A CHO cell line overexpressing feline parvovirus VP2 gene and a construction method thereof

Abstract

The invention provides a CHO cell line for overexpressing a feline parvovirus VP2 gene and a construction method thereof. The invention comprises the following steps: cloning a tandem sequence containing a CMV promoter, an EF1α promoter and an optimized feline parvovirus VP2 gene fragment into a Tol2 vector having a transposon element, constructing an FPV VP2 transposition system vector, and continuously and efficiently catalyzing the integration of the FPV VP2 gene into a gene locus of a CHO cell by a transposase, thereby obtaining a CHO cell line for overexpressing the feline parvovirus VP2 gene, realizing stable overexpression of FPV VP2 in the CHO cell, and proving through qPCR experiments and WB detection that the overexpression vector of the double promoter (CMV+EF1α) and FPV VP2 in series can significantly increase the expression amount of the FPV VP2 protein of the CHO cell line.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a CHO cell line over-expressing a feline parvovirus VP2 gene and a construction method thereof. Background Art

[0002] Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell expression system for producing recombinant proteins and are widely used in genetic engineering and biopharmaceuticals. However, there are also problems such as low recombinant protein expression and unstable glycosylated expression products, which restrict their application in vaccine and clinical therapeutic drug development. Therefore, using genetic engineering and cell engineering methods to study the expression of recombinant proteins in CHO cells to obtain cell lines with high expression levels and high expression stability is of great significance and application value for establishing a high-yield and stable recombinant protein CHO cell expression system.

[0003] Feline parvovirus (FPV) is also known as feline panleukopenia virus, feline distemper virus, and feline infectious enteritis virus. Feline panleukopenia is a highly contagious disease caused by feline parvovirus, with high morbidity and mortality. The virus infects a variety of cats and mustelids under natural conditions, causing significant economic losses to the animal breeding and pet industries.

[0004] FPV is a single-stranded negative-strand linear non-enveloped DNA virus with a diameter of 20 to 24 nm and icosahedral symmetry, which is highly resistant to physical and chemical factors. The FPV viral genome encodes two structural proteins (VP1, VP2) and two non-structural proteins (NS1, NS2). VP2 protein is the main component of the feline parvovirus shell, accounting for about 90% of all structural proteins. It is the main antigenic protein encoded by FPV and plays an important role in immune response, receptor recognition and tissue infection.

[0005] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for specific binding of RNA polymerase and transcription initiation. It is a key element in gene expression regulation and is generally located upstream of the transcription start site. In genetic engineering, promoters are widely used to construct gene expression vectors. By selecting a suitable promoter, the expression level and time of the target gene in the host cell can be regulated. However, whether the tandem of dual promoters can increase the expression yield of recombinant proteins in CHO cell lines has not been reported. Summary of the invention

[0006] The invention aims to provide a CHO cell line for overexpressing the feline parvovirus VP2 gene and a construction method thereof, construct an overexpression vector in which a double promoter (CMV+EF1α) and FPV VP2 are connected in series, a vector containing a Tol2 transposon element is used as a skeleton, an FPV VP2 transposition system vector is constructed, and FPV VP2 is integrated into a CHO cell gene locus through the catalysis of a transposase, so that stable overexpression of FPVVP2 in CHO cells can be achieved.

[0007] In the first aspect, the present invention first provides an overexpression vector of the feline parvovirus VP2 gene, which contains CMV and EF1α dual promoter elements, wherein the nucleic acid sequence of the EF1α promoter is as shown in SEQ ID NO.1, and the feline parvovirus VP2 gene is an optimized gene, and its nucleotide sequence is as shown in SEQ ID NO.3.

[0008] Preferably, the above-mentioned feline parvovirus VP2 gene overexpression vector is constructed by connecting the EF1α promoter and the optimized feline parvovirus VP2 gene fragment and then inserting the fragment into the multiple cloning site of the pIRES2-EGFP plasmid containing the CMV promoter.

[0009] In a second aspect, the present invention provides a transposition system vector of the feline parvovirus VP2 gene, which is constructed by double-digesting the above-mentioned feline parvovirus VP2 gene overexpression vector with AseI and ClaI to obtain a CMV-EF1α-FPV VP2 tandem sequence, and then inserting the CMV-EF1α-FPVVP2 tandem sequence into a Tol2 vector.

[0010] In a third aspect, the present invention provides a CHO cell line that overexpresses the feline parvovirus VP2 gene, comprising the above-mentioned transposition system vector of the feline parvovirus VP2 gene.

[0011] Preferably, the above-mentioned CHO cell line overexpressing the feline parvovirus VP2 gene is prepared by transfecting the transposition system vector of the feline parvovirus VP2 gene into CHO cells, so that the feline parvovirus VP2 gene is integrated into the CHO cell gene locus.

[0012] Preferably, the above-mentioned CHO cell line overexpressing the feline parvovirus VP2 gene, the CHO cells are CHO-S cells.

[0013] In a fourth aspect, the present invention provides a method for constructing a CHO cell line that overexpresses the feline parvovirus VP2 gene, wherein the transposition system vector of the feline parvovirus VP2 gene is integrated into the CHO cell locus under the action of the Tol2 transposon and the catalysis of the transposase, and CHO cells that stably overexpress the feline parvovirus VP2 are screened based on the mRNA expression level.

[0014] Preferably, in the above-mentioned method for constructing a CHO cell line overexpressing the feline parvovirus VP2 gene, the screening includes a qPCR detection process, and the qPCR primer set used in the qPCR detection process includes FPV VP2-F and FPV VP2-R; wherein the sequence of FPV VP2-F is shown in SEQ ID NO.4, and the sequence of FPVVP2-R is shown in SEQ ID NO.5.

[0015] Preferably, in the above-mentioned method for constructing a CHO cell line overexpressing the feline parvovirus VP2 gene, the qPCR primers for the internal reference β-actin used in the qPCR detection process include β-actin-F and β-actin-R, wherein the sequence of β-actin-F is such as SEQ ID NO.6, and the sequence of β-actin-R is such as SEQ ID NO.7.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The technical solution of the present invention clones a tandem sequence containing a CMV promoter, an EF1α promoter, and an optimized feline parvovirus VP2 gene fragment into a Tol2 vector with a transposon element to construct an FPV VP2 transposition system vector, and continuously and efficiently catalyzes the integration of the FPV VP2 gene into the gene locus of a CHO cell by a transposase, thereby obtaining a CHO cell line that overexpresses the feline parvovirus VP2 gene, and realizing stable overexpression of FPVVP2 in the CHO cells. qPCR experiments and WB detections prove that the overexpression vector of the present invention, in which the dual promoter (CMV+EF1α) and FPV VP2 are connected in series, can significantly increase the expression amount of the FPVVP2 protein in the CHO cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the structure of the overexpression vector of the dual promoter (CMV+EF1α)+FPV VP2 gene provided in an embodiment of the present invention;

[0019] Figure 2 A schematic diagram of the structure of the transposition system vector of the dual promoter (CMV+EF1α)+FPV VP2 gene provided in an embodiment of the present invention;

[0020] Figure 3 A schematic diagram of the transposition system vector structure of a single promoter (CMV) + FPV VP2 gene provided in an embodiment of the present invention;

[0021] Figure 4 A schematic diagram of the transposition system vector structure of a single promoter (EF1α) + FPV VP2 gene provided in an embodiment of the present invention;

[0022] Figure 5 The expression level of FPVVP2 mRNA in the CHO cell line overexpressing the FPV VP2 gene provided in the embodiment of the present invention;

[0023] Figure 6 The expression level of FPVVP2 protein in the CHO cell line overexpressing the FPV VP2 gene provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0024] The invention provides an overexpression vector of a feline parvovirus VP2 gene containing double promoters in series, which is constructed by connecting an EF1α promoter and an optimized feline parvovirus VP2 gene fragment and then inserting the fragment into a multiple cloning site (MCS) of a pIRES2-EGFP plasmid.

[0025] The sequence of the EF1α promoter selected in the present invention is shown in SEQ ID NO.1.

[0026] The present invention uses the VP2 sequence of the self-isolated FPV-JT02 strain as shown in SEQ ID NO. 2, and the FPV-JT02 strain has a high homology with the current popular strains in China. The present invention performs codon optimization according to the codon preference of Chinese hamster ovary cells.

[0027] The invention provides a transposition system vector of feline parvovirus VP2 gene containing double promoter series elements, which is constructed by inserting the series sequence of CMV promoter, EF1α promoter and optimized feline parvovirus VP2 gene fragment into Tol2 vector.

[0028] In the present invention, the feline parvovirus VP2 gene is regulated by the CMV promoter and EF1α promoter tandem elements.

[0029] In the present invention, the Tol2 vector is a vector having a transposon element. In the present invention, the feline parvovirus VP2 gene fragment together with the upstream CMV promoter and EF1α promoter tandem elements are inserted into the Tol2 vector.

[0030] The invention provides a CHO cell line over-expressing the feline parvovirus VP2 gene. The transposition system vector of the feline parvovirus VP2 gene is transfected into the CHO cells, so that the FPV VP2 gene is integrated into the CHO cell gene locus.

[0031] The present invention also provides a method for constructing the CHO cell line that overexpresses the feline parvovirus VP2 gene, wherein the transposition system vector of the FPVVP2 gene is transfected into CHO cells under the action of the Tol2 transposon and through the catalysis of the transposase, and the CHO cells that stably overexpress FPVVP2 are screened according to the mRNA expression level.

[0032] In the present invention, the screening includes a qPCR detection process, and the primer set in the qPCR detection process includes FPVVP2-F and FPV VP2-R. The sequence of FPV VP2-F of the present invention is shown in SEQ ID NO.4, and the specific sequence is CTGAGAACAGGCGATGAGT; the sequence of FPV VP2-R is shown in SEQ ID NO.5, and the specific sequence is TGGTGAGCGATGTAGGTGA.

[0033] In order to further illustrate the present invention, the test paper and the preparation method thereof of the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1 Optimization of Feline Parvovirus VP2 Gene

[0035] After the genome of the FPV strain was extracted and sequenced, the VP2 gene sequence was obtained. The present invention optimized the codons according to the codon preference of Chinese hamster ovary cells, replaced the rare codons, reduced translation sequences, and released translation sequences with common codons, and adjusted the CG content and unfavorable peaks to extend the half-life of mRNA. The codon preference was adjusted from 0.66 to 0.93, and the CG content was adjusted from 35.16% to 56.13%. The VP2 gene sequence before optimization is shown in SEQ ID NO.2, and the VP2 gene sequence after optimization is shown in SEQ ID NO.3.

[0036] Example 2 Synthesis of EF1α promoter tandem with feline parvovirus VP2 gene

[0037] The EF1α promoter gene sequence was connected to the optimized VP2 gene sequence, and the EF1α promoter gene sequence was shown in SEQ ID NO. 1. The constructed sequence was sent to the company for gene synthesis (commissioned to Sangon Biotech (Shanghai) Co., Ltd.), namely EF1α-FPVVP2.

[0038] Example 3 Construction of FPVVP2 overexpression vector

[0039] The synthesized EF1α-FPV VP2 gene was cloned into the pIRES2-EGFP plasmid (commissioned to Sangon Biotech (Shanghai) Co., Ltd.) to obtain the FPV VP2 overexpression vector. The schematic diagram of the structure of the vector before and after cloning is shown in Figure 1 shown.

[0040] Example 4 Construction of CMV+EF1α dual promoter FPVVP2 transposition system vector

[0041] The FPVVP2 overexpression vector constructed in Example 3 was double-digested with AseI and ClaI, and the excised fragment, namely CMV-EF1α-FPV VP2, was ligated into the transposition system vector pTol2 plasmid (purchased from Addgene) after double-digestion with AseI and ClaI by T4 ligase (purchased from Takara Biotechnology (Dalian) Co., Ltd.) to obtain the CMV+EF1α dual promoter FPVVP2 transposition system vector. The structural diagram of the vector is shown in FIG. Figure 2 shown.

[0042] Control Example 1 Construction of CMV single promoter FPVVP2 transposition system vector

[0043] The optimized VP2 gene sequence was synthesized (commissioned to Sangon Biotech (Shanghai) Co., Ltd.), and the synthesized FPV VP2 gene was cloned into the pIRES2-EGFP plasmid (commissioned to Sangon Biotech (Shanghai) Co., Ltd.). The constructed vector was double-digested with AseI and ClaI, and the excised fragment, namely CMV-FPV VP2, was ligated into the transposition system vector pTol2 plasmid (purchased from Addgene) double-digested with AseI and ClaI by T4 ligase (purchased from Takara Biotech (Dalian) Co., Ltd.) to obtain the CMV single promoter FPV VP2 transposition system vector. The structural schematic diagram of the vector is shown in the figure. Figure 3 shown.

[0044] Control Example 2 Construction of EF1α single promoter FPVVP2 transposition system vector

[0045] The AseI restriction site sequence, EF1α promoter gene sequence, optimized VP2 gene sequence, and ClaI restriction site sequence were concatenated and sent to the company for gene synthesis (commissioned by Sangon Biotech (Shanghai) Co., Ltd.), i.e., EF1α-FPVVP2, which was ligated into the transposition system vector pTol2 plasmid (purchased from Addgene) after double digestion with AseI and ClaI through T4 ligase (purchased from Takara Biotech (Dalian) Co., Ltd.), to obtain the EF1α single promoter FPV VP2 transposition system vector. The structural diagram of the vector is shown in the figure. Figure 4 shown.

[0046] Example 5 Construction of a CHO cell line overexpressing FPVVP2

[0047] CHO-S cells (purchased from Gibco) were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase. 5 The cells were inoculated into a 12-well cell culture plate for 24 hours and used for cell transfection when the cell confluence reached about 80%.

[0048] 50 μL of DMEM / F12 medium was added to 0.5 mL of serum-free medium (purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.), and then the CMV+EF1α dual promoter FPV VP2 transposition system vector (2.5 μg / well) constructed in Example 4 was transfected into the CHO-S cells obtained above under the action of transposase (purchased from Addgene) by liposome transfection to construct a CHO cell line overexpressing FPV VP2 as the overexpression (OE) group.

[0049] Similarly, the pTol2 plasmid, the CMV single promoter FPVVP2 transposition system vector constructed in Control Example 1, and the EF1α single promoter FPV VP2 transposition system vector constructed in Control Example 2 were transfected into the CHO-S cells obtained above under the action of transposase (purchased from Addgene) by liposome transfection method as blank control group (BC), single promoter control group C (Ctrl-C), and single promoter control group E (Ctrl-E).

[0050] Liposome transfection method is as follows:

[0051] (1) Taking a 24-well plate as an example, take a 1.5 mL centrifuge tube and mark the vector name, add 50 μL of basic DMEM / F12 medium into the tube, then take 0.6 μg of the CMV+EF1α dual promoter transposon vector expressing FPVVP2 constructed in Example 4 and 0.2 μg of transposase, add them into the tube respectively, mix well, and let stand at room temperature for five minutes;

[0052] (2) Take another centrifuge tube, mark it as the transfection reagent tube, add 50 μL of basic DMEM / F12 medium into the tube, then add 2 μL of transfection reagent Lipofectamine3000 into the tube, mix thoroughly, and let stand at room temperature for five minutes;

[0053] (3) Add the mixed solution in step (2) dropwise to the plasmid tube in step (1), mix thoroughly, and let stand at room temperature for 25 min;

[0054] (4) Take out the cells for transfection, discard the old culture medium, wash each well twice with 1× PBS buffer, and then add 500 μL of new DMEM / F12 reduced serum medium;

[0055] (5) Add the mixed solution in step (3) dropwise into the wells, gently shake the culture plate to evenly distribute the mixed solution, and then place it in an incubator for culture. After incubation for 5 hours, remove the culture plate, replace with fresh complete culture medium and continue culturing; use the optimal G418 concentration for cell screening 48 hours after cell transfection. After drug treatment, the cells no longer die, count the cell suspension, inoculate 1 cell per well into a 96-well plate, and wait for cell proliferation. When the cell fusion rate in the 96-well plate reaches about 90%, expand the culture, and then proceed to the subsequent passaging and screening steps.

[0056] Example 6 Detection of FPVVP2 overexpression efficiency

[0057] The wild-type CHO cells (BC) screened after transfection, CHO cells stably expressing FPV VP2 with a single CMV promoter (Ctrl-C), CHO cells stably expressing FPV VP2 with a single EF1α promoter (Ctrl-E), and CHO cells stably overexpressing FPV VP2 with dual promoters (OE) were collected and the expression level of FPV VP2 in each group was detected at the mRNA level by qPCR.

[0058] 1.qPCR Experiment

[0059] Primers for qPCR detection were designed for the target gene FPV VP2 and the internal reference gene β-actin. The CHO cell lines of the BC group, Ctrl-C group, Ctrl-E group, and OE group were collected and the total RNA of each group of cells was extracted with Trizol (Thermo Fisher Scientific), and then the total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Yubao Bioengineering (Dalian) Co., Ltd.). The cDNA was diluted at a ratio of 1:4 and used as a template. After qPCR detection, according to 2 -△△ The CT algorithm was used to calculate the relative expression of FPVVP2 mRNA.

[0060] The qPCR primers include FPV VP2-F and FPVVP2-R, the sequence of FPVVP2-F is shown in SEQ ID NO.4, specifically CTGAGAACAGGCGATGAGT; the sequence of FPV VP2-R is shown in SEQ ID NO.5, specifically TGGTGAGCGATGTAGGTGA.

[0061] The qPCR primers of the internal reference β-actin include β-actin-F and β-actin-R, the sequence of β-actin-F is such as SEQ ID NO.6, and the specific sequence is GTGCCCATTTATGAGGGCT; the sequence of β-actin-R is such as SEQ ID NO.7, and the specific sequence is AGCTGTGGTCACGAAGGAGT.

[0062] The total volume of the qPCR reaction system was 20.0 μL, including the following: 10.0 μL SYBR Green Mix, 2.0 μL diluted template, 1.0 μL each of primers (FPV VP2-F / FPV VP2-R), and 6.0 μL enzyme-free water.

[0063] The qPCR reaction conditions are: 95°C pre-denaturation for 30s, amplification phase (95°C for 10s, 55°C for 30s, 45 cycles), melting curve phase at 95°C for 15s, 60-95°C for 1min.

[0064] The results are as follows Figure 5 As shown, the mRNA expression level of FPVVP2 in the CHO cell line of the OE group was significantly increased, and was significantly better than that of the Ctrl-C and Ctrl-E groups and the sum of the expression levels of the Ctrl-C and Ctrl-E groups.

[0065] 2.WB detection

[0066] After taking out the four groups of cells (BC group, Ctrl-C group, Ctrl-E group, OE group) from the incubator, they were placed on an ice box, the original culture medium was discarded, and the cells were washed three times with pre-cooled PBS to remove the residual cell culture medium, and RIPA protein lysis buffer was added. The cells were shaken back and forth to allow the lysis solution to fully fuse with the cells in the culture bottle and fully lyse the cells. Use a high-speed refrigerated centrifuge to centrifuge at 12000rpm for 10 minutes and collect the supernatant. 5×SDS loading buffer was added to the supernatant and incubated at 100℃ for 10 minutes to fully denature it. 10μL of protein samples were taken respectively for separation with 10% SDS-PAGE, detected with Anti-FPVVP2 (1:1000) and Anti-β-actin (1:1000), developed with ECL chemiluminescent colorimetric solution (purchased from Biyuntian Biotechnology Co., Ltd.), and the expression level of the protein to be tested was observed and analyzed on the gel imaging analysis system.

[0067] The results are as follows Figure 6 As shown, compared with the CHO cells in the Ctrl-C group, Ctrl-E group and BC group, the expression level of FPVVP2 protein in the CHO cells in the OE group was significantly increased.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An overexpression vector of feline parvovirus VP2 gene, characterized in that: The overexpression vector contains CMV and EF1α double promoter elements, wherein the nucleic acid sequence of the EF1α promoter is shown in SEQ ID NO.1, and the feline parvovirus VP2 gene is an optimized gene, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. The overexpression vector of the feline parvovirus VP2 gene according to claim 1, characterized in that: The overexpression vector is constructed by connecting the EF1α promoter with the optimized feline parvovirus VP2 gene fragment and then inserting the fragment into the multiple cloning site of the pIRES2-EGFP plasmid containing the CMV promoter.

3. A transposition system vector of feline parvovirus VP2 gene, characterized in that: The method is constructed by double digesting the overexpression vector of the feline parvovirus VP2 gene according to claim 1 or 2 with AseI and ClaI to obtain a CMV-EF1α-FPVVP2 tandem sequence, and then inserting the CMV-EF1α-FPVVP2 tandem sequence into a Tol2 vector.

4. A CHO cell line overexpressing the feline parvovirus VP2 gene, characterized in that: A transposition system vector comprising the feline parvovirus VP2 gene according to claim 3.

5. The CHO cell line overexpressing the feline parvovirus VP2 gene according to claim 4, characterized in that The feline parvovirus VP2 gene is integrated into the CHO cell gene locus by transfecting the transposition system vector of the feline parvovirus VP2 gene into CHO cells.

6. The CHO cell line overexpressing the feline parvovirus VP2 gene according to claim 5, characterized in that: The CHO cells are CHO-S cells.

7. A method for constructing a CHO cell line overexpressing the feline parvovirus VP2 gene, characterized in that: The transposition system vector of the feline parvovirus VP2 gene described in claim 3 is integrated into the CHO cell locus under the action of the Tol2 transposon and catalyzed by the transposase, and CHO cells that stably overexpress feline parvovirus VP2 are screened based on the mRNA expression level.

8. The method for constructing a CHO cell line overexpressing the feline parvovirus VP2 gene according to claim 7, characterized in that: The screening includes a qPCR detection process, and the qPCR primer set used in the qPCR detection process includes FPV VP2-F and FPV VP2-R; wherein the sequence of FPV VP2-F is shown in SEQ ID NO.4, and the sequence of FPV VP2-R is shown in SEQ ID NO.

5.

9. The method for constructing a CHO cell line overexpressing the feline parvovirus VP2 gene according to claim 8, characterized in that: The qPCR primers for the internal reference β-actin used in the qPCR detection process include β-actin-F and β-actin-R, wherein the sequence of β-actin-F is such as SEQ ID NO.6, and the sequence of β-actin-R is such as SEQ ID NO.7.

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