CN112142827A - gB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application thereof - Google Patents
gB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application thereof Download PDFInfo
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- CN112142827A CN112142827A CN201910577700.XA CN201910577700A CN112142827A CN 112142827 A CN112142827 A CN 112142827A CN 201910577700 A CN201910577700 A CN 201910577700A CN 112142827 A CN112142827 A CN 112142827A
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Abstract
The invention provides gB subunit recombinant protein of porcine pseudorabies virus, a preparation method and application thereof, wherein the preparation method comprises the steps of 1) cloning an optimized gB gene sequence into a eukaryotic expression vector to obtain a recombinant plasmid containing a gB subunit protein coding gene of the pseudorabies virus; 2) then the recombinant plasmid containing the gB subunit protein coding gene of the pseudorabies virus is transfected into an expression cell; 3) culturing, screening and domesticating the expression cells in the step 2) to obtain a highly expressed cell strain; 4) fermenting and culturing the cell strain in the step 3), and purifying to obtain the gB subunit protein of the pseudorabies virus. The invention can provide the porcine pseudorabies virus gB subunit protein which can be industrially produced in a large scale, and has the advantages of simple preparation method, low cost and far higher yield than the prior baculovirus expression system.
Description
Technical Field
The invention belongs to the field of gene recombination expression in biotechnology. Relates to gB subunit recombinant protein of porcine pseudorabies virus, a preparation method and application thereof.
Background
The porcine Pseudorabies is an acute infectious disease which is caused by porcine Pseudorabies virus (PrV) and causes abortion of sows, central nervous system disorder of newborn piglets and respiratory symptoms and death of weaned piglets, and brings huge economic loss to the pig industry. At present, the main measure for preventing and controlling the disease is vaccination. The subunit vaccine does not contain nucleic acid, the generated immune response can be distinguished from the wild strains, continuous infection or latent infection can not be caused after inoculation, and the safety is better. However. Currently, subunit vaccines are limited in their use due to the high cost of protein production.
PRV belongs to the family of herpesviridae, and the virus is spherical, with a membrane and a fiber. The virus surface has identified 11 glycoproteins: gB (gII), gC (gIII), gD (gp50), gE, gG, gH, gI, gK, gL, gM, and gN. Among them, gB glycoprotein is encoded by UL27, also called gII glycoprotein, and consists of three proteins of gIIa, gIIb and gIIc, wherein gIIb and gIIc are obtained by hydrolysis of gIIa. The gB protein is found to be an important neutralizing protein of pseudorabies virus, and can promote the fusion of the cell outer membrane and the virus envelope. Therefore, the gB protein is a good protective antigen, and the subunit vaccine prepared by the gB protein can resist the attack of the strong virus of the pseudorabies virus.
gB is a glycosylated protein in order to better maintain its structural stability and immunogenicity. It is desirable to express the protein using eukaryotic systems. Patent CN 105693827A is a porcine pseudorabies virus subunit vaccine and a preparation method and application thereof. The method successfully expresses the pseudorabies virus gB protein by utilizing a baculovirus system, and provides a method for producing a gB subunit vaccine. However, the yield was only 6 mg/L. For veterinary vaccine applications, the yield is still relatively low. In addition, the glycosylated protein modification expressed by baculovirus of insect cells generally has only high-mannose type sugar chains or oligomannose sugar chains, and the end of the sugar chains has few residues such as galactose, sialic acid and the like, which is different from the glycosylation modification mode and degree of higher organisms. Only when expressed in mammalian cells, the glycoprotein has structural and physiological biochemical characteristics closer to those of the natural state. Therefore, the development of a production method of the gB protein which has low production cost, high production yield and a protein structure closer to the natural structure has important practical significance for application of pseudorabies subunit vaccines, development of diagnostic kits and the like.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a recombinant gB subunit protein on the surface of porcine pseudorabies virus, which can be industrially produced in a large scale.
In order to solve the problem, on the one hand, the inventor obtains a structurally stable gB subunit recombinant protein of the porcine pseudorabies virus by structurally analyzing the whole structure of the gB protein and removing a transmembrane region and an intracellular region without protective antigens in the gB protein on the basis of fully analyzing and researching the currently available porcine pseudorabies virus data, wherein the gB subunit recombinant protein is an extracellular region of the gB protein and has an amino acid sequence shown as SEQ ID No. 3.
According to the technical scheme of the invention, preferably, the gene sequence coded by the gB subunit recombinant protein is shown as SEQ ID NO. 2.
According to the technical scheme of the invention, preferably, the gene sequence encoded by the gB subunit recombinant protein can efficiently secrete and express an optimized gB gene sequence of the gB subunit protein in CHO cells, and the optimized gB gene sequence is shown as SEQ ID No. 1.
According to the technical scheme of the invention, preferably, the gB subunit recombinant protein can be connected with a detection or purification tag at the amino terminal or the carboxyl terminal of the amino acid sequence shown as SEQ ID NO.3, wherein the tag is one selected from ploy-Arg, ploy-His, FLAG, c-myc and HA. Here, in order to make the subunit gB protein convenient for detection or purification, one skilled in the art can attach a tag as shown in table one to the amino terminus or carboxy terminus of the amino acid sequence shown in SEQ ID No.3, specifically exemplified by Poly-His in this example, to the carboxy terminus of the amino acid sequence shown in SEQ ID No.3 by conventional technical means.
TABLE-TAGS AND AMINO ACID SEQUENCES THEREOF
According to the technical scheme of the invention, preferably, the porcine pseudorabies virus subunit protein comprises a derived protein which is substituted, deleted or added with one amino acid or a plurality of amino acids in the amino acid sequence shown as SEQ ID NO3 and has immunogenicity.
According to the technical scheme of the invention, preferably, the amino acid of the porcine pseudorabies virus gB is from a strain comprising porcine pseudorabies virus bartha, HeN1, NVDC-PRV-BJ, PRV-TJ, Fa and Becker; porcine pseudorabies virus variant strains PRV-ZJ01, HN1201, HN1202 and the like. The porcine pseudorabies virus gB subunit protein is the glycoprotein of the herpes virus members that is most conserved.
According to the technical scheme of the invention, preferably, the expression system of the porcine pseudorabies virus subunit gB protein is a mammalian cell.
According to the technical solution of the present invention, preferably, the mammalian cell is a CHO cell.
According to another aspect of the present invention, there is provided a method for preparing porcine pseudorabies virus gB protein, comprising: 1) cloning the optimized gB gene sequence shown as SEQ ID NO.1 into a eukaryotic expression vector to obtain a recombinant plasmid containing a gB subunit protein coding gene of the pseudorabies virus; 2) then the recombinant plasmid containing the gB subunit protein coding gene of the pseudorabies virus is transfected into an expression cell; 3) culturing, screening and domesticating the expression cells in the step 2) to obtain a highly expressed cell strain; 4) fermenting and culturing the cell strain in the step 3), and purifying to obtain gB subunit protein of the pseudorabies virus, wherein the amino acid sequence of the gB subunit protein is shown as SEQ ID NO. 3.
In the technical solution of the present invention, preferably, in step 1), the eukaryotic expression vector may be pee6.4, pee12.4, pgl4.13, pcdna3.1; preferably, the eukaryotic expression vector is pEE12.4 or pcDNA3.1.
In the technical solution of the present invention, preferably, in step 2), the expression cell is a mammalian cell.
In the technical solution of the present invention, preferably, the mammalian cell is a CHO cell or 293T cell.
In the technical scheme of the invention, preferably, the CHO cell comprises DG44, DXB11, CHO-K1 and CHO-S cell strain, and preferably, the CHO cell is CHO-K1 cell.
According to a further aspect of the present invention, the present invention provides a use of a recombinant protein of gB subunit of porcine pseudorabies virus for the preparation of a vaccine for the diagnosis, prevention and treatment of porcine pseudorabies.
The invention constructs and screens the suspension stable high-efficiency CHO cell strain secreting and expressing the pseudorabies virus gB protein, the cell strain expresses the pseudorabies virus gB protein with high yield (the yield is up to 0.8-1.2g/L), is far higher than the prior expression technology, is easy to purify (as shown in figure 4, the purity of the target protein can reach more than 90 percent only by one-step affinity chromatography, and the invention far meets the requirements of subunit vaccines and diagnostic reagents), and is easy for large-scale production. Therefore, the problem of high production cost of the protein, which is a defect of the gB subunit vaccine of the pseudorabies virus, is solved. In addition, the CHO cell strain for production has high controllability during culture, easy quality control, stable production protein batch-to-batch, and high biosafety (no virus, no risk of virus dispersion).
Drawings
FIG. 1 shows the alignment results before and after optimization of the gB gene sequence.
FIG. 2 shows a map of the pEE12.4-OPTI-gB plasmid.
FIG. 3 shows the results of the double-restriction enzyme identification of pEE12.4-OPTI-gB: m is a DNA Marker: DL10000 Marker; 1 is the result of pEE12.4-OPTI-gB double-enzyme digestion electrophoresis, and the enzyme digestion sizes are 8746bp and 2172bp respectively.
FIG. 4 shows SDS-PAGE purification assay of recombinant gB: 1 is a protein Marker, and 2 is a purified glycosylated gB protein with the molecular weight of about 130Kd (the loading buffer does not contain reducing agents such as beta-mercaptoethanol and the like).
FIG. 5 shows the West-blot assay of recombinant gB proteins: 1 is maker, 2 is purified gB protein, and the western results show that the band is approximately 130Kd, which is consistent with the size of SDS-PAGE.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.
The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.
The sources of the reagent and the medicine of the invention are listed as follows:
the CHO-K1 cells are derived from cell banks of China academy of sciences type culture Collection cell banks, Shanghai Life sciences research institute of China academy of sciences;
cell culture medium and serum were purchased from gibco, usa;
the eukaryotic expression vector pEE12.4 is purchased from Shanghai Linyuan Biotech, Inc.;
lipofectamine LTX was purchased from Thermo Fisher, USA;
methionine sulfoxide iminium (L-methionine sulfoximine, MSX) was purchased from Sigma company;
BCA protein quantification kit was purchased from Thermo Fisher, USA.
Example 1gB protein expression and preparation
1.1 selection of porcine pseudorabies gB protein
The porcine pseudorabies virus surface glycoprotein gB is a segment of polypeptide encoded by an UL27 gene, amino acids 1-800 are predicted to be the extracellular region of the gB protein, a transmembrane region is arranged at the amino acid position of 801-821, and the amino acid position of 822-916 is the intracellular region. The gB protein extracellular region has stable structure and good immunogenicity, and is a main antigen for producing gB subunit vaccines. At present, the protein can be expressed and purified in a large scale in a mammalian system, which is an important technical problem to be solved by the invention.
1.2 codon optimization of gB protein of porcine pseudorabies virus
The gB protein is the most conserved protein on the surface of the porcine pseudorabies virus, the homology is high, the classical strain Bartha strain (GenBank: JF797217.1) is taken as a template in the laboratory, the gB gene is optimally synthesized, the gB expression vector is constructed, and the gB protein is expressed and purified. Thus, gB proteins from other strains can also be prepared by this method. Combining the experience of the previous research on the envelope protein of the expressed virus, through the research on the protein structure prediction and the structure of the amino acid, the partial sequence of the N-terminal extracellular region and the C-terminal transmembrane region and intracellular region which are not beneficial to recombinant expression are removed, and the extracellular region with stable structure of the porcine pseudorabies virus protein is selected as the immunogenic protein, namely the amino acid of 41V-754N. Since the amino acids encoded by the start codon of eukaryotes are all methionine (M), we add one methionine (M) before 41V in designing expression, i.e., designing the amino acid sequence of the recombinant expression gB protein 40M-754N. In order to facilitate the better stable and efficient expression of the porcine pseudorabies virus gB gene in a CHO system, the nucleotide sequence for coding the gB protein is subjected to codon optimization as shown in SEQ ID NO.2 to obtain an OPTI-gB sequence as shown in SEQ ID NO.1, and the sequence synthesis work is entrusted to Nanjing Kingsry Biotech Co. As shown in FIG. 2, the nucleotide sequences before and after the optimization were different by 19.3%.
Example 2: construction of pEE12.4-OPTI-gB recombinant plasmid
2.1PCR amplification of the fragment of interest OPTI-gB
2.1.1PCR reaction
(1) Primer design and Synthesis
Upstream primer 5'-cgaAGCTTGCCGCCACCATGGTGGCTC-3'
Downstream primer 5'-cgcGAATTCTCAATGGTGATGGTGATGGTGATTATGATCCACCTT-3'
(2) Sample loading system 50 μ L, as shown in the following table:
PCR amplification procedure:
2.1.2PCR products for gel recovery
(1) Marking a sample collection EP tube, an adsorption column and a collection tube;
(2) weighing the weight of the marked empty EP pipe, and recording the numerical value;
(3) a single DNA band of interest was carefully excised from the agarose gel on a gel cutter with a scalpel and placed into a clean 1.5mL centrifuge tube;
(4) adding 600 mu L of PC buffer into the 1.5mL centrifuge tube in the step (3), placing in a water bath at 50 ℃ for about 5min, and turning the centrifuge tube up and down continuously and gently to ensure that the gel block is fully dissolved;
(5) column balancing: adding 500 μ L of balance liquid BL into adsorption column CB2 (the adsorption column is placed into the collection tube in advance), centrifuging at 12,000rpm/min for 1min, pouring off waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(6) adding the solution obtained in the step (5) into an adsorption column CB2, standing for 2min at 10,000rpm/min, centrifuging for 30s, pouring out waste liquid in a collecting pipe, and then putting the adsorption column CB2 into the collecting pipe;
(7) adding 600 mu L of rinsing liquid PW buffer into the adsorption column, standing for 3min, centrifuging at 10,000rpm/min for 30s, pouring off waste liquid in the collecting tube, and putting the adsorption column CB2 into the collecting tube;
(8) repeating the step (7);
(9) centrifuging with an empty adsorption column at 12,000rpm/min for 2min, removing rinsing liquid as much as possible, standing the adsorption column at room temperature for 10min, and completely air drying;
(10) placing adsorption column CB2 in a collecting tube, suspending and dropwise adding 50 μ L of precipitation buffer (preheated at 65 ℃) to the middle position of an adsorption film, standing for 3min, centrifuging at 12,000rpm/min for 2 min;
(11) taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, covering the centrifuge tube with a cover, and keeping the DNA sample in the centrifuge tube;
(12) and (3) storing the DNA sample in the step 11 at 4 ℃, and preparing an agarose gel electrophoresis identification gel to recover the DNA fragment.
2.2 double digestion of PCR products and vectors
(1) The required 1.5mL EP tube was labeled, and the sample was loaded and mixed in the 1.5mL EP tube according to the following table: 50 μ L reaction System
(2) And (3) placing the 1.5mL EP tube in the step (1) into a corresponding enzyme constant-temperature water bath kettle with the optimal temperature, and carrying out water bath for 2-3 h.
Recovering the double enzyme digestion product gel: taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment in the double enzyme digestion system by the same method as that of the PCR product gel recovery in the 1.2.1.
2.3 ligation reaction
(1) A plurality of clean 1.5mL EP tubes are prepared, marked and placed on an EP tube frame for standby.
(2) The sample was loaded and mixed in a 1.5mL EP tube as described in the following table.
(3) After sample adding is finished according to the table in the step (2), placing each 10 mu l reaction system in a low-temperature cooling liquid circulator at the temperature of 16 ℃ for water bath for 10-16 h;
(4) taking out the EP tube in the step (3), placing the EP tube in a water bath kettle at 65 ℃, and carrying out water bath for 15 min;
(5) taking out the EP tube in the step (4), and storing at 4 ℃.
2.4 conversion reaction
(1) Quickly adding 10 μ L of the ligation reaction solution into 100 μ L of competent cells, uniformly mixing by blowing, and carrying out ice bath for 30 min;
(2) taking out the sample tube, placing in water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2 min;
(3) taking out the sample tube, adding 600 mu L of liquid LB culture medium into the sample tube in a super-clean workbench, then placing the sample tube in a constant temperature shaking table at 37 ℃, and culturing for 1h at 220 rpm/min;
(4) coating a plate: and (4) taking out the sample tube in the step (3), centrifuging at room temperature for 8,000rpm/min for 2min, removing 600 mu L of supernatant liquid, re-suspending the thalli at the bottom of the tube by the residual supernatant liquid, putting the re-suspended bacterial liquid into the center of a corresponding transformation plate, and uniformly spreading the bacterial liquid in the center of the transformation plate by using a bacteria coating rod.
(5) The flat plate in the transformation step (4) is placed in a biochemical constant-temperature incubator, and is cultured for 1h at 37 ℃, and then the transformation flat plate is inverted and cultured for 15 h;
(6) the transformation results were observed.
2.5 plasmid extraction and double restriction enzyme identification
2.5.1 plasmid extraction
(1) Picking the monoclonals from the conversion plate by using a 10-microliter pipette tip to 5mL of LB liquid culture medium containing the benzyl resistance, and shaking the bacteria at 37 ℃ and 220rpm/min overnight;
(2) transferring the bacterial liquid into a 1.5mL EP tube, centrifuging at room temperature at 12,000rpm/min for 2min, and removing the supernatant;
(3) adding 250 mu L of plasmid extraction reagent P1buffer into the EP tube in the step (2) to completely suspend the thalli;
(4) adding 250 mu L P2buffer into the solution in the step (3), immediately and gently inverting the centrifuge tube for 5-10 times, uniformly mixing, and standing at room temperature for 2-4 min;
(5) adding 350 mu L P3buffer into the solution in the step (4), immediately and gently inverting the centrifuge tube for 5-10 times and uniformly mixing; standing at room temperature for 2-4 min;
(6) centrifuging the solution in the step (5) at room temperature, and carrying out centrifugation at 14,000rpm/min for 10 min;
(7) transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging at room temperature for 30s at 12,000rpm/min, and pouring out liquid in a collecting pipe;
(8) adding 500 μ L Buffer DW1 into the center of the adsorption column, centrifuging at room temperature at 12,000rpm/min for 30s, and pouring off the liquid in the collection tube;
(9) adding 500 μ L wash solution into the center of the adsorption column, centrifuging at room temperature, 12,000rpm/min for 30s, pouring off the liquid in the collection tube, and repeating once;
(10) the column was air-adsorbed, centrifuged at room temperature, 12,000rpm, 2 min.
(11) The column was placed in a clean 1.5mL centrifuge tube, 30. mu.L of Elutionbuffer was added to the center of the adsorption membrane, allowed to stand at room temperature for 5min, and centrifuged at room temperature at 12,000rpm for 2 min. The DNA solution in the tube was preserved.
2.5.2 double restriction enzyme identification
(1) The 1.5mL EP tubes were labeled for use and loaded as follows: 20 μ L reaction System
(2) Putting the EP tube 20 mu L reaction system in the step (1) into a constant-temperature water bath kettle at 37 ℃ and carrying out water bath for 2 h.
(3) Carrying out agarose gel electrophoresis on the double enzyme digestion system sample in the step (2), and checking whether the size of the inserted fragment is correct; the results are shown in FIG. 2: the construction is correct by enzyme digestion identification.
(4) Clones with correct inserts were selected for sequencing by the sequencing company. And (4) storing the plasmid with the correct sequencing result for later use.
Example 3: establishment of pEE12.4-OPTI-gB recombinant plasmid transfection CHO-K1 cell and monoclonal screening
3.1CHO-K1 cell transfection
(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; DMEM/F12 (containing 10% serum and 1% double antibody), DMEM/F12 and PBS were preheated to 37 ℃ in a 37 ℃ water bath.
(2) The cells (10cm cell culture dish) were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8mL PBS, and the PBS was discarded.
(3) Adding 1-2mL of 0.25% trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and displaying the cells as single cells.
(4) Digestion was stopped by adding 4mL of DMEM/F12 (10% serum, 1% double antibody) and the cells were pipetted off.
(5) The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 5min at 200 g.
(6) Cells were resuspended in DMEM/F12 (10% serum, 1% double antibody) and counted.
(7) Dilute cells to 2 × 1052mL of the mixed cells were added to a six-well plate, which was placed at 37 ℃ with 5% CO2Incubate overnight in a cell incubator.
(8) Taking out the cell culture dish in the step (7), and observing the cell state: transfection was initiated when cell confluence reached 80% -90%, and the medium was changed to antibiotic-free and serum-free DMEM/F12, 2 mL/well before transfection.
(9) Plasmid dilution: the plasmid was diluted with OPTI-MEM, and 2.5. mu.g of the plasmid was added to 125. mu.L of OPTI-MEM, followed by 2.5. mu.L of plus, followed by mixing, and the mixture was allowed to stand at room temperature for 5 min.
(10) Dilution of Lipofectamine LTX: mu.L of OPTI-MEM was added with 9. mu.L of Lipofectamine LTX, followed by 2.5. mu.L of plus, gently mixed, and allowed to stand at room temperature for 5 min.
(11) And (4) lightly mixing the mixture obtained in the step (10) and the step (11). Standing at room temperature for 5min, and then dropwise adding into a six-hole plate for uniform distribution.
(12) Placing the six-hole plate at 37 ℃ and 5% CO2Culturing in a cell culture box for 4-6 h.
(13) Liquid changing: the supernatant medium was discarded, 2mL of DMEM/F12 (10% serum in 1% double antibody) was added, and the six well plate was placed at 37 ℃ and 5% CO2Culturing in a cell culture box.
3.2 pressure screening
Pressurization was started 24h after transfection: six well plate cells were removed from the 37 ℃ incubator, supernatant medium was discarded, 2mL of DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added, pressurized for 7days, cells were observed in the middle, and the dead cells were replaced with more fluid.
3.3 monoclonal screening
(1) The monoclonal selection was initiated at approximately 7days when the negative control cells were essentially dead by pressure selection.
(2) The six well plate was removed, the medium was discarded, PBS was washed once, then 300. mu.L of 0.25% trypsin-EDTA was added, the digestion was carried out at room temperature for about 2min, 2mL of DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added to stop the digestion reaction, and the cells were blown off by pipette.
(3) The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 5min at 200 g.
(4) Cells were resuspended in DMEM/F12 (containing 10% serum + 25. mu.M MSX) and counted.
(5) Plate paving: diluting the cells to 5/mL, adding 200. mu.L of the mixed cells to a 96-well plate, standing at 37 ℃ with 5% CO2And incubating for 4-6h in the cell incubator.
(6) Wells of individual cells were recorded.
(7) When the wells of the individual cells in the 96-well plate were grown up, the medium was discarded, PBS was washed once, 100. mu.L of 0.25% trypsin-EDTA was added, digestion was carried out at room temperature for about 2min, 2mL of DMEM/F12 (containing 10% serum + 25. mu.M MSX) was added to terminate the digestion reaction, and the cells were blown off by pipette. And transferring the cell sap to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by dot-blot, and continuously performing amplification culture and freezing storage on the high-efficiency expression positive clone.
Example 4: CHO-K1 cell strain acclimatized to suspension culture
(1) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; DMEM/F12 (containing 10% serum, 25. mu.M MSX) was preheated to 37 ℃ in a 37 ℃ water bath.
(2) The cells (10cm cell culture dish) were removed from the 37 ℃ incubator, the supernatant medium was discarded, the cells were washed once with pre-warmed 8mL PBS, and the PBS was discarded.
(3) Adding 1-2mL of 0.25% trypsin-EDTA into each 10cm cell culture dish, digesting at room temperature for about 2min, observing the cells under a microscope to shrink and become round, and displaying the cells as single cells.
(4) Digestion was stopped by adding 4mL of DMEM/F12 (10% serum, 25. mu.M MSX) and the cells were blown off with a pipette gun.
(5) The digested cells were transferred to a 15mL centrifuge tube and centrifuged at room temperature for 5min at 200 g.
(6) Cells were suspended in 100% DMEM/F12 (containing 10% serum, 25. mu.M MSX) and counted.
(7) Dilute cells to 5 × 105cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO2Incubate overnight on an orbital shaker in a cell incubator at 120 rpm/min.
(8) Wiping the biological safety cabinet table top with 75% alcohol for sterilization, and irradiating with ultraviolet for 30 min.
(9) Cell density and viability were counted every 24 h.
(10) And performing second-generation culture when the cell survival rate reaches 94-97% after the first-generation cell culture is performed once.
(11) Preparing: sterilizing the biological safety cabinet for 30min by ultraviolet; 100% DMEM/F12 (containing 10% serum, 25. mu.M MSX), EX-CELL 302 in CO2The cell incubator was preheated to 37 ℃.
(12) The cells were removed from the 37 ℃ incubator, transferred to a 50mL centrifuge tube, and centrifuged at 200g for 5min at room temperature.
(13) DMEM/F12 (containing 10% serum, 25. mu.M MSX) and EX-CELL 302 were mixed as 1:1 mixing, resuspending the cells, and counting.
(14) Dilute cells to 5 × 105cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO2Incubate overnight on an orbital shaker in a cell incubator at 120 rpm/min.
(15) Wiping the biological safety cabinet table top with 75% alcohol for sterilization, and irradiating with ultraviolet for 30 min.
(16) Cell density and viability were counted every 24 h.
(17) The survival rate of the cells obtained after the second generation culture is twice is more than 95 percent; the cell survival rate obtained after three times of culture of the third to the sixth generation is more than 95 percent. After 7 weeks, the cells were seeded for 3 days and propagated for three generations with a density of 1X 106Individual cells/mL with a cell viability of 95%, which cells are considered to have been adapted to suspension culture. The inoculation density is reduced to 3 x 105one/mL.
(18) After domestication, the monoclonal cell strains of 32E2, 37A4, 39A12, 40B10, 4C2 and 6A3 all meet the requirements, which indicates successful domestication.
Example 5: cell shake flask fermentation
(1) Preparation of a subculture medium: 60% CD-CHO + 40% Ex-cell 302 was preheated to 37 ℃ in a 37 ℃ water bath.
(2) From CO2Taking out the shake flask cells by a constant temperature shaking table, and counting.
(3) The cells of the 32E2 and 40B10 strains obtained in example 4 were diluted to 2.5 to 3.5X 105cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO2Incubate overnight in a constant temperature shaker at 100 rpm/min.
(4) Counting the cell density and activity every 24h, measuring glucose, and adding the glucose to 4g/L when the blood sugar is lower than 2 g/L; samples were taken at 1mL per day and the supernatant was used to detect protein expression.
(5) Feeding (about day four): 70g/L of CB5 was supplemented, and 10% of the basal medium was added.
(6) Beginning on day 5, CO was added2The incubator temperature was adjusted to 32 ℃.
(7) On day nine, 70g/L CB5 was supplemented and 10% of the basal medium was added.
(8) On the fourteenth day, cell supernatants were harvested.
Example 6 construction and domestication of recombinant CHO-S cell lines stably expressing gB protein
The present inventors also easily constructed a stable cell line expressing recombinant gB protein according to the procedures of examples 1-5, and thus, it is expected that a stable cell line expressing recombinant gB protein can be easily constructed using the method for a commonly-used engineered mammalian cell line, thereby producing the protein on a large scale. Therefore, the present invention is also within the scope of protection.
Example 7: protein purification
The cell culture medium of example 5 (approximately 100mL per batch) was collected, centrifuged at 8,000g for 30min at 4 ℃ and the supernatant was filtered through a 0.8 μm filter and loaded, leaving 80 μ L of sample and 20 μ L of 5 SDS-sample buffer for SDS-PAGE detection.
Column balancing: balancing 2-3 CV (column volume) with ultrapure water, and discharging ethanol preservation solution; then using BufferA (20mM NaH)2PO4(pH 7.4) and 500mM NaCl, and balancing for 2-3 CV, 4-7 mL/min.
Loading: if one is used for 5mL pre-packed columns, loading is carried out at 1mL/min (the loading Flow rate is adjusted according to the volume of the pre-packed column, the retention time is 5min), Flow Through (FT) is collected, and 80. mu.L of sample is added to 20. mu.L of 5 XSDS-sample buffer for SDS-PAGE detection.
Washing: with 4% bufferB (20mM NaH)2PO4(pH 7.4), 500mM NaCl, 20mM imidazole) and a flow rate of 4mL/min, the protein not bound to the column and the hetero-protein with weak binding ability were washed clean until the baseline OD280nm was stabilized.
And (3) elution: 50% buffer B (20mM NaH)2PO4(pH 7.4), 500mM NaCl, 250mM imidazole) until the baseline is flat, 2mL/min, and collecting: 10 mL/tube; after sample mixing (Elutethregh-ET) 80. mu.L of sample was added to 20. mu.L of 5 XSDS-sample buffer for SDS-PAGE detection.
Washing: 100% buffer B (20mM NaH2PO4(pH 7.4), 500mM NaCl, 500mM imidazole), 4mL/min, no collection, 2-3 column volumes washed until UV baseline was washed flat. The ultra-pure water has a balance of 2-3 CV. The HisTrap excel column can be preserved with 20% ethanol preservation solution to balance 2-3 CV.
And (3) dialysis liquid change: the imidazole eluate containing the target protein was poured into a dialysis bag, dialyzed with 1 XPBS for at least 1,000 times, and 80. mu.l of the eluate was detected.
And (3) degerming and filtering: in a biosafety cabinet, a 0.22 μm low protein binding needle filter, or a Nalgene filter with a 0.22 μm membrane sterilized with a large volume of protein solution was passed through, and the filtered protein solution sample was stored in a freezer at-80 ℃.
Protein concentration determination: determining the protein concentration by using a BCA method, wherein the protein concentration of the batch is 1.6mg/mL and 2.4mg/mL respectively, and the volume is about 50 mL; by calculation (protein yield ═ protein concentration ═ protein volume/volume of fermentation supernatant taken), the protein yields of 32E2 and 40B10 strains were both about 0.8-1.2 g/L.
Example 8: identification of gB proteins
8.1SDS-PAGE electrophoretic detection
The protein purified in example 6 was subjected to SDS-PAGE, and the concentration of gB protein in the sample was 2. mu.g/well, as shown in FIG. 4: from the figure, it can be calculated that the purified gB protein has an SDS-PAGE purity of 94% and the glycosylated protein has a molecular weight of about 130 kD.
8.2Western-blot detection
The purified protein of example 6 was subjected to western-blot assay with a membrane-transfer time of 1h, the antibody used was the positive serum of a swine inoculated with pseudorabies attenuated vaccine at a dilution ratio of 1:500, and the incubation time was 1h, the results are shown in fig. 5: as can be seen from the results in the figure, the purified gB protein was able to bind to the antibody efficiently.
8.3ELISA detection
(1) Coating: diluting purified gB protein to 0.5 μ g/ml with coating solution (50mM carbonate buffer, pH 9.5), coating each antigen with 8 wells (4 wells plus PRV positive serum sample, 4 wells plus PRV negative serum as control), adding each antigen with 100 μ l/well, sealing with sealing film, and standing in refrigerator at 4 deg.C overnight;
(2) washing: after the ELISA plate was removed from the refrigerator, the plate was washed 5 times with PBST;
(3) and (3) sealing: adding 200 μ l of sealing liquid (5% skimmed milk) into each well, sealing with sealing film, and incubating at 37 deg.C for 2 hr;
(4) serum dilution: diluting the positive serum (14 days after immunization) of PRV (porcine PRV) attenuated vaccine immunized pig by 500 times with blocking solution (such as adding 1 μ l serum into 499 μ l diluent, mixing uniformly), and diluting the negative serum by 500 times;
(5) washing: the same (2);
(6) sample adding: adding diluted positive serum and negative serum, and incubating at 37 deg.C for 1 h;
(7) washing: the same (2);
(8) adding a secondary antibody: adding 100 mu l of a diluted (dilution ratio of 1:5000) HRP-labeled rabbit anti-pig IgG secondary antibody into each hole, and incubating at 37 ℃ for 0.5 h;
(9) washing: the same (2);
(10) color development: adding 100 mul of TMB color development solution into each hole under the condition of keeping out of the sun, and incubating for 10min at 37 ℃;
(11) and (4) terminating: add 50. mu.l stop solution (2M H) to each well2SO4) Terminating the reaction;
(12) and (3) detection: measuring the OD value of the sample at the wavelength of 450nm, and analyzing the data;
(13) the results are shown in the following table: the coated gB protein can be specifically combined with positive serum, and the OD450 mean value is 1.867; neither the coated gB protein specifically bound to negative sera, with an OD450 mean of 0.171. The gB protein can be used as an antigen of an Elisa kit, and a diagnostic kit for detecting the infection and the immunity of the porcine pseudorabies virus can be developed after the appropriate coating concentration and serum dilution ratio are searched.
Example 9 preparation of a gB subunit vaccine
9.1 vaccine preparation
Preparing an aqueous phase: according to the content of the gB protein in the vaccine, PBS (or normal saline) is used for diluting the gB protein to a proper concentration, and the gB protein is an aqueous phase;
preparing an oil phase: according to the total amount of the prepared vaccine, a proper amount of ISA 201VG adjuvant is measured according to the weight ratio of 1:1 and the volume ratio of 46:54 of the antigen phase and the adjuvant;
emulsification: preheating the water phase and the oil phase to 33 ℃, slowly adding the water phase into the oil phase, stirring at 200-500rpm for 20-30min, standing at 20 ℃ for 1h, and standing at 4 ℃ overnight;
subpackaging and storing: subpackaging as required, and storing at 4 deg.C for use after qualified inspection.
9.2 vaccine detection
Observing the physical properties by adopting an eye-watching method to observe the appearance (whether the emulsion is milky white or not);
sucking a small amount of vaccine by using a clean straw and dripping the vaccine into cold water, observing (except for the 1 st drop), wherein the vaccine is dispersed in a cloud form and is judged to be a water-in-oil-in-water dosage form;
adding 10mL of vaccine into a centrifuge tube, centrifuging for 15min at 3000r/min, and judging the vaccine to be stable if the water separated out from the tube bottom is less than or equal to 0.5 mL;
and (4) performing viscosity detection on the vaccine by using a viscometer, wherein the viscosity detection is required to be within 20-50cp, and the vaccine is judged to be qualified.
9.3gB subunit vaccine for mouse safety experiment
A safety experiment was performed on 16 healthy SPF female mice (purchased from Zhejiang university of medicine) 16 in 4 groups of 4 mice each at random according to the following method.
Single dose one-time immunization group: each group was inoculated with 100. mu.L (30. mu.g/mouse) of 4 mice intramuscularly and continuously observed for 2 weeks.
Single dose secondary immunization group: each group was inoculated with 100. mu.L (30. mu.g/mouse) of 4 mice intramuscularly and continuously observed for 2 weeks. After 2 weeks, another vaccination was performed at the same dose for 2 additional weeks.
Overdose one-time immunization group: each group was inoculated intramuscularly with 100. mu.L (300. mu.g/mouse, 10-fold normal immunization dose) of 4 mice per group, and observed for 2 weeks.
Control group: each group was inoculated intramuscularly with 100. mu.L of 4 mice (vaccine in PBS) and observed for 2 weeks. During the experiment, the animal's spirit, feeding, activity, drinking, inflammation change at injection site, excretion and other clinical changes were observed every day, and the abnormal conditions of the animal were recorded.
Through continuous observation, clinical symptoms of mice injected with the gB protein are compared, and a single dose, a secondary immune dose, an overdose immune group and a control group are normal in diet, free of adverse changes in spirit and normal in excretion, free of inflammation at an injection part and dead mice, and inoculated animals do not suffer any adverse reaction. The vaccine protein prepared by the invention has no obvious side effect even if injected at high dose (300 mu g), and is a safe immune protein.
The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the particular examples and embodiments described herein. These specific examples and embodiments are included to assist those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention and, accordingly, it is intended that the invention be limited only by the terms of the appended claims, along with the full scope of equivalents to which such terms are entitled.
<110> Zhejiang Hilon Biotechnology Ltd
<120> construction and preparation method of porcine pseudorabies virus gB protein recombinant expression vector
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ATGGTGGCTCTGGCCCTGCTGCTGCTGGCTCTGGCTGCTGCTCCACCTTGCGGAGCTGCTGCTGTGACCAGGGCTGCTTCCGCCAGCCCAACACCAGTGCCTGGATCCCCAGGACTGACCCCAAACGACGTGTCCGCTGAGGCCAGCCTGGAGGAGATCGAGGCTTTCACCCCAGGACCATCTGAGGCTCCAGACGGAGAGTACGGCGACCTGGATGCTAGGACAGCCGTGCGGGCTGCTGCTACCGAGAGAGATAGGTTCTACGTGTGCCCCCCTCCATCTGGCTCCACAGTGGTGAGGCTGGAGCCTGAGCAGGCTTGTCCAGAGTACTCCCAGGGCCGGAACTTCACCGAGGGCATCGCCGTGCTGTTTAAGGAGAATATCGCCCCCCACAAGTTCAAGGCTCATATCTACTATAAGAACGTGATCGTGACCACAGTGTGGAGCGGCTCTACATACGCTGCCATCACAAATAGGTTCACCGACCGCGTGCCTGTGCCAGTGCAGGAGATCACCGACGTGATCGATAGGCGGGGCAAGTGCGTGTCCAAGGCTGAGTATGTGAGGAACAATCACAAGGTGACAGCCTTCGACCGGGATGAGAACCCCGTGGAGGTGGACCTGAGACCTAGCCGCCTGAACGCTCTGGGAACCAGGGGATGGCACACCACAAATGATACCCATACAAAGATCGGCGCTGCCGGCTTTTACCATACCGGCACAAGCGTGAATTGTATCGTGGAGGAGGTGGAGGCTAGGTCCGTGTACCCTTATGACTCTTTCGCCCTGTCCACCGGCGATATCGTGTACATGAGCCCCTTCTACGGACTGAGAGAGGGAGCTCACGGAGAGCATATCGGATATGCTCCAGGCCGCTTCCAGCAGGTGGAGCACTACTATCCTATCGACCTGGATTCTAGGCTGCGGGCCTCCGAGAGCGTGACAAGGAACTTCCTGCGGACACCACACTTCACCGTGGCCTGGGACTGGGCTCCCAAGACCAGACGCGTGTGCTCCCTGGCCAAGTGGAGAGAGGCTGAGGAGATGATCAGAGACGAGACACGCGATGGCAGCTTCAGGTTTACCTCTCGGGCTCTGGGAGCCTCCTTCGTGTCCGACGTGACACAGCTGGATCTGCAGAGAGTGCACCTGGGCGACTGCGTGCTGAGGGAGGCTTCTGAGGCCATCGATGCTATCTACCAGAGGCGGTATAACAATACCCATGTGCTGGCCGGCGATAGACCAGAGGTGTACCTGGCTAGGGGAGGATTCGTGGTGGCTTTTCGCCCCCTGATCAGCAATGAGCTGGCCCAGCTGTATGCTAGAGAGCTGGAGCGCCTGGGACTGGCTGGAGTGGTGGGACCAGCTTCTCCTGCTGCTGCTAGAAGGGCTAGGAGGGCTGCTGGACAGGCTGGAACACCTGAGCCACCTGCTGTGAACGGAACCGGACACCTGAGAATCACCACAGGATCCGCCGAGTTCGCTAGGCTGCAGTTTACCTACGACCACATCCAGGCCCATGTGAATGATATGCTGGGAAGGATCGCTGCTGCTTGGTGCGAGCTGCAGAACAAGGACAGGACACTGTGGAGCGAGATGTCTCGGCTGAATCCATCCGCCGTGGCTACCGCTGCCCTGGGACAGAGGGTGTCTGCTCGGATGCTGGGCGATGTGATGGCCATCTCCAGATGCGTGGAGGTGCGCGGAGGCGTGTACGTGCAGAACTCCATGAGGGTGCCTGGAGAGAGGGGAACATGTTATAGCAGACCACTGGTGACATTCGAGCACAACGGAACCGGCGTGATCGAGGGACAGCTGGGCGACGATAATGAGCTGCTGATCTCCCGCGACCTGATCGAGCCTTGTACCGGCAACCATAGACGCTACTTTAAGCTGGGCTCCGGCTACGTGTACTATGAGGATTACAGCTATGTGAGAATGGTCGAGGTGCCCGAGACCATCAGCACACGCGTGACCCTGAATCTGACCCTGCTGGAGGACAGGGAGTTCCTGCCTCTGGAGGTGTACACAAGGGAGGAGCTGGCTGACACCGGACTGCTGGATTATTCTGAGATCCAGAGGCGGAACCAGCTGCACGCCCTGAAGTTTTACGACATCGATAGGGTGGTGAAGGTGGATCATAAT
<210>4
<211>2145
<212>DNA
<213> nucleotide sequence of gB protein before codon optimization
<400>2
ATGGTCGCGCTAGCGCTGCTGCTGCTGGCGCTCGCCGCGGCCCCGCCGTGCGGCGCGGCGGCCGTGACGCGGGCCGCCTCGGCCTCGCCGACGCCCGTCCCGGGCAGCCCCGGCCTCACCCCCAACGACGTCTCCGCGGAGGCGTCCCTCGAGGAGATCGAGGCGTTCACCCCCGGCCCCTCGGAGGCCCCCGACGGCGAGTACGGCGACCTGGACGCGCGCACGGCCGTGCGCGCGGCCGCGACCGAGCGGGACCGCTTCTACGTCTGCCCGCCGCCGTCCGGCTCCACGGTGGTGCGCCTGGAGCCCGAGCAGGCCTGCCCCGAGTACTCGCAGGGGCGCAACTTCACGGAGGGGATCGCCGTGCTCTTCAAGGAGAACATCGCCCCGCACAAGTTCAAGGCCCACATCTACTACAAGAACGTCATCGTCACGACCGTGTGGTCCGGGAGCACGTACGCGGCCATCACGAACCGCTTCACGGACCGCGTGCCCGTCCCCGTGCAGGAGATCACGGACGTGATCGACCGCCGCGGCAAGTGCGTCTCCAAGGCCGAGTACGTGCGCAACAACCACAAGGTGACCGCCTTCGACCGCGACGAGAACCCCGTCGAGGTGGACCTGCGCCCCTCGCGCCTGAACGCGCTCGGCACCCGCGGCTGGCACACCACCAACGACACCCACACCAAGATCGGCGCCGCGGGCTTCTACCACACGGGCACCTCCGTCAACTGCATCGTCGAGGAGGTGGAGGCGCGCTCCGTGTACCCCTACGACTCCTTCGCCCTGTCCACGGGGGACATTGTGTACATGTCCCCCTTCTACGGCCTGCGCGAGGGGGCCCACGGGGAGCACATCGGCTACGCGCCCGGGCGCTTCCAGCAGGTGGAGCACTACTACCCCATCGACCTGGACTCGCGCCTCCGCGCCTCCGAGAGCGTGACGCGCAACTTTCTGCGCACGCCGCACTTCACGGTGGCCTGGGACTGGGCCCCCAAGACGCGGCGCGTGTGCAGCCTGGCCAAGTGGCGCGAGGCCGAGGAGATGATCCGCGACGAGACGCGCGACGGGTCCTTCCGCTTCACGTCGCGGGCCCTGGGCGCCTCCTTCGTCAGCGACGTCACGCAGCTGGACCTGCAGCGCGTGCACCTGGGCGACTGCGTCCTCCGCGAGGCCTCGGAGGCCATCGACGCCATCTACCAGCGGCGCTACAACAACACGCACGTGCTGGCCGGCGACAGGCCCGAGGTGTACCTCGCCCGCGGGGGCTTCGTGGTGGCCTTCCGCCCGCTGATCTCGAACGAGCTGGCGCAGCTGTACGCGCGCGAGCTCGAGCGCCTCGGCCTCGCCGGCGTCGTGGGCCCCGCGTCCCCCGCGGCGGCCCGGCGGGCCCGGCGCGCCGCCGGACAGGCGGGGACGCCCGAGCCGCCGGCCGTCAACGGCACGGGGCACCTGCGCATCACCACGGGCTCGGCGGAGTTTGCGCGCCTGCAGTTCACCTACGACCACATCCAGGCGCACGTGAACGACATGCTGGGCCGCATCGCGGCCGCCTGGTGCGAGCTGCAGAACAAGGACCGCACCCTGTGGAGCGAGATGTCGCGCCTGAACCCCAGCGCCGTGGCCACGGCCGCGCTCGGCCAGCGCGTCTCGGCGCGCATGCTCGGCGACGTGATGGCCATCTCGCGGTGCGTGGAGGTGCGCGGCGGCGTGTACGTGCAGAACTCCATGCGCGTGCCCGGCGAGCGCGGCACGTGCTACAGCCGCCCGCTGGTCACCTTCGAGCACAACGGCACGGGCGTGATCGAGGGCCAGCTCGGCGACGACAACGAGCTCCTCATCTCGCGCGACCTCATCGAGCCCTGCACCGGCAACCACCGGCGCTACTTTAAGCTGGGGAGCGGGTACGTGTACTACGAGGACTACAGCTACGTGCGCATGGTGGAGGTGCCCGAGACGATCAGCACGCGGGTGACCCTGAACCTGACGCTGCTGGAGGACCGCGAGTTCCTGCCCCTCGAGGTGTACACGCGCGAGGAGCTCGCCGACACGGGCCTCCTGGACTACAGCGAGATCCAGCGCCGCAACCAGCTGCACGCGCTCAAGTTCTACGACATCGACCGCGTGGTCAAGGTGGACCACAAC
<210>4
<211>715
<212>PRT
<213> amino acid sequence of gB protein
<400>3
MVALALLLLALAAAPPCGAAAVTRAASASPTPVPGSPGLTPNDVSAEASLEEIEAFTPGPSEAPDGEYGDLDARTAVRAAATERDRFYVCPPPSGSTVVRLEPEQACPEYSQGRNFTEGIAVLFKENIAPHKFKAHIYYKNVIVTTVWSGSTYAAITNRFTDRVPVPVQEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDENPVEVDLRPSRLNALGTRGWHTTNDTHTKIGAAGFYHTGTSVNCIVEEVEARSVYPYDSFALSTGDIVYMSPFYGLREGAHGEHIGYAPGRFQQVEHYYPIDLDSRLRASESVTRNFLRTPHFTVAWDWAPKTRRVCSLAKWREAEEMIRDETRDGSFRFTSRALGASFVSDVTQLDLQRVHLGDCVLREASEAIDAIYQRRYNNTHVLAGDRPEVYLARGGFVVAFRPLISNELAQLYARELERLGLAGVVGPASPAAARRARRAAGQAGTPEPPAVNGTGHLRITTGSAEFARLQFTYDHIQAHVNDMLGRIAAAWCELQNKDRTLWSEMSRLNPSAVATAALGQRVSARMLGDVMAISRCVEVRGGVYVQNSMRVPGERGTCYSRPLVTFEHNGTGVIEGQLGDDNELLISRDLIEPCTGNHRRYFKLGSGYVYYEDYSYVRMVEVPETISTRVTLNLTLLEDREFLPLEVYTREELADTGLLDYSEIQRRNQLHALKFYDIDRVVKVDHN
Sequence listing
<110> Zhejiang Hilon Biotechnology Ltd
<120> gB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application thereof
<160> 4
<170> PatentIn version 3.3
<210> 4
<211> 2145
<212> DNA
<213> codon-optimized nucleotide sequence (DNA) of gB protein
<400> 1
ATGGTGGCTCTGGCCCTGCTGCTGCTGGCTCTGGCTGCTGCTCCACCTTGCGGAGCTGCTGCTGTGACCAGGGCTGCTTCCGCCAGCCCAACACCAGTGCCTGGATCCCCAGGACTGACCCCAAACGACGTGTCCGCTGAGGCCAGCCTGGAGGAGATCGAGGCTTTCACCCCAGGACCATCTGAGGCTCCAGACGGAGAGTACGGCGACCTGGATGCTAGGACAGCCGTGCGGGCTGCTGCTACCGAGAGAGATAGGTTCTACGTGTGCCCCCCTCCATCTGGCTCCACAGTGGTGAGGCTGGAGCCTGAGCAGGCTTGTCCAGAGTACTCCCAGGGCCGGAACTTCACCGAGGGCATCGCCGTGCTGTTTAAGGAGAATATCGCCCCCCACAAGTTCAAGGCTCATATCTACTATAAGAACGTGATCGTGACCACAGTGTGGAGCGGCTCTACATACGCTGCCATCACAAATAGGTTCACCGACCGCGTGCCTGTGCCAGTGCAGGAGATCACCGACGTGATCGATAGGCGGGGCAAGTGCGTGTCCAAGGCTGAGTATGTGAGGAACAATCACAAGGTGACAGCCTTCGACCGGGATGAGAACCCCGTGGAGGTGGACCTGAGACCTAGCCGCCTGAACGCTCTGGGAACCAGGGGATGGCACACCACAAATGATACCCATACAAAGATCGGCGCTGCCGGCTTTTACCATACCGGCACAAGCGTGAATTGTATCGTGGAGGAGGTGGAGGCTAGGTCCGTGTACCCTTATGACTCTTTCGCCCTGTCCACCGGCGATATCGTGTACATGAGCCCCTTCTACGGACTGAGAGAGGGAGCTCACGGAGAGCATATCGGATATGCTCCAGGCCGCTTCCAGCAGGTGGAGCACTACTATCCTATCGACCTGGATTCTAGGCTGCGGGCCTCCGAGAGCGTGACAAGGAACTTCCTGCGGACACCACACTTCACCGTGGCCTGGGACTGGGCTCCCAAGACCAGACGCGTGTGCTCCCTGGCCAAGTGGAGAGAGGCTGAGGAGATGATCAGAGACGAGACACGCGATGGCAGCTTCAGGTTTACCTCTCGGGCTCTGGGAGCCTCCTTCGTGTCCGACGTGACACAGCTGGATCTGCAGAGAGTGCACCTGGGCGACTGCGTGCTGAGGGAGGCTTCTGAGGCCATCGATGCTATCTACCAGAGGCGGTATAACAATACCCATGTGCTGGCCGGCGATAGACCAGAGGTGTACCTGGCTAGGGGAGGATTCGTGGTGGCTTTTCGCCCCCTGATCAGCAATGAGCTGGCCCAGCTGTATGCTAGAGAGCTGGAGCGCCTGGGACTGGCTGGAGTGGTGGGACCAGCTTCTCCTGCTGCTGCTAGAAGGGCTAGGAGGGCTGCTGGACAGGCTGGAACACCTGAGCCACCTGCTGTGAACGGAACCGGACACCTGAGAATCACCACAGGATCCGCCGAGTTCGCTAGGCTGCAGTTTACCTACGACCACATCCAGGCCCATGTGAATGATATGCTGGGAAGGATCGCTGCTGCTTGGTGCGAGCTGCAGAACAAGGACAGGACACTGTGGAGCGAGATGTCTCGGCTGAATCCATCCGCCGTGGCTACCGCTGCCCTGGGACAGAGGGTGTCTGCTCGGATGCTGGGCGATGTGATGGCCATCTCCAGATGCGTGGAGGTGCGCGGAGGCGTGTACGTGCAGAACTCCATGAGGGTGCCTGGAGAGAGGGGAACATGTTATAGCAGACCACTGGTGACATTCGAGCACAACGGAACCGGCGTGATCGAGGGACAGCTGGGCGACGATAATGAGCTGCTGATCTCCCGCGACCTGATCGAGCCTTGTACCGGCAACCATAGACGCTACTTTAAGCTGGGCTCCGGCTACGTGTACTATGAGGATTACAGCTATGTGAGAATGGTCGAGGTGCCCGAGACCATCAGCACACGCGTGACCCTGAATCTGACCCTGCTGGAGGACAGGGAGTTCCTGCCTCTGGAGGTGTACACAAGGGAGGAGCTGGCTGACACCGGACTGCTGGATTATTCTGAGATCCAGAGGCGGAACCAGCTGCACGCCCTGAAGTTTTACGACATCGATAGGGTGGTGAAGGTGGATCATAAT
<210> 4
<211> 2145
<212> DNA
<213> nucleotide sequence (DNA) of gB protein before codon optimization
<400> 2
ATGGTCGCGCTAGCGCTGCTGCTGCTGGCGCTCGCCGCGGCCCCGCCGTGCGGCGCGGCGGCCGTGACGCGGGCCGCCTCGGCCTCGCCGACGCCCGTCCCGGGCAGCCCCGGCCTCACCCCCAACGACGTCTCCGCGGAGGCGTCCCTCGAGGAGATCGAGGCGTTCACCCCCGGCCCCTCGGAGGCCCCCGACGGCGAGTACGGCGACCTGGACGCGCGCACGGCCGTGCGCGCGGCCGCGACCGAGCGGGACCGCTTCTACGTCTGCCCGCCGCCGTCCGGCTCCACGGTGGTGCGCCTGGAGCCCGAGCAGGCCTGCCCCGAGTACTCGCAGGGGCGCAACTTCACGGAGGGGATCGCCGTGCTCTTCAAGGAGAACATCGCCCCGCACAAGTTCAAGGCCCACATCTACTACAAGAACGTCATCGTCACGACCGTGTGGTCCGGGAGCACGTACGCGGCCATCACGAACCGCTTCACGGACCGCGTGCCCGTCCCCGTGCAGGAGATCACGGACGTGATCGACCGCCGCGGCAAGTGCGTCTCCAAGGCCGAGTACGTGCGCAACAACCACAAGGTGACCGCCTTCGACCGCGACGAGAACCCCGTCGAGGTGGACCTGCGCCCCTCGCGCCTGAACGCGCTCGGCACCCGCGGCTGGCACACCACCAACGACACCCACACCAAGATCGGCGCCGCGGGCTTCTACCACACGGGCACCTCCGTCAACTGCATCGTCGAGGAGGTGGAGGCGCGCTCCGTGTACCCCTACGACTCCTTCGCCCTGTCCACGGGGGACATTGTGTACATGTCCCCCTTCTACGGCCTGCGCGAGGGGGCCCACGGGGAGCACATCGGCTACGCGCCCGGGCGCTTCCAGCAGGTGGAGCACTACTACCCCATCGACCTGGACTCGCGCCTCCGCGCCTCCGAGAGCGTGACGCGCAACTTTCTGCGCACGCCGCACTTCACGGTGGCCTGGGACTGGGCCCCCAAGACGCGGCGCGTGTGCAGCCTGGCCAAGTGGCGCGAGGCCGAGGAGATGATCCGCGACGAGACGCGCGACGGGTCCTTCCGCTTCACGTCGCGGGCCCTGGGCGCCTCCTTCGTCAGCGACGTCACGCAGCTGGACCTGCAGCGCGTGCACCTGGGCGACTGCGTCCTCCGCGAGGCCTCGGAGGCCATCGACGCCATCTACCAGCGGCGCTACAACAACACGCACGTGCTGGCCGGCGACAGGCCCGAGGTGTACCTCGCCCGCGGGGGCTTCGTGGTGGCCTTCCGCCCGCTGATCTCGAACGAGCTGGCGCAGCTGTACGCGCGCGAGCTCGAGCGCCTCGGCCTCGCCGGCGTCGTGGGCCCCGCGTCCCCCGCGGCGGCCCGGCGGGCCCGGCGCGCCGCCGGACAGGCGGGGACGCCCGAGCCGCCGGCCGTCAACGGCACGGGGCACCTGCGCATCACCACGGGCTCGGCGGAGTTTGCGCGCCTGCAGTTCACCTACGACCACATCCAGGCGCACGTGAACGACATGCTGGGCCGCATCGCGGCCGCCTGGTGCGAGCTGCAGAACAAGGACCGCACCCTGTGGAGCGAGATGTCGCGCCTGAACCCCAGCGCCGTGGCCACGGCCGCGCTCGGCCAGCGCGTCTCGGCGCGCATGCTCGGCGACGTGATGGCCATCTCGCGGTGCGTGGAGGTGCGCGGCGGCGTGTACGTGCAGAACTCCATGCGCGTGCCCGGCGAGCGCGGCACGTGCTACAGCCGCCCGCTGGTCACCTTCGAGCACAACGGCACGGGCGTGATCGAGGGCCAGCTCGGCGACGACAACGAGCTCCTCATCTCGCGCGACCTCATCGAGCCCTGCACCGGCAACCACCGGCGCTACTTTAAGCTGGGGAGCGGGTACGTGTACTACGAGGACTACAGCTACGTGCGCATGGTGGAGGTGCCCGAGACGATCAGCACGCGGGTGACCCTGAACCTGACGCTGCTGGAGGACCGCGAGTTCCTGCCCCTCGAGGTGTACACGCGCGAGGAGCTCGCCGACACGGGCCTCCTGGACTACAGCGAGATCCAGCGCCGCAACCAGCTGCACGCGCTCAAGTTCTACGACATCGACCGCGTGGTCAAGGTGGACCACAAC
<210> 4
<211> 715
<212> PRT
<213> amino acid sequence (PRT) of gB protein
<400> 3
MVALALLLLALAAAPPCGAAAVTRAASASPTPVPGSPGLTPNDVSAEASLEEIEAFTPGPSEAPDGEYGDLDARTAVRAAATERDRFYVCPPPSGSTVVRLEPEQACPEYSQGRNFTEGIAVLFKENIAPHKFKAHIYYKNVIVTTVWSGSTYAAITNRFTDRVPVPVQEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDENPVEVDLRPSRLNALGTRGWHTTNDTHTKIGAAGFYHTGTSVNCIVEEVEARSVYPYDSFALSTGDIVYMSPFYGLREGAHGEHIGYAPGRFQQVEHYYPIDLDSRLRASESVTRNFLRTPHFTVAWDWAPKTRRVCSLAKWREAEEMIRDETRDGSFRFTSRALGASFVSDVTQLDLQRVHLGDCVLREASEAIDAIYQRRYNNTHVLAGDRPEVYLARGGFVVAFRPLISNELAQLYARELERLGLAGVVGPASPAAARRARRAAGQAGTPEPPAVNGTGHLRITTGSAEFARLQFTYDHIQAHVNDMLGRIAAAWCELQNKDRTLWSEMSRLNPSAVATAALGQRVSARMLGDVMAISRCVEVRGGVYVQNSMRVPGERGTCYSRPLVTFEHNGTGVIEGQLGDDNELLISRDLIEPCTGNHRRYFKLGSGYVYYEDYSYVRMVEVPETISTRVTLNLTLLEDREFLPLEVYTREELADTGLLDYSEIQRRNQLHALKFYDIDRVVKVDHN
Claims (10)
1. A gB subunit recombinant protein of porcine pseudorabies virus is characterized in that the gB subunit recombinant protein is an extracellular region of gB protein, and the amino acid sequence of the gB subunit recombinant protein is shown as SEQ ID NO. 3.
2. The porcine pseudorabies virus gB subunit recombinant protein according to claim 1, wherein the gene sequence encoded by the gB subunit recombinant protein is shown in SEQ ID No. 2.
3. The gB subunit recombinant protein of claim 1, wherein the gB subunit recombinant protein encodes a gene sequence capable of efficiently secreting and expressing an optimized gB gene sequence of the gB subunit protein in CHO cells, and the optimized gB gene sequence is shown in SEQ ID No. 1.
4. The gB subunit recombinant protein according to claim 1, wherein the gB subunit recombinant protein is linked to a detection or purification tag at the amino terminus or the carboxy terminus of the amino acid sequence shown in SEQ ID No.3, wherein the tag is selected from one of ploy-Arg, ploy-His, FLAG, c-myc, and HA.
5. A method for preparing the gB subunit recombinant protein of porcine pseudorabies virus according to any one of claims 1 to 4, wherein the method comprises the following steps:
1) cloning the optimized gB gene sequence shown as SEQ ID NO.1 into a eukaryotic expression vector to obtain a recombinant plasmid containing a gB subunit protein coding gene of the pseudorabies virus;
2) then the recombinant plasmid containing the gB subunit protein coding gene of the pseudorabies virus is transfected into an expression cell;
3) culturing, screening and domesticating the expression cells in the step 2) to obtain a highly expressed cell strain;
4) fermenting and culturing the cell strain in the step 3), and purifying to obtain gB subunit protein of the pseudorabies virus, wherein the amino acid sequence of the gB subunit protein is shown as SEQ ID NO. 3.
6. The method of claim 5, wherein in step 1), the eukaryotic expression vector is pEE12.4 or pcDNA3.1.
7. The method according to claim 5, wherein in step 2), the expression cells are mammalian cells.
8. The method of claim 7, wherein the mammalian cell is a CHO cell or a 293T cell.
9. The method of claim 8, wherein the CHO cell comprises DG44, DXB11, CHO-K1, a CHO-S cell strain.
10. Use of a recombinant protein of the gB subunit of porcine pseudorabies virus according to any one of claims 1 to 4 for the preparation of a vaccine for the diagnosis, prevention and treatment of porcine pseudorabies.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113173976A (en) * | 2021-06-03 | 2021-07-27 | 北京市农林科学院 | Canine influenza virus H3 subtype HA protein, and preparation method and application thereof |
| CN114908056A (en) * | 2022-05-18 | 2022-08-16 | 华中农业大学 | Recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105693827A (en) * | 2015-06-29 | 2016-06-22 | 普莱柯生物工程股份有限公司 | Porcine pseudorabies virus subunit vaccine and preparation method and application thereof |
| CN106267182A (en) * | 2015-06-29 | 2017-01-04 | 普莱柯生物工程股份有限公司 | The preparation method of a kind of porcine pseudorabies virus subunit vaccine and vaccine combination and application |
| CN107485712A (en) * | 2017-08-09 | 2017-12-19 | 扬州优邦生物药品有限公司 | A kind of PRV subunit vaccine and its preparation method and application |
| CN109206491A (en) * | 2017-07-07 | 2019-01-15 | 浙江海隆生物科技有限公司 | Preparation method of porcine pseudorabies virus gD protein, porcine pseudorabies virus subunit vaccine and application |
-
2019
- 2019-06-28 CN CN201910577700.XA patent/CN112142827B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105693827A (en) * | 2015-06-29 | 2016-06-22 | 普莱柯生物工程股份有限公司 | Porcine pseudorabies virus subunit vaccine and preparation method and application thereof |
| CN106267182A (en) * | 2015-06-29 | 2017-01-04 | 普莱柯生物工程股份有限公司 | The preparation method of a kind of porcine pseudorabies virus subunit vaccine and vaccine combination and application |
| CN109206491A (en) * | 2017-07-07 | 2019-01-15 | 浙江海隆生物科技有限公司 | Preparation method of porcine pseudorabies virus gD protein, porcine pseudorabies virus subunit vaccine and application |
| CN107485712A (en) * | 2017-08-09 | 2017-12-19 | 扬州优邦生物药品有限公司 | A kind of PRV subunit vaccine and its preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| BO YEON KIM等: "Production of Aujeszky"s disease (pseudorabies) virus envelope glycoproteins gB and gC as recombinant polyhedra in baculovirus-infected silkworm larvae", 《JOURNAL OF ASIA-PACIFIC ENTOMOLOGY》 * |
| MARÍA SOLEDAD SERENA等: "Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 王雨等: "伪狂犬病病毒gB基因的原核表达及间接ELISA方法的建立", 《动物医学进展》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113173976A (en) * | 2021-06-03 | 2021-07-27 | 北京市农林科学院 | Canine influenza virus H3 subtype HA protein, and preparation method and application thereof |
| CN113173976B (en) * | 2021-06-03 | 2022-07-12 | 北京市农林科学院 | Canine influenza virus H3 subtype HA protein, and preparation method and application thereof |
| CN114908056A (en) * | 2022-05-18 | 2022-08-16 | 华中农业大学 | Recombinant CHO cell line for expressing porcine PRV gDFc or gBFc fusion protein and construction method and application thereof |
| CN114908056B (en) * | 2022-05-18 | 2024-04-16 | 华中农业大学 | Recombinant CHO cell line expressing pig PRV gDFc or gBFc fusion protein and construction method and application thereof |
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