresis and Western Blot to detect the expression condition of the recombinant protein. Protein expression in cell lysate and cell culture supernatant is shown through BCA protein quantification and Western Blot results, which indicates that CHO cell strains capable of secreting and expressing Cap protein are obtained through screening. Through calculation, under the condition of adherent culture in a laboratory, the concentration of the recombinant Cap protein after nickel column purification is 0.6 mg/L; under the condition of shake flask culture, the yield can reach more than 1.0 g/L.
8 strains of CHO-K1 cells obtained by screening are respectively cultured in an expansion mode, and the cells are collected to obtain the target protein after cracking for identification. By using anti-Cap (Beijing Boolsen biotechnology Co., Ltd., cat # bs-10057R) as a primary antibody and detecting the expression of the recombinant protein by Western Blot method, as shown in FIG. 7A, a band was observed at 25-35kDa, which is consistent with the theoretical size of 26.1kDa, and based on this, SP4-1, SP4-2, SP4-3, SP4-5, SP4-6, SP4-7, SP4-9, SP4-11 of 8 cell lines (SP4-1, SP4-2, SP4-3, SP4-5, SP4-9 and SP4-11 all stably expressed Cap protein.
In order to obtain cell strains with stronger cell proliferation capacity, SP4-1, SP4-2, SP4-3 and SP4-54 strain CHO-K1 cells with higher expression levels are screened by MTT experiments. In the MTT experiment, a blank control group (without cells and G418, only complete culture medium) and an experimental group (with cells and complete culture medium, G418 are added) are arranged; 3 multiple wells were set for each group, and after continuous culture for 96 hours, MTT assay was performed, and it was found that SP4-1 cell line had a strong proliferation potency, as shown in FIG. 7B. And (3) amplifying the SP4-1 monoclonal cell strain with stronger proliferation capacity, collecting cell lysate, purifying Cap protein through a nickel column, and storing the Cap protein in a refrigerator at the temperature of-80 ℃ for later use.
Example 3 analysis of immunogenicity of recombinant Cap proteins expressed by CHO cells
24 female BALB/c mice 6-8 weeks old were randomly divided into the following three groups: a PBS negative control group; cap protein experimental group; ③ positive control group of PCV2 vaccine (Yunliyou, jin Yu Biotechnology Co., Ltd.), 8 vaccine groups. The immunization was performed at 0, 14 and 28d, respectively, using the subcutaneous route. Wherein, the PBS group is injected with 200 mu L sterile PBS per mouse, the positive vaccine group is used for immunizing 100 mu L commercial Yueliou vaccine per mouse, and the experimental group is used for immunizing 25 mu g Cap protein per mouse. Tail bleeds were performed at 7, 14, 28, 35 and 42d, respectively, and splenic lymphocyte proliferation assays were performed on 3 mice per group at 14, 28, 35 and 42d, respectively.
The specific detection steps are as follows:
(1) antibody level detection: in order to evaluate the humoral immune effect of the mouse on antigen stimulation, an indirect ELISA method is adopted, the mouse is coated with Cap antigen protein which is diluted by buffer solution and has the concentration of 1 mug/mL prokaryotic expression, ELISA confining liquid is sealed, serum collected in the above 7, 14, 28, 35 and 42d five time periods is respectively incubated, secondary antibody is incubated, and color development liquid is added for color development, wherein the plate is washed 4 times by ELISA washing liquid in every two steps, and each time is 1 min. And finally adding an ELISA stop solution to stop the color reaction, and detecting the OD value at the wavelength of 450 nm. The results are shown in fig. 8, the experimental group of mice showed significant specific IgG response (P <0.001) to the antigen, indicating that the recombinant Cap protein expressed by CHO cells has good immunogenicity.
(2) SpleenLymphocyte proliferation assay: in order to evaluate the cellular immune effect of the mice on antigen stimulation, splenic lymphocytes of groups of 14, 28, 35 and 42d of mice were isolated by using a mouse lymphocyte separation solution (Beijing Dake is Biotechnology Co., Ltd.), and the specific operation flow is shown in the product specification. The isolated groups of mice were counted for splenic lymphocytes and diluted to a density of 4.5X 10 per well in a 96-well plate6cells/mL 100. mu.L of cell suspension. After the cells are attached to the wall, 100. mu.L of diluted recombinant Cap antigen protein (10. mu.g/mL final concentration), 1640 culture medium and positive control (concanavalin, 10. mu.g/mL final concentration) are added to each well. Placing the cells in CO2After culturing in an incubator for 42h, 20 μ g of MTT (5mg/mL) was added to each well, the culture was continued for 4h, the cell culture supernatant was discarded, 100 μ L of MSO solution was added to each well, the mixture was shaken and mixed on a shaker for 5min, and finally the OD value at 490nm was measured and the stimulation index (SI ═ stimulated well OD value/unstimulated well OD value) was calculated. As a result, the 28d starting after immunization was found to be significantly higher than that of the PBS group and the positive vaccine group (P)<0.05、P<0.01 or P<0.001), as shown in fig. 9, where NS indicates no significant difference, and indicates P<0.05, represents P<0.01, represents P<0.001. Compared with a PBS negative control group and a commercial vaccine positive control group, the mice of the Cap immunization experiment group show higher Stimulation Index (SI) for antigen stimulation, and the mice can trigger specific cellular immune effect in the mice after being immunized by the recombinant Cap protein expressed by the CHO cells.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Sequence listing
<110> Zhejiang Hongshan Cheng Biotechnology GmbH, Zhejiang university of science and technology, Inc
<120> high-efficiency expression system for stably expressing foreign protein CHO cells, screening method thereof and application of high-efficiency expression system in PCV2Cap protein
<160>5
<170>PatentIn version 3.5
<210> 1
<211>57
<212>DNA
<213> SP1 sequence
<400> 1
atgaagtggg tgaccttcat ctccctgctg ttcctgtttt ccagctcttc cagggcc 57
<210> 2
<211>57
<212>DNA
<213> SP2 sequence
<400> 2
atgaccaggc tgacagtgct ggccctgctg gctggactgc tggcctccag ccgggct 57
<210>3
<211>60
<212>DNA
<213> SP3 sequence
<400>3
atggagaccg acacactgct gctgtgggtg ctgctgctgt gggtgccagg ctccaccggc 60
<210>4
<211>69
<212>DNA
<213> SP4 sequence
<400>4
atggccctga ccttcgctct gctggtggcc ctgctggtgc tgtcttgcaa gtccagctgt 60
tccgtgggc 69
<210>5
<211>60
<212>DNA
<213> SP5 sequence
<400>5
atgaggcgga tgcagctgct gctgctgatc gccctgtccc tggctctggt gaccaacagc 60
<210>6
<211>579
<212>DNA
<213> Cap sequence
<400>6
aacggcatct tcaatacaag actgtctcgc acatttggct ataccgtgaa gaagaccaca 60
gtgaggaccc catcctggaa ggtggacatg atgcggttca acatcaatga ttttctgcca 120
cctggaggag gctccaaccc actgagcgtg cccttcgagt actataggat caggaaggtg 180
aaggtggagt tttggccctg ctcccctatc acccagggcg acaggggagt gggctccagc 240
gccgtgatcc tggacgataa cttcgtgacc aaggccacag ctctgaccta cgatccttat 300
gtgaattact cttccagaca caccatcaca cagccattca gctatcattc tcgctacttt 360
acacctaagc cagtgctgga cagcaccatc gattatttcc agcccaacaa taagaggaac 420
cagctgtggc tgcggctgca gaccacaggc aatgtggacc acgtgggcct gggcacagct 480
tttgagaact ctatctatga tcaggagtac aatatcagag tgaccatgta cgtgcagttc 540
cgcgagttta acctgaagga cccacccctg aatcctaag 579
<210>7
<211>659
<212>DNA
<213> CAG promoter Gene sequence
<400>7
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420
cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480
tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540
gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600
cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcg 659
Claims (10)
1. A CHO cell high-efficiency expression system is characterized in that the CHO cell contains a eukaryotic expression vector of CAG promoter gene-human interferon-alpha 2 signal peptide gene-Luc gene.
2. The system of claim 1, wherein the CAG promoter gene is set forth in SEQ ID No. 7; the human interferon-alpha 2 signal peptide gene is shown in SEQ ID NO. 4.
3. The system according to claim 1, wherein the Luc gene is as set forth in SEQ ID No. 8.
4. The system of claim 1, wherein the CHO cell is a CHO-K1 cell; the eukaryotic expression vector is pcDNA3.1.
5. The method for screening the CHO cell high expression system according to claim 1, wherein the method comprises the following steps:
1) screening of promoters: cloning the promoter gene and the Luc gene into a eukaryotic expression vector to obtain a recombinant eukaryotic expression vector containing the promoter gene-Luc gene; transfecting the recombinant eukaryotic expression vector into a CHO cell, culturing the CHO cell, detecting the expression condition of the Luc protein by using a chemiluminescence instrument and a wersternblot, and screening a promoter with high Luc expression quantity for screening subsequent signal peptides;
2) screening of signal peptides: cloning the signal peptide gene into the optimal recombinant eukaryotic expression vector containing the promoter gene-Luc gene screened in 1) to obtain the recombinant eukaryotic expression vector containing the promoter gene-signal peptide gene-Luc gene, wherein the sequence of the genes in the recombinant eukaryotic expression vector is the promoter gene-signal peptide gene-Luc gene; by culturing CHO cells, detecting the expression condition of Luc protein by using a chemiluminescence apparatus and a wertern blot, and screening signal peptide with high Luc expression quantity, a CHO cell high-efficiency expression system is obtained.
6. The method of claim 5, wherein the eukaryotic expression vector is pcDNA3.1 and the CHO cell is CHO-K1 cell.
7. The application of the CHO cell high-efficiency expression system in PCV2Cap protein expression according to claim 1, wherein the Luc gene is replaced by the gene of PCV2-Cap protein, and the obtained CHO cell contains the eukaryotic expression vector of CAG promoter-human interferon-alpha 2 signal peptide-PCV 2-Cap protein gene.
8. The use according to claim 7, wherein the gene sequence of PCV2-Cap protein is shown in SEQ ID No. 6.
9. The use according to claim 7, wherein the sequence of nucleotides in the eukaryotic expression vector is SEQ ID No.7-SEQ ID No.4-SEQ ID No. 6.
10. The use of claim 7, wherein the CHO cell line is CHO-K1 cell line; the eukaryotic expression vector is pcDNA3.1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115044613A (en) * | 2022-06-20 | 2022-09-13 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Genetically engineered cell line for expressing cap protein of porcine circovirus, and construction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554973A (en) * | 2015-09-30 | 2017-04-05 | 北京吉尚立德生物科技有限公司 | A kind of Chinese hamster ovary celI secretion capacity evaluation system |
-
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106554973A (en) * | 2015-09-30 | 2017-04-05 | 北京吉尚立德生物科技有限公司 | A kind of Chinese hamster ovary celI secretion capacity evaluation system |
Non-Patent Citations (3)
| Title |
|---|
| RAM´ON 等: ""Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide"", 《JOURNAL OF BIOTECHNOLOGY》 * |
| 李琴 等: ""不同启动子和MAR组合对重组CHO细胞转基因表达的影响"", 《四川大学学报》 * |
| 苗杨利 等: ""促进米黑根毛霉脂肪酶分泌的毕赤酵母内源信号肽的筛选"", 《现代食品科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115044613A (en) * | 2022-06-20 | 2022-09-13 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Genetically engineered cell line for expressing cap protein of porcine circovirus, and construction method and application thereof |
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