CHO stably transfected cell strain expressing recombinant antibody capable of specifically recognizing CSFV2 type Erns protein

Abstract

The invention relates to the technical field of bioengineering, and aims to provide a CHO stably transfected cell strain capable of expressing a recombinant antibody capable of specifically recognizing CSFV2 type Erns protein. The cell strain is preserved in the China Center for Type Culture Collection, the preservation name is Chinese hamster ovary cell gs weak strain CHO-9B1-C, and the preservation number is CCTCC NO: C2024393. The invention also provides application of the cell strain in preparation of a diagnostic kit for specifically detecting the CSFV2 type hog cholera virus. The CHO stably transfected cell strain provided by the invention has the advantages of short monoclonal antibody production cycle, high consistency and high repeatability, is suitable for large-scale production, and is easy to transform.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and specifically relates to a method for expressing a gene that can specifically identify the 2nd type E gene of classical swine fever virus. rns A CHO stable cell line of protein antibody and a preparation method and application method thereof. Background Art

[0002] Classical swine fever (CSF) is an important viral disease that infects domestic pigs and wild boars. It is highly contagious and pathogenic and is one of the diseases that must be reported by the World Organization for Animal Health (OIE). The disease is caused by the classical swine fever virus (CSFV) of the genus Pestivirus in the family Flaviviridae and is a Category II animal infectious disease in my country.

[0003] CSFV contains three genotypes (type 1, type 2 and type 3), CSFV type 1 is more classic, and most of the strains isolated since the 1980s belong to CSFV type 2. The CSFV attenuated C strain (hereinafter referred to as the C strain) is obtained by continuously attenuating the CSFV type 1 strong strain in rabbits. Pigs will not have a temperature reaction after being inoculated with the C strain, and only mild viremia will occur. The C strain is recognized as a safe and effective CSF vaccine, but it does not have the ability to provide relevant detection kits with the ability to identify CSFV wild virus infection serum antibodies and vaccine C strain immune serum antibodies. In addition, the CSFV strains prevalent in the main pig-producing areas in China are mainly CSFV type 2, such as QZ14 strain, HZ08 strain, HuN23 strain, HLJ1 strain, etc. The prevalence of different types of strains such as CSFV type 2 may affect the immune protection effect of the C strain vaccine based on genotype 1.

[0004] Therefore, it is necessary to propose new solutions to solve the above problems. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for expressing a gene that can specifically identify CSFV2 type E rns CHO stable cell line for protein recombinant antibody.

[0006] To solve the above technical problems, the solution of the present invention is:

[0007] Provide a method for expressing a gene that can specifically recognize CSFV2 type E rns The CHO stable cell line of protein recombinant antibody is deposited in the China Center for Type Culture Collection, the deposit name is Chinese hamster ovary cell gs weak strain CHO-9B1-C, and the deposit number is CCTCC NO: C2024393.

[0008] As a preferred embodiment of the present invention, the monoclonal antibody secreted by the cell line belongs to the IgG1 subtype of κ light chain.

[0009] The present invention further provides a method for constructing the aforementioned CHO stable transfection cell line, including:

[0010] (1) For the hybridoma cell line 9B1 against the CSFV type 2 E rns protein, total RNA extraction, reverse transcription, codon optimization and PCR amplification were carried out to obtain the nucleotide sequences of the heavy chain gene and the light chain gene encoding the 9B1 monoclonal antibody; then the two nucleotide sequences were respectively inserted into the pEE12.4 plasmid to obtain two recombinant plasmids;

[0011] (2) Using the dual plasmid co-transfection system, the two recombinant plasmids were electrotransfected into CHO-K1 cells; then through recovery culture, pressure screening and monoclonalization treatment, a CHO stable transfection cell line expressing a recombinant antibody that can specifically recognize the CSFV type 2 E rns protein was finally obtained.

[0012] As a preferred embodiment of the present invention, the nucleotide sequence of the heavy chain gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the light chain gene is as shown in SEQ ID NO.2; the amino acid sequences of the two recombinant plasmids are as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0013] The present invention also provides the application of the aforementioned CHO stable transfection cell line in the preparation of a diagnostic kit for specifically detecting CSFV type 2 classical swine fever virus.

[0014] The present invention also provides a diagnostic kit for specifically detecting CSFV type 2 classical swine fever virus. This kit is a blocking ELISA kit, including: an enzyme-labeled plate coated with CtE rns protein, a monoclonal antibody secreted by the CHO stable transfection cell line as claimed in claim 1 or 2 labeled with HRP, positive control serum and negative control serum.

[0015] As a preferred embodiment of the present invention, the kit further includes PBS buffer, PBST buffer, blocking solution, antibody diluent, coating solution and termination solution.

[0016] As a preferred embodiment of the present invention, the PBS buffer is prepared by taking 8 g NaCl, 0.2 g KCl, 3.58 g Na2HPO4·12H2O, and 0.27 g KH2PO4, adding deionized water to 800 mL, adjusting the pH to 7.4, and fixing the volume to 1 L; the final concentration is 10 mM, pH 7.4; the PBST buffer is prepared by taking the PBS buffer, adding 500 μL tween-20 per liter; the final concentration is 10 mM, pH 7.4; the blocking solution is prepared by taking the sterilized PBS buffer, adding components to make the final concentrations of sucrose 0.05 g / mL, proclin-300 0.1%, and BSA 0.02g / mL; filter sterilize, and aseptically aliquot; the preparation method of the antibody diluent is: take the sterilized PBS buffer, add the components and make the final concentrations of mannitol 0.05g / mL, sucrose 0.05g / mL, proclin-300 0.1%, and BSA 0.01g / mL; filter sterilize, and aseptically aliquot; the preparation method of the coating solution is: take 1.59g Na2CO3 and 2.93gNaHCO3, dissolve them in 900mL pure water, adjust the pH value to 9.6±0.2, and make up to 1000mL; the preparation method of the stop solution is: take 111mL of 98% concentrated sulfuric acid, add 899mL of deionized water.

[0017] Compared with the prior art, the technical effects of the present invention are:

[0018] 1. Short monoclonal antibody production cycle: Using conventional technology, recombinant antibodies can be produced using CHO stable cell lines within a few weeks.

[0019] 2. High consistency and high repeatability: The sequence of the recombinant antibody secreted by the CHO stable cell line in the present invention is determined, the entire production process is a standardized process, and the results are controllable, so that the consistency and repeatability between batches are good.

[0020] 3. Suitable for large-scale production: CHO stable cell lines can be used to quickly and massively prepare recombinant antibodies without the use of animals, thus overcoming the ethical issues of using animals.

[0021] 4. Easy to modify: For example, by adjusting the variable region sequence, chimeric antibodies, and antibody humanization of CHO stable cell lines, customization of antibody characteristics can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the map of pEE12.4-9B1-H.

[0023] Figure 2 This is the map of pEE12.4-9B1-L.

[0024] Figure 3 Schematic diagram of the construction process of the 9B1 recombinant antibody stable transfection cell line.

[0025] Figure 4 Morphological observation of the 9B1 recombinant antibody stable transfection cell line.

[0026] Figure 5 PAGE electrophoresis identification and immunoblotting identification diagrams of the 9B1 recombinant antibody before and after purification.

[0027] Figure 6 Fitting the titer of the 9B1 recombinant antibody with a non - linear regression model.

[0028] Figure 7 Application of the 9B1 recombinant antibody in blocking ELISA to detect the reactivity of sera from different sources with CSFV type 2 E rns protein (QZ14 - 1, QZ14 - 2, HZ08 - 1, and HZ08 - 2 are sera from pigs infected with wild strains, RecC - M1 - 1 and RecC - M1 - 2 are sera from pigs immunized with the marked vaccine strain, BVDV1 - 1 and BVDV1 - 2 are sera from pigs artificially infected with BVDV1 type). Detailed implementation mode

[0029] For the construction of most immunoassay systems, monoclonal antibodies are the key materials. After obtaining hybridoma cells that can secrete specific antibodies, ascites are mainly prepared in mice to obtain a sufficient amount of antibodies. However, this method has risks such as animal welfare issues, and with the increase in the number of passages, there are risks of mycoplasma contamination or unstable antibody secretion in hybridoma cells.

[0030] Recombinant antibodies are prepared by using recombinant DNA technology to construct the nucleotide sequence encoding a specific monoclonal antibody into a eukaryotic expression plasmid, which is then transfected into suitable mammalian cells for expression. The obtained protein is the specific monoclonal antibody. Compared with traditional monoclonal antibodies, recombinant antibodies have many advantages: permanent preservation of the specific antibody sequence in the form of an expression plasmid; such antibodies can be stably and reliably expressed in vitro with good batch - to - batch consistency; they can be engineered; the expression process does not require animals, avoiding animal ethics issues; and the production cost is low, etc.

[0031] The basic research material of the present invention is a hybridoma cell line 9B1 secreting CSFV type 2 E rns protein, which is preserved in the applicant's internal laboratory (already publicly reported in Chinese invention patent document CN 114874995 A, with the preservation number of CCTCC NO: C202247, and the public can obtain it through legal channels). The monoclonal antibody secreted by this cell line can bind to CSFV type 2 E rnsThe protein (also described in Chinese Patent Literature CN 114874995 A) has a specific reaction and does not react with the E rns protein (i.e., the E rns ) from bovine viral diarrhea virus type 1 [BVDV1]. Therefore, the monoclonal antibody secreted by this cell line can be used for the differential detection of serum antibodies in pigs infected with CSFV type 2 strains and pigs immunized with the RecC-M1 or E2 protein subunit vaccine.

[0032] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] Example 1 Construction of recombinant plasmids pEE12.4-9B1-H and pEE12.4-9B1-L

[0034] 1. Total RNA extraction

[0035] The hybridoma cell line 9B1 of classical swine fever virus type 2 E rns protein preserved in our laboratory is a publicly known technology, and its preservation number is CCTCC NO: C202247. One 9B1 hybridoma cell line with a confluence of about 80% in a 25-cm 2 cell culture flask was selected for total RNA extraction.

[0036] 2. Antibody gene amplification

[0037] The extracted RNA was sent to Nanjing GenScript Co., Ltd. for reverse transcription and antibody gene amplification. After optimization with CHO-preferred codons, the heavy chain gene sequence of the 9B1 monoclonal antibody-encoding gene is shown in SEQ ID NO.1, and the light chain gene sequence is shown in SEQ ID NO.2.

[0038] 3. Construction of antibody expression vector

[0039] The genes of the antibody heavy chain and antibody light chain were amplified respectively. Using the above 9B1 monoclonal antibody-encoding gene as a template for PCR, the purified PCR products were inserted into the pEE12.4 eukaryotic expression vector by homologous recombination method to obtain recombinant plasmids pEE12.4-9B1-H and pEE12.4-9B1-L respectively. The results are shown in Figure 1 and Figure 2 . The amino acid sequence of 9B1-H is shown in SEQ ID NO.3, and the amino acid sequence of 9B1-L is shown in SEQ ID NO.4.

[0040] Example 2 Construction of 9B1 recombinant antibody stable transfection cell line

[0041] 1. Electroporation of plasmid

[0042] Take 1×10 7 CHO-K1 cells in suspension culture, add 15 μg of pEE12.4-9B1-H vector and 15 μg of pEE12.4-9B1-L vector, mix evenly and transfer them into an electroporation cuvette for electroporation. The parameters are set as voltage 150 V and capacitance 950 μF.

[0043] 2. Recovery culture

[0044] Transfer the electroporated cells above to a shake flask containing CM-012A medium with 4 mM glutamine, and place them in a shaking incubator at 37 °C, 5% CO2, and 120 rpm for 48 h of recovery culture. Then change the medium to CM-012A medium and continue culturing for 7 days.

[0045] 3. Pressure screening

[0046] Change the cell medium above to CM-012A medium containing 25 μM MXS, and culture for 7 days for pressure screening.

[0047] 4. Monoclonalization

[0048] 1) Count the cells after pressure screening, and inoculate the cells into a 96-well plate by the limited dilution method. After 2 weeks, take the supernatant and screen for high-expression CHO cells by ELISA detection method.

[0049] Coat with CtE rns protein at a concentration of 1.5 μg / mL overnight at 4 °C. The next day, discard the coating solution and wash 3 times with PBST, 5 min each time.

[0050] CtE rns The preparation method of the protein can refer to the description in Chinese invention patent document CN 114874995 A. In this example, the preparation method is illustrated as follows: Clone the protein coding region sequence of amino acids 104 to 170 of CSFV-QZ14-E rns into the vector pFastBacHTB to obtain the recombinant transfer plasmid pFastBac-CtE rns ; pFastBac-CtE rns is transposed into E. coli DH10Bac competent cells to obtain the recombinant bacmid Bacmid-CtE rns ; This recombinant bacmid is transfected into Sf9 cells to obtain a recombinant baculovirus expressing CSFV type 2 E rns protein, and its expression product is abbreviated as CtE rns .

[0051] 2) Add 200 μL of blocking solution to each well, discard it after blocking at 37 °C for 2 h, and wash 3 times with PBST, 5 min each time.

[0052] 3) Add the CHO cell culture supernatant to the corresponding wells of the detection ELISA plate, 100 μL per well. Incubate at 37 °C for 1 h, then discard the liquid, and wash 3 times with PBST, 5 min each time.

[0053] 4) Dilute the ascites of monoclonal antibody 9B1 and CM-012A medium at 1:1000 respectively as positive and negative controls. Dilute the goat anti-mouse enzyme-labeled secondary antibody at 1:5000, and add 100 μL per well to the ELISA plate. React at 37 °C for 1 h, and wash 3 times with PBST, 5 min each time.

[0054] 5) Add 100 μL of TMB chromogenic solution to each well and react in the dark at 37 °C for 10 min. Add 50 μL of 2M H2SO4 to each well to terminate the chromogenesis, and measure the OD 450 nm value. Screen the cells in the positive wells as positive monoclonal cell lines, and expand the culture and then preserve them.

[0055] Finally, a CHO stable transfection cell line stably expressing a specific antibody against CSFV type 2 E rns protein was obtained, and the process is as Figure 3 shown.

[0056] The applicant deposited the cell line, and the deposit information is as follows: the deposit name is Chinese hamster ovary cell gs weak type strain CHO-9B1-C, the deposit unit is China Center for Type Culture Collection; the deposit address is Wuhan, China, Wuhan University; the deposit number is CCTCC NO: C2024393, and the deposit date is December 11, 2024.

[0057] Example 3 Expression and purification of 9B1 recombinant antibody

[0058] 1. Expression of 9B1 recombinant antibody

[0059] Take 1×10 6 of the above monoclonal 9B1 recombinant antibody stable transfection cell lines, inoculate them into CM-013 medium containing 25 μM MXS, and culture for 3 days. Then add 1.5% DF-013 feed and 0.15% DF-003 feed every day. Sample every day to observe the cell state and viability (see Figure 4 ), and collect the samples when the cell viability ≤ 60%. Centrifuge to collect the supernatant for protein purification.

[0060] 2. Purification of 9B1 recombinant antibody

[0061] 1) Add 1 / 10 volume of equilibration solution (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) to adjust the pH value of the cell supernatant containing the antibody to 7.0.

[0062] 2) Pass the antibody solution through a Protein A gravity column and record the volume of the gravity column.

[0063] 3) Wash with 10 - 15 column volumes of washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0).

[0064] 4) Elute the gravity column with 0.1 M glycine (pH 3.0), adding approximately 1 / 2 column volume each time and adding in portions.

[0065] 5) Collect the eluate in a test tube containing 1 / 10 column volume of neutralization buffer (1 M Tris - HCl, pH 8.5), gently shake each tube to restore the pH value to neutral, which is the target protein fraction.

[0066] Example 4 Identification of 9B1 Recombinant Antibody

[0067] 1. SDS - PAGE Identification

[0068] 1) Prepare the SDS - PAGE gel. Take 40 μL of cell supernatant, cell pellet, sample before loading onto the column, flow - through after passing through the column, and eluates at different pH values respectively, add 10 μL of 5×Loading Buffer, pipette and mix well, and heat in a boiling water bath for 10 min;

[0069] 2) Assemble the electrophoresis tank, fill it with electrophoresis buffer, load the samples, and perform electrophoresis according to the program of 150 V for 65 min;

[0070] 3) After electrophoresis, place the gel in Coomassie Brilliant Blue staining solution and shake on a shaker at 37°C for 2 h for staining;

[0071] 4) Place it in the decolorizing solution for decolorization, change the decolorizing solution every 1 h, and place it on a gel imager to take pictures and observe the results after the bands are clearly visible; The results are as Figure 5 shown. The recombinant antibody shows two protein bands with molecular weights of approximately 50 kD and 25 kD respectively, which is consistent with the electrophoresis band pattern of IgG purified by Protein A affinity.

[0072] 2. Immunoblotting Identification

[0073] 1) Perform SDS - PAGE electrophoresis on cell supernatant, cell pellet, sample before loading onto the column, flow - through after passing through the column, and eluates at different pH values;

[0074] 2) Cut a PVDF membrane the same size as the polyacrylamide gel to be transferred. Activate the PVDF membrane in methanol for 1 min before transfer and then transfer it to the wet transfer buffer for soaking. Soak the filter paper and sponge directly in the wet transfer buffer;

[0075] 3) In the order of (+) thick sponge - thin filter paper - PVDF membrane - glue - thin filter paper - thick sponge (-), perform wet transfer at 100 V for 1 h; after the transfer is completed, place the PVDF membrane into a newly prepared 5% skim milk blocking solution and block it on a shaker at 37 °C for 1 h;

[0076] 4) After blocking, wash the PVDF membrane 3 times with TBST, 5 min each time. Add HRP-labeled goat anti-mouse IgG diluted 1:5000 and incubate it on a shaker at 37 °C for 1 h. Wash the PVDF membrane 3 times with TBST, 5 min each time;

[0077] 5) Prepare the chromogenic solution for color development, and place it in a gel imager to photograph and record the experimental results. The results are as Figure 5 shown, specific bands appear at 50 kD and 25 kD.

[0078] Example 5 HRP Labeling and Titer Determination of 9B1 Recombinant Antibody

[0079] 1. HRP Labeling of 9B1 Recombinant Antibody

[0080] The purified 9B1 recombinant antibody was labeled with horseradish peroxidase (HRP) by Shanghai Youningwei Biotechnology Co., Ltd., and the antibody concentration after labeling was 1.3 mg / mL.

[0081] 2. Titer Determination of HRP-9B1 Recombinant Antibody

[0082] Using CtE rns protein as the coating antigen to perform direct ELISA, the HRP-9B1 recombinant antibody was serially diluted 2-fold from the initial concentration of 1:1500, and the same concentration of HRP-9B1 ascites IgG was set in parallel; using log 10 (dilution factor) as the abscissa, and the ratio P / N value of the sample OD 450 nm to the negative OD 450 nm as the ordinate, a four-parameter Logistic regression model was made. The results are as Figure 6 shown, the titer of the HRP-9B1 recombinant antibody was determined to reach 1:5.6×10 6 , and the titer of HRP-9B1 ascites IgG was about 1:1.4×10 5 , indicating that the affinity of this recombinant antibody is about 40 times that of HRP-9B1 ascites IgG and can be used for the construction of the subsequent blocking ELISA system.

[0083] Example 6: Specific Application Example of 9B1 Recombinant Antibody

[0084] Prepare the reagents for the blocking ELISA kit according to the following formula:

[0085] 1) 10 mM PBS buffer at pH 7.4

[0086] 8 g of NaCl, 0.2 g of KCl, 3.58 g of Na2HPO4·12H2O, 0.27 g of KH2PO4. Add deionized water to 800 mL, adjust the pH to 7.4, and make up to 1 L.

[0087] 2) 10 mM PBST buffer at pH 7.4

[0088] 10 mM PBS buffer at pH 7.4, add 500 μL of tween-20 per liter.

[0089] 3) Blocking solution

[0090] Take the sterilized 10 mM PBS buffer at pH 7.4, and add components to a final concentration of 0.05 g / mL of sucrose, 0.1% of proclin-300, and 0.02 g / mL of BSA. Filter and sterilize, and dispense aseptically.

[0091] 4) Antibody diluent

[0092] Take the sterilized 10 mM PBS buffer at pH 7.4, and add components to a final concentration of 0.05 g / mL of mannitol, 0.05 g / mL of sucrose, 0.1% of proclin-300, and 0.01 g / mL of BSA. Filter and sterilize, and dispense aseptically.

[0093] 5) Coating solution

[0094] 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve in 900 mL of pure water, adjust the pH value to 9.6 ± 0.2, and make up to 1000 mL.

[0095] 6) Stop solution

[0096] 111 mL of 98% concentrated sulfuric acid, 899 mL of deionized water.

[0097] The kit is used in the following manner:

[0098] 1) CtE protein diluted with carbonate buffer at pH 9.6, coat the ELISA plate at 100 μL / well, and incubate at 4 °C overnight; rns 2) Discard the coating solution, wash the plate 3 times with PBST, 5 min each time, and pat dry for the last time;

[0099] 3) Add the blocking solution, 200 μL / well, place in a humid box and block at 37 °C for 2 h; discard the blocking solution, wash the plate 3 times with PBST, 5 min each time, and pat dry for the last time;

[0100] 4) Add the antibody diluent, 100 μL / well, place in a humid box and incubate at 37 °C for 1 h; discard the antibody diluent, wash the plate 3 times with PBST, 5 min each time, and pat dry for the last time;

[0101] 4) Add equal volume mixture (50 μl + 50 μl) of diluted HRP-9B1 recombinant antibody and serum sample, 100 μL / well, set up positive control and negative control at the same time, place in a wet box and incubate at 37°C for 60 min; discard the liquid in the well, wash the plate 3 times with PBST, 5 min each time, and pat dry for the last time;

[0102] 5) Add TMB colorimetric solution, 100 μL / well, and color at room temperature in the dark for 10 min;

[0103] 6) Add the reaction stop solution, 50 μL / well, mix well and measure the OD immediately 450 nm value.

[0104] Eight pig sera (including 2 infected with CSFV2 strain QZ14, 2 infected with CSFV2 strain HZ08, 2 immunized with RecC-M1 labeled vaccine strain, and 2 infected with BVDV1 artificial inoculation) and one CSFV / BVDV negative pig serum (Negative) were tested using the blocking ELISA system established above. The results are as follows: Figure 7 As shown in the figure, the blocking rate of CSFV2 QZ14 and HZ08 strains challenged pig sera reached more than 60%, and BVDV1 sera and BVDV1 E rns The blocking rates of RecC-M1-immunized pig sera were less than 30%.

[0105] The above results show that the CtE-based rns The blocking ELISA system of protein and 9B1 recombinant antibody can specifically detect serum antibodies infected with CSFV2 type QZ14 and HZ08 strains, and does not cause obvious competitive blocking with serum antibodies infected with BVDV1 type and serum antibodies immunized with RecC-M1 vaccine. The blocking ELISA system can be used in conjunction with RecC-M1 labeled vaccine or E2 subunit vaccine, and is suitable for the identification and detection of serum antibodies of pigs infected with CSFV2 type strains and pigs immunized with RecC-M1 labeled vaccine or E2 subunit vaccine, and can also be used to distinguish serum antibodies of pigs infected with CSFV2 type strains and pigs infected with BVDV1 type virus.

[0106] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

[0107]

[0108]

Claims

1. A CHO stable transfection cell line expressing a recombinant antibody that can specifically recognize the E protein of CSFV genotype 2, characterized in that, rns This cell line is deposited with the China Center for Type Culture Collection under the deposit name of Chinese Hamster Ovary Cell gs Weak Strain CHO-9B1-C, and the deposit number is CCTCC NO: C2024393. ​ 2. The CHO stable transfection cell line according to claim 1, wherein The monoclonal antibody secreted by this cell line belongs to the IgG1 subtype of κ light chain.

3. The method for constructing the CHO stable transfected cell line according to claim 1, characterized in that, Including: (1)For the hybridoma cell line 9B1 of CSFV type 2 E protein with the preservation number of CCTCC NO: C202247, total RNA extraction, reverse transcription, codon optimization and PCR amplification were carried out to obtain the nucleotide sequences of the heavy chain gene and the light chain gene encoding the 9B1 monoclonal antibody; then the two nucleotide sequences were respectively inserted into the pEE12.4 plasmid to obtain two recombinant plasmids; rns For the hybridoma cell line 9B1 of CSFV type 2 E protein with the preservation number of CCTCC NO: C202247, total RNA extraction, reverse transcription, codon optimization and PCR amplification were carried out to obtain the nucleotide sequences of the heavy chain gene and the light chain gene encoding the 9B1 monoclonal antibody; then the two nucleotide sequences were respectively inserted into the pEE12.4 plasmid to obtain two recombinant plasmids; (2) Using a dual-plasmid co-transfection system, electrotransfer the two recombinant plasmids into CHO-K1 cells; After recovery culture, pressure screening, and monoclonalization, a CHO stable transfection cell line expressing a recombinant antibody that can specifically recognize the CSFV type 2 E protein was finally obtained. rns protein was obtained.

4. The method according to claim 3, characterized in that The nucleotide sequence of the heavy chain gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the light chain gene is as shown in SEQ ID NO.2; the amino acid sequences of the two recombinant plasmids are as shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

5. Use of the stably transfected CHO cell line according to claim 1 in the preparation of a diagnostic kit for specifically detecting classical swine fever virus type 2 (CSFV2).

6. A diagnostic kit for specifically detecting classical swine fever virus type 2, characterized in that, The kit is a blocking ELISA kit, comprising: an enzyme-labeled plate coated with CtE rns protein, a monoclonal antibody secreted by the CHO stable transfected cell line described in claim 1 or 2 labeled with HRP, positive control serum, and negative control serum.

7. The kit according to claim 6, wherein This kit also includes PBS buffer, PBST buffer, blocking solution, antibody diluent, coating solution and termination solution.

8. The kit according to claim 7, characterized in that The preparation method of the PBS buffer: Take 8 g of NaCl, 0.2 g of KCl, 3.58 g of Na2HPO4·12H2O, and 0.27 g of KH2PO4, add deionized water to 800 mL, adjust the pH to 7.4, and make up the volume to 1 L; the final concentration is 10 mM, pH 7.4; The preparation method of the PBST buffer: Take the PBS buffer and add 500 μL of tween-20 per liter; the final concentration is 10 mM, pH 7.4; The preparation method of the blocking solution: Take the sterilized PBS buffer, add components and make the final concentrations sucrose 0.05 g / mL, proclin-300 0.1%, BSA 0.02 g / mL; filter and sterilize, and dispense aseptically; The preparation method of the antibody diluent: Take the sterilized PBS buffer, add components and make the final concentrations mannitol 0.05 g / mL, sucrose 0.05 g / mL, proclin-300 0.1%, BSA 0.01 g / mL; filter and sterilize, and dispense aseptically; The preparation method of the coating solution: Take 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in 900 mL of pure water, adjust the pH value to 9.6 ± 0.2, and make up the volume to 1000 mL; The preparation method of the termination solution: Take 111 mL of 98% concentrated sulfuric acid and add 899 mL of deionized water.

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