WO2018024153A1 - Preparation method and use of recombinant swine fever e2 protein and subunit vaccine thereof - Google Patents

Abstract

Disclosed are a preparation method and use of a recombinant swine fever E2 protein and subunit vaccine thereof. The method for preparing the recombinant swine fever E2 protein comprises the following steps: 1) cloning the swine fever E2 protein coding gene into an eukaryotic expression vector to obtain a recombinant plasmid containing the swine fever E2 protein coding gene; 2) transfecting the recombinant plasmid containing the swine fever E2 protein coding gene into a CHO cell strain; 3) obtaining a highly expressed cell strain by culturing, screening and domestication of the CHO cell strain described in step 2); and 4) fermenting and culturing the cell strain described in step 3), and obtaining the recombinant swine fever E2 protein after purification. The method can harvest the target protein from the cell culture supernatant with a yield of up to 1 g/L, and not only shortens the protein purification time and simplifies the vaccine production steps, but also reduces the vaccine production cost.

Description

Preparation method and application of recombinant swine fever E2 protein and subunit vaccine thereof Technical field

The invention relates to a CHO cell strain for suspending and stably expressing the swine fever E2 protein, a method for constructing and screening the same, and a preparation method and application of the swine fever E2 protein subunit vaccine, belonging to the technical field of animal vaccines and veterinary biological products. .

Background technique

Classical Swine Fever (CSF) is a kind of acute, heat and lethal disease caused by Classical Swine Fever Virus (CSFV) in Europe. Hog Cholera is characterized by high levels of contagious, rapid onset, high heat retention and small vessel wall degeneration resulting in extensive hemorrhage, infarction and necrosis. Domestic and wild pigs are their only natural hosts. The World Organisation for Animal Health (OIE) has designated it as a Class A infectious disease, which is classified as a type of infectious disease in China's Animal Epidemic Prevention Law. Hog is one of the main diseases that currently harm the development of China's pig industry.

The swine fever virus belongs to the family Flaviviridae and prion, and is a single-stranded linear RNA virus. The virions are slightly round and have a lipoprotein capsule with a fragile fiber structure on the surface of the virion. The CSFV genome is approximately 12.5 kb in length and contains only one large open reading frame (ORF) encoding a polyprotein of approximately 3898 amino acid residues with a molecular weight of approximately 438 kDa. This polyprotein is processed into 12 mature proteins by translation of the virus and host cell proteases after translation, followed by N ^pro , C, E ^rns , E1 , E2 , p7 , NS2 , NS3 , NS4A , NS4B , NS5A . , NS5B, wherein C, E ^rns , E1 and E2 are structural proteins, and the rest are non-structural proteins. E2 is the most important immunogenic protein of CSFV, which can induce the body to produce neutralizing antibodies and protect pigs against CSFV virulent strains. It is also an important target protein for studying genetic engineering vaccines of classical swine fever.

Pig swines are popular all over the world and are extremely harmful to the pig industry. The current effective prevention measures for this disease are vaccine immunization. After many years of application, there are three safe and effective attenuated vaccine strains: 1 Chinese rabbit attenuated vaccine; 2 Japanese GPE (-) cell attenuated vaccine; 3 French "Thiveosal" cold variant attenuated strain. Among them, the Chinese Department (C Department) pigs and rabbits have developed attenuated vaccines. Since 1957, they have been widely used in many countries in Europe and Asia, and have helped these countries control or eliminate piglets. The vaccine is recognized as the ideal swine fever vaccine in the world. At present, there are three kinds of swine fever vaccines commonly used in the market in China, namely live swine cell vaccine (cell seedlings), live swine fever vaccine (tissue seedlings), and swine spleen spleen vaccine (spleen seedlings). The production of tissue seedlings and spleen seedlings requires a large number of healthy animals. The unfavorable factors such as high labor intensity, high cost and inoculation side effects in the production process have affected the wide application of such seedlings. Cell seedlings are also plagued by many factors, such as batch differences; cell-free lesions, difficult to control virus production; The material involves yak testicular cells and bovine serum, leading to the risk of BVDV contamination during its preparation. BVDV-infected pigs can cause symptoms of suspected swine fever. In recent years, pig diseases caused by BVDV-contaminated swine fever vaccine have been reported in China. In addition, due to the long-term use of live vaccines, it is impossible to fundamentally purify piglets.

In view of the defects and shortcomings of the existing swine fever vaccine, and with the development of modern genetic engineering technology and cell technology, many researchers have tried to develop a novel swine fever vaccine that can overcome the existing vaccine defects by modern molecular biology. These novel swine fever vaccines include viral live vector vaccines, synthetic peptide vaccines, DNA vaccines, subunit vaccines for E. coli expression proteins, and E2 protein submonotherapy vaccines for insect baculovirus expression. Among them, the E2 protein subunit vaccine expressed by insect baculovirus developed by European researchers has been applied to the present, and the vaccine is not affected by the maternal antibody, and can be differentially diagnosed with the virus infection. However, the vaccine is expressed by an insect baculovirus expression system. Compared with the prokaryotic expression system, the expression system can be post-translationally processed and modified to a certain extent, but there is still some difference from the structure of the virus natural antigen protein. Protein expression is post-folded and modified less than mammalian cell expression systems. Moreover, when the system produces antigens, the cells will be lysed and killed after virus infection, which will increase the difficulty of downstream purification and other production processes. In addition, the yield of the protein produced by the system is not very high, generally only 200-300 mg / L, it is difficult to achieve a yield of 500 mg / L.

CHO cells were obtained from the ovary of a female female hamster in 1957 by Dr. Theodore T. Puck of the University of Colorado, USA, and are epithelial adherent cells. The cell is immortal and can be passaged for generations or more. It is a cell widely used in bioengineering. Compared with other expression systems, it has the following advantages: (1) It has an accurate post-transcriptional modification function, and the expressed protein is closest to the natural protein molecule in terms of molecular structure, physical and chemical properties and biological function; (2) Wall growth, suspension culture, and high tolerance to shear and osmotic pressure; (3) efficient amplification and expression of recombinant genes, integration of foreign proteins; (4) with product cells Exocrine function, and rarely secrete its own endogenous protein, facilitate the separation and purification of downstream products; (5) can achieve high-density culture in suspension culture or in serum-free medium, and the culture volume can reach more than 1,000L, which can be large Scale production.

There are many types of CHO cells, such as DG44, DXB11, CHO K1 and CHO-S. Since the 1980s and 1990s, the DHFR (dihydrofolate reductase-deficient) gene amplification screening system has been used earlier in the industry, and the host cell strain is DG44. When the cell culture medium contains methotrexate (MTX), dihydrofolate reductase is inhibited, and the gene is amplified by feedback regulation. The genes in the range of 100-1,000 kb upstream and downstream will follow. Amplification, thus inserting the gene of interest into this range of sites to obtain amplification. Many systems of monoclonal antibody production are still the DHFR system of DG44. The GS (Glutamine Synthetase) Amplification System, which uses CHO-K1 as a host cell, is a novel gene amplification screening system developed in recent years. It has obvious superiority over the DHFR system and is currently available internationally. Wide recognition. The principle is that GS synthesizes glutamine using intracellular ammonia and glutamic acid while ATP hydrolyzes to provide energy. Adding the GS inhibitor lysine sulfoximine (MSX) to the medium lacking exogenous glutamine can effectively expand the GS gene and the target gene linked to it. Increase, so as to achieve the purpose of improving the level of expression of the target gene. The advantages of this system are mainly: no need for gene-deficient CHO-K1 cell line as host cell; CHO-K1 cell is easy to culture and stronger; no glutamine is added in the medium, which can avoid glutamine decomposition and cause culture system The problem of high ammonia level reduces the difficulty of process control and can effectively increase cell fermentation density and prolong cell survival time.

However, when the present inventors first used CHO cells to express the hog E2 protein, it was found that the CHO cell gene does not express the hog E2 protein when it is not codon-optimized, and therefore, CHO cells are used to express hog cholera. Codon optimization is a very important step in E2 proteins.

Summary of the invention

The problem to be solved by the present invention is to provide a preparation method and application of a large-scale industrial production of a recombinant subunit vaccine of classical swine fever virus.

According to an aspect of the present invention, the present invention provides a method for producing a recombinant hog cholera E2 protein, which comprises the following steps: 1) cloning a codon-optimized hog cholera E2 protein encoding gene into a eukaryotic expression vector Recombinant plasmid containing the gene encoding the hog E2 protein; 2) transfecting the recombinant plasmid containing the gene encoding the hog E2 protein into the CHO cell line; 3) CHO as described in the culture, screening and domestication 2) The cell strain is highly expressed cell line; and 4) the cell strain described in the fermentation culture 3) is purified to obtain recombinant hog cholera E2 protein.

In a preferred embodiment of the present invention, preferably, the nucleotide sequence of the codon-optimized Hog Cholera E2 protein encoding gene is as shown in SEQ ID NO.

In a preferred embodiment of the present invention, preferably, the eukaryotic expression vector is pEE6.4, pEE12.4, pGL4.13, pcDNA3.1. More preferably, the eukaryotic expression vector is pEE12.4.

In a preferred embodiment of the present invention, preferably, the CHO cell strain is a DG44, DXB11, CHO-K1, CHO-S cell strain. More preferably, the CHO cell line is CHO-K1.

According to another aspect of the present invention, the present invention also provides a preparation method comprising a recombinant swine fever E2 protein subunit vaccine, which fully mixes the recombinant swine fever E2 protein obtained by the above preparation method with a pharmaceutically acceptable adjuvant. , get the piglet E2 recombinant subunit vaccine.

In a preferred embodiment of the present invention, preferably, the adjuvant used in the recombinant subunit vaccine is ISA 201 VG.

In a preferred embodiment of the present invention, preferably, the recombinant swine fever E2 protein and the adjuvant ISA 201 VG are emulsified by mixing at a volume ratio of 46:54.

According to still another aspect of the present invention, the present invention also provides a use of a recombinant swine fever E2 protein and a classical swine fever virus virus subunit vaccine for the preparation of a related diagnostic reagent.

The invention constructs and screens a CHO cell strain which stably and stably expresses the E2 protein of Porcine scorpion E2 protein, and the cell line expresses E2 protein with high yield, is easy to be purified, is easy to be mass-produced, and the expressed E. coli antigen can induce the immune pig. Good immune response and can tolerate the virulent challenge of the porcine sphagnum; the subunit vaccine prepared from the recombinant E2 protein has the following advantages: 1. large-scale production, sufficient supply, and easy quality control; High integrity, good immunogenicity; 3. Stable between batches; 4. Low production costs; 5. No risk of BVDV contamination.

The system for selecting the expression of the swine fever E2 protein of the present invention is a CHO expression system, and the system research is relatively clear, and the cell strain expressed by the present inventors is suitable for suspension culture, and the yield is as high as 1 g/L, so it is very easy to enlarge in industry. It is especially suitable for large-scale industrial production. Compared with other eukaryotic expression systems, the production cost is very low. In addition, the intestine E2 protein expressed by the present inventors using CHO cells is secreted in the cell supernatant because of the fermentation of CHO cells. It is a serum-free medium, so the amount of miscellaneous protein in the cell supernatant is small, so it is convenient and quick to purify. The inventors have found that the purity of the E2 protein expressed in the cell supernatant can be purified on SDS-PAGE. More than 80% (see the SDS-PAGE results of the stock solution in Figure 6), so only a simple nickel column purification purity can reach more than 95%, far enough to meet the requirements of the piglet E2 subunit vaccine preparation.

DRAWINGS

Figure 1 shows the plasmid map of pEE12.4-OPTI-E2.

Figure 2 shows the plasmid map of pCDNA3.1-OPTI-E2.

Figure 3 shows the results of double enzyme digestion of pEE12.4-OPTI-E2: 3 is DNA Marker: 1 kb ladder; 1 is the result of pEE12.4-OPTI-E2 undigested electrophoresis; 2 is pEE12.4-OPTI-E2 double enzyme Cut the electrophoresis results.

Figure 4 shows the results of double enzyme digestion of pCDNA3.1-OPTI-E2: 3 is DNA Marker: DL10000; 1, 2 are the results of double digestion with pEE12.4-OPTI-E2.

Figure 5 shows the results of cell shake flask fermentation.

Figure 6 shows the nickel column affinity chromatogram and SDS-PAGE results.

Figure 7 shows the results of antibody titer after detection of recombinant swine fever E2 protein subunit vaccine by IDEXX kit.

Figure 8 shows a comparison of nucleotide sequences before and after codon optimization: E2 indicates the sequence before codon optimization; OPTI-E2 indicates the sequence after codon optimization.

detailed description

The present invention will be further described with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only intended to illustrate the technical solutions of the present invention.

The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

The list of sources of reagents and drugs of the present invention is as follows:

CHO-K1 cells and CHO/dhfr ^- cells are derived from the Cell Bank of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences, Cell Culture Bank of the Chinese Academy of Sciences;

Cell culture medium was purchased from Gibco, USA, and fetal bovine serum was purchased from Hyclone;

The eukaryotic expression vector pEE12.4 was purchased from Shanghai Linyuan Biotechnology Co., Ltd.;

The eukaryotic expression vector pCDNA3.1 (+) was purchased from Invitrogen, USA;

Lipofectamine LTX was purchased from Thermo Fisher Company, USA;

Methotrexate (MTX) was purchased from Sigma;

Methionine sulfoximine (MS-) was purchased from Sigma;

The BCA protein quantification kit was purchased from Thermo Fisher Company, USA;

The ISA 201 VG was purchased from the French company Sabic.

Example 1: Choerodon E2 protein codon optimization

The OP-E2 sequence was obtained by codon-optimizing the nucleotide sequence of the swine fever E2 protein, as shown in SEQ ID NO. 2, and the work was completed by Suzhou Jinweizhi Biotechnology Co., Ltd.

The sequence of SEQ ID NO. 2 and SEQ ID NO. 3 was aligned, and it was found that the codon-optimized nucleotide sequence of the swine fever E2 protein and the nucleotide sequence of the codon-optimized swine fever E2 protein were 21.3%. The difference is that there are 222 nucleotides out of 1044 nucleotides. See Figure 8 for details.

Example 2: Construction of pEE12.4-OPTI-E2 recombinant plasmid

2.1 PCR amplification target segment OPTI-E2

2.1.1 PCR reaction

(1) Primer design and synthesis

Upstream primer: 5'-CCAAGCTTGCCGCCACCATGAAAGTGCTGAGGGGCCAG-3'

Downstream primer: 5'-CCGGAATTCTTAGTGATGGTGATGGTGATGAGC-3'

(2) 50 μL of the sample loading system, as shown in the following table:

PCR amplification procedure:

2.1.2 PCR product for gel recovery

(1) Mark the sample collection EP tube, adsorption column and collection tube;

(2) Weigh the weight of the marked empty EP tube and record the value;

(3) Carefully cut a single DNA strip of interest onto a gelatinizer from agarose gel using a scalpel and place it in a clean 1.5 mL centrifuge tube;

(4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3), and leave it in a 50 ° C water bath for about 5 minutes, during which time the tube is gently inverted up and down to ensure that the gel is fully dissolved;

(5) Column balance: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube in advance), centrifuge 12,000 rpm/min for 1 min, pour off the waste liquid in the collection tube, and put the adsorption column back. In the collection tube;

(6) The solution obtained in the step (5) is added to the adsorption column CB2, allowed to stand for 2 min, 10,000 rpm / min, centrifuged for 30 s, the waste liquid in the collection tube is drained, and the adsorption column CB2 is placed in the collection tube;

(7) Add 600 μL of rinse liquid PW buffer to the adsorption column, let stand for 3 min, centrifuge 10,000 rpm / min, 30 s, pour off the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube;

(8) repeating step (7);

(9) Centrifugation on an empty adsorption column, 12,000 rpm/min, 2 min, remove the rinse solution as much as possible, place the adsorption column at room temperature for 10 min, and dry thoroughly;

(10) The adsorption column CB2 was placed in a collection tube, and 50 μL of Elution buffer (preheating at 65 ° C) was suspended from the middle of the adsorption film, allowed to stand for 3 min, centrifuged at 12,000 rpm/min for 2 min;

(11) taking out the centrifuge tube in step (10) from the centrifuge, discarding the intermediate adsorption column CB2, capping the centrifuge tube cover, and retaining the DNA sample in the centrifuge tube;

(12) The DNA sample in step 11 was stored at 4 ° C, and agarose gel electrophoresis was performed to identify the gel-recovered DNA fragment.

2.2 PCR product and vector double digestion reaction

(1) Mark the 1.5mL EP tube that needs to be used, and add and mix in the 1.5mL EP tube according to the following table: 50μL reaction system

(2) The 1.5 mL EP tube in the step (1) is placed in a constant temperature water bath of the corresponding enzyme, and the water bath is 2-3 hours.

Double-digested product gel recovery: The above double-digestion system was taken out and subjected to agarose gel electrophoresis to recover the DNA fragment therein, and the method was the same as the PCR product gel recovery in 1.2.1.

2.3 connection reaction

(1) Prepare a number of clean 1.5mL EP tubes, mark them, and place them on the EP tube rack for use.

(2) Add and mix the 1.5 mL EP tube according to the following table.

(3) After completing the loading according to the table in step (2), each 10 μl reaction system is placed in a 16 ° C low temperature coolant circulation machine, water bath 10-16 h;

(4) taking out the EP tube in step (3), placing it in a 65 ° C water bath, water bath for 15 min;

(5) Take out the EP tube in step (4) and store at 4 °C.

1.2.4 Conversion reaction

(1) 10 μl of ligation reaction solution was quickly added to 100 μl of competent cells, and mixed by pipetting, ice bath for 30 min;

(2) Take out the sample tube, place it in a water bath at 42 ° C for 100 s, then immediately ice bath for 2 min;

(3) take out the sample tube, in the ultra-clean workbench, add 600 μL of liquid LB medium to the sample tube, and then place the sample tube on a 37 ° C constant temperature shaker, 220 rpm / min, culture for 1 h;

(4) Coating: Take out the sample tube in step (3), centrifuge at 8,000 rpm/min for 2 min at room temperature, remove 600 μl of the supernatant liquid, and resuspend the supernatant to resuspend the cells at the bottom of the tube, and place the resuspended bacteria solution. In the corresponding conversion plate center, spread the bacterial liquid in the center of the conversion plate evenly with a stick.

(5) The transformation step (4) plate is placed in a biochemical constant temperature incubator, and cultured at 37 ° C for 1 h, the transformation plate is inverted and cultured for 15 h;

(6) Observe the transformation results.

2.5 plasmid extraction and double enzyme digestion identification

2.5.1 Plasmid extraction

(1) Using a 10 μL pipette tip, pick a single clone from the transformation plate to 5 ml of ampicillin-resistant LB liquid medium, and shake at 37 ° C, 220 rpm / min overnight;

(2) The bacterial liquid was transferred to a 1.5 ml EP tube, centrifuged at room temperature, 12,000 rpm/min, 2 min, and the supernatant was discarded;

(3) adding 250 μL of plasmid extraction reagent P1 buffer to the EP tube of step (2) to completely suspend the cells;

(4) Add 250 μL of P2 buffer to the solution of step (3), and immediately invert the centrifuge tube 5-10 times. Mix and let stand at room temperature for 2-4 min;

(5) Add 350 μL of P3 buffer to the solution of step (4), immediately invert the centrifuge tube 5-10 times to mix; stand at room temperature for 2-4 min;

(6) The step (5) solution, centrifugation at room temperature, 14,000 rpm / min, 10 min;

(7) moving the supernatant solution in the step (6) to the center of the adsorption column, centrifuging at room temperature, 12,000 rpm / min, 30 s, and draining the liquid in the collection tube;

(8) Add 500 μL Buffer DW1 to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm/min, 30 s, and drain the liquid in the collection tube;

(9) Add 500 μL of wash solution to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm/min, 30 s, and drain the liquid in the collection tube, repeating once;

(10) Empty adsorption column, centrifuged at room temperature, 12,000 rpm, 2 min.

(11) Place the adsorption column in a clean 1.5 ml centrifuge tube, add 30 μL of Elution buffer to the center of the adsorption membrane, let stand at room temperature for 5 min, centrifuge at room temperature, 12,000 rpm, 2 min. Store the DNA solution in the tube.

2.5.2 Double enzyme digestion identification

(1) Mark the 1.5mL EP tube that needs to be used and add it according to the following table: 20μL reaction system

(2) The 20 μL reaction system of the EP tube in the step (1) was placed in a 37 ° C constant temperature water bath, and the water was bathed for 2 hours.

(3) The double-cut system sample in step (2) was subjected to agarose gel electrophoresis to check whether the size of the insert was correct; the experimental results are shown in Fig. 3.

(4) Select the correct clone of the insert and send it to the sequencing company for sequencing.

2.6 endotoxin-free plasmid

2.6.1 Endotoxin-free plasmid extraction

(1) sequencing the correct clone inoculated into 100ml of ampicillin-resistant medium, shaken at 37 ° C, 220rpm / min, cultured for 15h;

(2) Transfer the bacterial solution cultured in the step (1) to a 50 mL centrifuge tube, centrifuge at room temperature, 8,000 rpm/min for 5 min, collect the cells, and discard the supernatant medium;

(3) adding 8 mL of solution P1 to the centrifuge tube of step (2), and fully resuspending the cells with a pipette;

(4) Add 8 mL of solution P2 to the centrifuge tube of step (3), immediately invert the centrifuge tube 6-8 times, and let stand at room temperature for 5 min;

(5) Add 8 mL of solution P4 to the centrifuge tube of step (4), immediately invert 6-8 times, mix well until the solution appears white flocculent precipitate, and leave it at room temperature for about 10 minutes. Centrifuge at 8,000 rpm/min for 5-10 min at room temperature to allow the white precipitate to leave the bottom of the tube;

(6) Carefully move all the supernatant in step (5) into the filter CS1, slowly push the filter, and collect the filtrate in a clean 50 mL centrifuge tube;

(7) Column balance: Add 2.5 mL of equilibration liquid BL to the adsorption column CP6 (adsorbing column into 50 mL collection tube), centrifuge at 8,000 rpm/min for 2 min at room temperature, pour off the waste liquid in the collection tube, and re-adsorb the adsorption column. Put back into the collection tube;

(8) To the filtrate of the step (6), 0.3 times of a filtrate volume of isopropanol is added, mixed upside down, and transferred to the adsorption column CP6. Centrifuge at 8,000 rpm/min for 2 min at room temperature, pour out the liquid in the collection tube, and re-inject the adsorption column CP6 into the same collection tube;

(9) adding 10 mL of the rinsing liquid PW to the adsorption column CP6 in step (8), centrifuging at 8,000 rpm/min for 2 min at room temperature, discarding the waste liquid in the collection tube, and returning the adsorption column to the collection tube;

(10) repeating the operation step (9) once;

(11) adding 3 mL of absolute ethanol to the adsorption column CP6 in step (10), centrifuging at room temperature 8,000 rpm/min for 2 min, and draining the waste liquid;

(12) The adsorption column CP6 of step (11) was returned to the collection tube and centrifuged at 8,000 rpm/min for 5 min at room temperature. Open the adsorption column CP6, leave it at room temperature for several minutes to dry;

(13) Put the adsorption column in step (12) into a clean 50mL centrifuge tube, add 1-2mL buffer TB in the center of the adsorption membrane, let stand at room temperature for 5min, centrifuge at 8,000rpm/min for 2min at room temperature, and put it into a 50mL centrifuge tube. The eluates were all transferred to a clean 1.5 mL centrifuge tube and the concentration was measured and stored at -20 °C.

(14) 1-2 μL of the obtained plasmid DNA solution was subjected to agarose gel electrophoresis and the electrophoresis result data was saved.

Example 3: Construction of pCDNA3.1-OPTI-E2 recombinant plasmid

The recombinant plasmid pCDNA3.1-OPTI-E2 was constructed by the procedure of Example 2, and the results of double enzyme digestion were shown in Fig. 4.

Example 4: Establishment of pEE12.4-OPTI-E2 Recombinant Plasmid Transfected with CHO-K1 Cells and Monoclonal Screening

4.1 CHO-K1 cell transfection

(1) Preparation: Biosafety cabinet was UV-sterilized for 30 min; DMEM/F12 (containing 10% serum, 1% double antibody), DMEM/F12 and PBS were placed in a 37 ° C water bath to preheat to 37 °C.

(2) Remove the cells (10 cm cell culture dish) from the 37 ° C incubator, discard the supernatant medium, and use Wash the cells once in pre-warmed 8 ml PBS and discard the PBS.

(3) Add 1-2ml of 0.25% trypsin-EDTA to each 10cm cell culture dish, digest at room temperature for about 2min, observe the cell shrinkage and roundness under the microscope, and present a single cell.

(4) The digestion reaction was terminated by adding 4 ml of DMEM/F12 (containing 10% serum, 1% double antibody), and the cells were blown off with a pipette.

(5) Transfer the digested cells to a 15 ml centrifuge tube, centrifuge at room temperature, 200 g, 5 min.

(6) The cells were resuspended in DMEM/F12 (containing 10% serum, 1% double antibody) and counted.

(7) Dilute the cells to 2 × 10 ⁵ /ml, and add 2 ml of the mixed cells to a six-well plate, and place the six-well plate at 37 ° C, and incubate overnight in a 5% CO ₂ cell incubator.

(8) Take out the step (7) cell culture dish and observe the cell state: when the cell confluence reaches 80%-90%, the transfection can be started. Before the transfection, the medium is replaced with the antibiotic-free serum-free DMEM/F12. 2 mL / well.

(9) Dilution of plasmid: The plasmid was diluted with OPTI-MEM, 2.5 μg of plasmid was added to 125 μl of OPTI-MEM, then 2.5 μl of plus was added, mixed, and allowed to stand at room temperature for 5 min.

(10) Diluted Lipofectamine LTX: 125 μl OPTI-MEM was added with 9 μl of Lipofectamine LTX, then 2.5 μl of plus was added, gently mixed, and allowed to stand at room temperature for 5 min.

(11) Mix the mixture of step (10) and step (11) gently. It was allowed to stand at room temperature for 5 min and then uniformly added to a six-well plate.

(12) Six-well plates were placed in a 37 ° C, 5% CO ₂ cell incubator for 4-6 h.

(13) Liquid exchange: The supernatant medium was discarded, 2 ml of DMEM/F12 (containing 10% serum 1% double antibody) was added, and the six-well plate was placed in a 37 ° C, 5% CO ₂ cell incubator.

4.2 Pressure screening

Pressurization was started 24 h after transfection: Six wells of cells were removed from the incubator at 37 ° C, the supernatant medium was discarded, 2 ml of DMEM/F12 (containing 10% serum + 25 μM MSX) was added, and the cells were observed for 7 days. Change the dead cells.

4.3 monoclonal screening

(1) When the filter was pressed until the negative control cells were substantially dead, about 7 days, monoclonal screening was started.

(2) Take out the six-well plate, discard the medium, wash once with PBS, then add 300μl 0.25% trypsin-EDTA, digest at room temperature for about 2min, add 2ml DMEM/F12 (containing 10% serum + 25μM MSX) to terminate the digestion reaction, and use The pipette blows the cells away.

(3) Transfer the digested cells to a 15 ml centrifuge tube, centrifuge at room temperature, 200 g, 5 min.

(4) The cells were resuspended in DMEM/F12 (containing 10% serum + 25 μM MSX) and counted.

(5) Plating: Dilute the cells to 5/ml, and add 200 μL of the mixed cells to a 96-well plate, place them at 37 ° C, and incubate for 4-6 h in a 5% CO ₂ cell incubator.

(6) Record the wells of a single cell.

(7) When the pores of a single cell in a 96-well plate are grown, discard the medium, wash once with PBS, and add 100 μl of 0.25% trypsin-EDTA, digested at room temperature for about 2 min, and the digestion reaction was terminated by adding 2 ml of DMEM/F12 (containing 10% serum + 25 μM MSX), and the cells were blown off with a pipette. The cell fluid was transferred to a 12-well plate. When the 12-well plate was full, the supernatant was taken, and the clone was positive by ELISA. The highly expressed positive clones were further expanded and frozen.

(8) After screening, a total of 2 cell lines were harvested, numbered 133 and 54 strains.

Example 5: Establishment of transfection of pCDNA3.1-OPTI-E2 recombinant plasmid with CHO/dhfr ^- cells and monoclonal screening

CHO/dhfr ^- cell transfection and monoclonal screening were performed as described in Example 4 (25 μM MSX was replaced with 25 nM MTX at screening). After screening, a total of 1 cell line was harvested, numbered 251.

Example 6: CHO-K1 cell line domesticated into suspension culture

(1) Preparation: The biosafety cabinet was UV-sterilized for 30 min; DMEM/F12 (containing 10% serum, 25 μM MSX) was preheated to 37 ° C in a 37 ° C water bath.

(2) The cells (10 cm cell culture dish) were taken out from the 37 ° C incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8 ml PBS, and PBS was discarded.

(3) Add 1-2ml of 0.25% trypsin-EDTA to each 10cm cell culture dish, digest at room temperature for about 2min, observe the cell shrinkage and roundness under the microscope, and present a single cell.

(4) The digestion reaction was terminated by adding 4 ml of DMEM/F12 (containing 10% serum, 25 μM MSX), and the cells were blown off with a pipetting gun.

(5) Transfer the digested cells to a 15 ml centrifuge tube, centrifuge at room temperature, 200 g, 5 min.

(6) The cells were suspended in 100% DMEM/F12 (containing 10% serum, 25 μM MSX) and counted.

(7) Dilute the cells to 5 × 10 ⁵ cells/ml and inoculate 30 ml of the medium in a 125 ml shake flask. Cell culture flask placed in 37 ℃, ₂ cell culture incubator in 5% CO orbital shaker 120rpm / min incubated overnight.

(8) The biosafety countertop is wiped and disinfected with 75% alcohol and irradiated with ultraviolet light for 30 minutes.

(9) Count cell density and vigor every 24 hours.

(10) The second generation culture was carried out when the cell survival rate reached 94-97% after the first generation of the cells were cultured once.

(11) Preparation: Biosafety cabinet was UV-sterilized for 30 min; 100% DMEM/F12 (containing 10% serum, 25 μM MSX), and EX-CELL 302 was placed in a CO ₂ cell incubator to preheat to 37 °C.

(12) The cells were removed from the 37 ° C incubator and transferred to a 50 ml centrifuge tube, and centrifuged at 200 g for 5 min at room temperature.

(13) DMEM/F12 (containing 10% serum, 25 μM MSX) and EX-CELL 302 were mixed by 1:1, and the cells were resuspended and counted.

(14) Dilute the cells to 5 x 10 ⁵ cells/ml and inoculate 30 ml of the medium in a 125 ml shake flask. The cell culture flask was placed in an orbital shaker in a 5% CO ₂ cell incubator at 37 ° C overnight at 120 rpm / min.

(15) The biosafety countertop is wiped and disinfected with 75% alcohol and irradiated with ultraviolet light for 30 minutes.

(16) Cell density and viability were counted every 24 hours.

(17) The cell survival rate obtained after the second generation culture was twice greater than 95%; the cell survival rate after the third to sixth generation cultures was three times greater than 95%. After 7 weeks, 3 days after cell inoculation three generations of breeding, density of 1 × 10 ⁶ cells / ml, while the 95% cell viability, the cell is considered to have been adapted to suspension culture. The seeding density was reduced to 3 × 10 ⁵ /ml.

(18) After acclimation, 133 strains and 54 strains all met the requirements, indicating that 133 strains and 54 strains were domesticated successfully.

Example 7: CHO/dhfr ^- cell strain acclimated into suspension culture

Domestication was carried out in accordance with the operation of Example 6. After acclimation (in which 25 μM MSX was replaced with 25 nM MTX), 251 strains met the requirement, indicating that 251 strains were successfully acclimated.

Example 8: Cell shake flask fermentation

(1) Preparation of subculture medium: 60% of CD-CHO + 40% of Ex-cell 302 was preheated to 37 ° C in a 37 ° C water bath.

(2) The shake flask cells were taken out from the CO ₂ constant temperature shaker and counted.

(3) Dilute the cells to 2.5-3.5 × 10 ⁵ cells/ml and inoculate 30 ml of the medium in a 125 ml shake flask. The cell culture flask was placed at 37 ° C and incubated overnight at 100 rpm/min in a 5% CO ₂ shaker.

(4) Count cell density and vigor every 24 hours, measure glucose, add glucose to 4g/L when it is less than 2g/L; take 1ml sample every day, and use supernatant to detect protein expression.

(5) Feed (about the fourth day): supplement 70g/LCB5, add 10% of the basal medium.

(6) Starting from the fifth day, the temperature of the CO ₂ incubator was adjusted to 32 °C.

(7) On the ninth day, supplement 70g/L CB5 and add 10% of the basal medium.

(8) On the twelfth day, the cell supernatant was harvested.

(9) As shown in Fig. 4, by werstern detection, 133 strains have the highest expression yield and are suitable for large-scale production.

Example 9: Protein purification

The cell culture supernatant of Example 8 was collected, centrifuged at 8,000 g for 30 min at 4 ° C, the supernatant was taken, passed through a 0.8 μm filter, and loaded, and 80 μl of the sample was added to add 20 μl of 5×SDS-sample buffer for SDS- PAGE detection.

Column equilibration: equilibrate 2 to 3 CV (column volume column volume) with ultrapure water, and drain the ethanol preservation solution; then equilibrate 2 to 3 CV with 4 to 7 ml/ with Buffer A (20 mM NaH ₂ PO ₄ (pH 7.4), 500 mM NaCl). Min.

Loading: If 5ml pre-packed column, 1ml/min for loading (adjust the loading flow rate according to the pre-packed column volume, retention time 5min), collect Flow through (FT), take 80μl sample and add 20μl 5×SDS-sample Buffer for SDS-PAGE detection.

Washing: Wash the column with 4% buffer B (20 mM NaH ₂ PO ₄ (pH 7.4), 500 mM NaCl, 20 mM imidazole) at a flow rate of 4 ml/min. Rinse the unbound protein and the weakly bound heteroprotein. , until the OD280nm baseline is stable.

Elution: 50% buffer B (20 mM NaH ₂ PO ₄ (pH 7.4), 500 mM NaCl, 500 mM imidazole) eluted the protein of interest, washed to baseline, 2 ml/min, collected: 10 ml/tube; collected samples after mixing (Elutethrough) - ET) 80 μl of sample was added to 20 μl of 5×SDS-sample buffer for SDS-PAGE detection. See Figure 5.

Washing: 100% buffer B (20 mM NaH ₂ PO ₄ (pH 7.4), 500 mM NaCl, 500 mM imidazole), 4 ml/min, not collected, rinsed 2-3 column volumes, and washed to UV baseline. Ultrapure water balance 2 ~ 3CV. The HisTrap excel column can be equilibrated with 2 to 3 CV in a 20% ethanol preservation solution.

Ultrafiltration liquid exchange: Millipore 30KD ultrafiltration membrane package for ultrafiltration liquid exchange, PBS 10 times diluted and concentrated to the original volume of the method of liquid exchange, repeated three times, a total of 1000 times diluted solution.

Sterilization filtration: In a biosafety cabinet, a 0.22 μm low protein binding needle filter, or a large amount of protein solution sterilized 0.22 μm filter Nalgene filter, the filtered protein solution sample is stored in a -80 ° C refrigerator.

Determination of protein concentration and purity: The protein concentration after purification was determined by BCA method, and the total amount of protein obtained after purification was calculated according to the total volume of protein obtained after purification, and then the protein yield was calculated according to the volume of the supernatant taken, and 133 strains of protein were calculated. The yield was 1g/L, and the others were less than 500mg/L. The purity was determined by HPLC method and the purity was over 95%.

Example 10: Vaccine Preparation and Immunoassay Test

10.1 Vaccine Preparation

The appropriate amount of CHO cell-expressed swine fever E2 protein was added to ISA 201 VG adjuvant (volume ratio: 46:54) to a final protein concentration of 30 μg/ml, which was emulsified, passed the quality test and stored at 4 ° C.

The appropriate amount of the baculovirus E2 protein expressed by the insect baculovirus was added to the ISA 201 VG adjuvant (volume ratio: 46:54) to make the final concentration of the protein 140 μg/ml, and the emulsification, quality inspection, and storage at 4 ° C.

10.2 Immunization challenge test (in the national engineering experiment of veterinary vaccine of Jinyu Baoling Biotechnology Co., Ltd.)

Screening 3-4 week old Landrace pigs (negative antibody to hog cholera, negative for ring antigen, negative for blue ear antigen, 5 in each group), each time immunized with 1 ml of CHO cell expressed H. sinensis E2 protein subunit vaccine ( 30μg/ml) and the porcine E2 protein subunit vaccine (140μg/ml) expressed by the insect baculovirus. The booster was boosted once after three weeks of priming, and the blood was taken once a week after immunization. The serum was separated and determined by IDXEE kit. Antibody titer.

Three weeks after the second exemption, the pig scorpion strain blood poison (purchased from the China Veterinary Drug Control Institute) was used for the challenge. Each pig was intramuscularly injected with 2 ml (1/2) of the blood of the pig scutellaria, and observed continuously for 16 days. The trial was conducted with more than 4/5 deaths in the control group.

The results in Figure 7 show that the blocking rate of the two groups of immunization groups after the second immunization has been about 70%, but the E2 protein content of CHO cells expressing E2 protein vaccine is only 30μg/ml, which is far lower than the baculovirus expression E2 protein vaccine. The E2 protein content, which indicates that the low dose of CHO cells expressed E2 protein immunization was equivalent to the antibody titer produced by high dose of baculovirus-expressing E2 protein.

In addition, after the challenge, the control pigs died within 8 days after the challenge, indicating that the challenge experiment was established; the CHO cell expression E2 protein immunization group did not die during the challenge, and the 5 pigs did not have any clinical Symptoms (such as elevated body temperature, depression, loss of appetite, etc.), so the overall protection rate is 100%; baculovirus expression E2 protein immunization group did not die during the attack, but a pig was in the process of attack The body temperature rises above 40.5 °C, and the mental depression and appetite are obviously reduced. It can be judged as the symptoms of swine fever after the attack, so the comprehensive protection rate is 80%. Therefore, from the results of the protection of the challenge, the CHO cell expression E2 protein immunization group is better than the baculovirus expression E2 protein immunization group.

The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the specific examples and embodiments described herein. The specific examples and embodiments are included herein to assist those skilled in the art in practicing the invention. Further modifications and improvements may be made by those skilled in the art without departing from the spirit and scope of the invention, and the invention is intended to be limited only by the scope and scope of the invention. Alternatives and equivalents within the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A method for preparing a recombinant piglet E2 protein, characterized in that the preparation method comprises the following steps:

   1) cloning the codon-optimized swine fever E2 protein coding gene into a eukaryotic expression vector to obtain a recombinant plasmid containing the hog E2 protein-encoding gene;

   2) transfecting the recombinant plasmid containing the gene encoding the hog E2 protein into CHO cells;

   3) obtaining a highly expressed cell strain by culturing, screening, and acclimating the CHO cell strain described in 2);

   4) Fermentation of the cell strain described in 3), and purifying to obtain recombinant swine fever E2 protein.
2. The method according to claim 1, wherein the nucleotide sequence of the codon-optimized swine fever E2 protein-encoding gene is as shown in SEQ ID NO.
3. The method according to claim 1, wherein the eukaryotic expression vector is pEE6.4, pEE12.4, pGL4.13, pcDNA3.1.
4. The method according to claim 3, wherein the eukaryotic expression vector is pEE12.4.
5. The method according to claim 1 or 2, wherein the CHO cell strain is a DG44, DXB11, CHO-K1, CHO-S cell strain.
6. The method according to claim 5, wherein the CHO cell strain is CHO-K1.
7. A preparation method comprising a recombinant swine fever E2 protein subunit vaccine, characterized in that the recombinant hog cholera E2 protein obtained by the preparation method according to claim 1 is thoroughly mixed with a pharmaceutically acceptable adjuvant to obtain a hog cholera E2 protein recombinant subunit vaccine.
8. The method of claim 7, wherein the adjuvant used in the recombinant subunit vaccine is ISA 201 VG.
9. The preparation method according to claim 7, wherein the recombinant hog cholera E2 protein and the adjuvant ISA 201 VG are emulsified by mixing at a volume ratio of 46:54.
10. Use of a recombinant swine fever E2 protein and a classical swine fever virus recombinant subunit vaccine according to any one of claims 1 to 7 for the preparation of a related diagnostic reagent.

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