A subunit F protein of bovine parainfluenza virus, a preparation method thereof, and applications thereof

Abstract

The present invention relates to a subunit F protein of bovine parainfluenza virus, a preparation method thereof and applications. The amino acid sequence of the subunit F protein is as follows: 1) the amino acid sequence shown in SEQ ID NO.2; 2) an immunogenic derivative amino acid sequence obtained by substituting, deleting or adding one or several amino acids to SEQ ID NO.2. The subunit F protein of the present invention is mainly prepared by constructing a recombinant plasmid, transfecting the recombinant plasmid into a cell line, screening a cell line with high expression, and purifying the subunit F protein of bovine parainfluenza virus. It can be preferably applied to subunit vaccines or diagnostic reagents for bovine parainfluenza virus, and has the characteristics of high secretion expression efficiency, high protein purity, easy purification, reduced production cost, high safety performance, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal vaccines and veterinary biologics, and particularly relates to the subunit F protein of bovine parainfluenza virus, its preparation method and application. Background Art

[0002] Bovine parainfluenza virus type 3 (BPIV3) is a highly contagious viral disease. It can cause cattle to have fever, cough, runny nose, decreased mental appetite, etc., resulting in a decline in productivity. It often co-infects with bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus, Pasteurella multocida in cattle, etc., causing calf respiratory syndrome. This disease is prevalent globally, bringing huge economic burdens and production losses to the cattle industry and seriously affecting the development of the cattle industry.

[0003] At present, there is no effective treatment for this disease, and prevention and control mainly rely on vaccines. There is no vaccine on the market in China. There are traditional inactivated vaccines and attenuated live vaccines for bovine parainfluenza abroad, but there are certain limitations in terms of immune effect and safety. In order to control the infection of bovine parainfluenza virus, new safe and effective vaccines need to be developed.

[0004] BPIV3 virus belongs to the family Paramyxoviridae. The virus particles are polymorphic, ranging from spherical to filamentous structures, with a diameter of 150 - 300 nm. The BPIV3 genome encodes 6 major structural proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large polymerase protein (L) protein. Among them, F protein and HN protein are the main proteins of the virus envelope, which can stimulate the body to produce neutralizing antibodies and are the first glycoproteins for subunit vaccines. The F protein subunit vaccine does not contain nucleic acid substances and will not produce persistent infection or latent infection after inoculation; the immune response produced can be distinguished from wild virus infection, which is conducive to the control and elimination of the disease. However, the F subunit vaccine also has obvious defects: the expression yield is relatively low, the pre-fusion conformation is unstable, the production cost is high, and its application is limited.

[0005] The cost of subunit vaccines is mainly in the production of subunit proteins. The F protein is a glycosylated modified trimeric protein that is essential for bovine parainfluenza virus to enter cells. It generally consists of 540 amino acids and is called protein F0 before cleavage. In order to obtain the biologically active subunit F protein, F0 needs to be hydrolyzed by cellular proteases into a polypeptide F1 and F2 connected by disulfide bonds. Therefore, in order to ensure that the expressed protein can have glycosylation modification and form a complete and active F protein, it must be achieved in animal cells.

[0006] Engineered cells are currently widely used expression cells in biopharmaceutical engineering. The proteins expressed in this system are closest to natural protein molecules in terms of biological functions such as molecular structure, physical and chemical properties, and post-transcriptional modification. Moreover, they can be cultured to a high density in suspension culture, and the culture volume can reach more than 2,000 L, enabling large-scale production.

[0007] Research has shown that the F protein has two conformations, pre-fusion and post-fusion. The pre-fusion conformation contains a large number of epitopes that induce neutralizing antibodies, while the post-fusion conformation has fewer neutralizing epitopes. However, when using engineered cells to express the F protein, when the coding gene sequence of the F protein is not mutated and optimized, the basic expression level of the F protein in engineered cells is low, and mostly post-fusion proteins are obtained. As a result, it is difficult to obtain an F protein with excellent immunogenicity and stability for the prevention and control of bovine parainfluenza virus. Therefore, when using engineered cells to express the F protein, optimizing the mutated coding gene sequence of the F protein is a necessary process.

[0008] Chinese patent application CN202011013669.6 discloses a bovine parainfluenza recombinant antigen, whose antigen is a heterodimer formed by a truncated F protein and an HN protein, that is, bovine antibody Fc fragments are added to the C-terminals of both the F protein fragment and the HN protein fragment, so that a more stable heterodimer can be formed when the two protein fragments are expressed in the same cell simultaneously. However, a very small fragment of the F protein is used, which does not have the pre-fusion conformation and no separate F protein is obtained. Summary of the Invention

[0009] Aiming at the deficiencies of the existing technology, the first object of the present invention is to provide a subunit F protein of bovine parainfluenza virus, which, based on the original F protein, has excellent immunogenicity and stability of the F protein of bovine parainfluenza virus, and is also convenient for stable and efficient secretion and expression in CHO cell lines.

[0010] The second object of the present invention is to provide a preparation method of the subunit F protein of bovine parainfluenza virus, which is convenient for large-scale industrial production of the subunit F protein and reduces the production cost of the F protein.

[0011] The third object of the present invention is to provide an application of the subunit F protein of bovine parainfluenza virus, which can be preferably applied to subunit vaccines and diagnostic reagents for bovine parainfluenza virus, thus facilitating people's prevention and control of bovine parainfluenza virus.

[0012] To achieve the above first object, the present invention provides a subunit F protein of bovine parainfluenza virus, and the amino acid sequence of the subunit F protein is:

[0013] 1) The amino acid sequence shown in SEQ ID NO.2;

[0014] 2) An immunogenic derivative amino acid sequence obtained by substituting, deleting or adding one or several amino acids to SEQ ID NO.2.

[0015] The subunit F protein of bovine parainfluenza virus according to the present invention, preferably, a trimeric motif GCN4 protein is further linked to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.2, the amino acid sequence of the trimeric motif GCN4 protein is shown in SEQ ID NO.7, to obtain a fusion protein I of the subunit F protein and the trimeric motif GCN4 protein, and the amino acid sequence of the fusion protein I is shown in SEQ ID NO.3.

[0016] The subunit F protein of bovine parainfluenza virus according to the present invention, preferably, the amino acid sequence of the subunit F protein is mutated by Q162C, L168C, I213C, G230C, A463V, I474Y to obtain a mutant of the subunit F protein, and the amino acid sequence of the mutant of the subunit F protein is shown in SEQ ID NO.4.

[0017] The subunit F protein of bovine parainfluenza virus according to the present invention, preferably, a trimeric motif GCN4 protein is further linked to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.4, the amino acid sequence of the trimeric motif GCN4 protein is shown in SEQ ID NO.7, to obtain a fusion protein II of the mutant of the subunit F protein and the trimeric motif GCN4 protein, and the amino acid sequence of the fusion protein II is shown in SEQ ID NO.5.

[0018] The subunit F protein of bovine parainfluenza virus according to the present invention, preferably, one of the tags poly-His, FLAG, c-myc, HA, poly-Arg is linked to the amino terminus or carboxyl terminus of the amino acid sequence of the fusion protein II.

[0019] The subunit F protein of bovine parainfluenza virus according to the present invention, preferably, the tag is poly-His, to obtain the subunit F protein of bovine parainfluenza virus, and its amino acid sequence is shown in SEQ ID NO.6.

[0020] The subunit F protein of bovine parainfluenza virus according to the present invention, preferably, the coding gene sequence of the subunit F protein is shown in SEQ ID NO.8, and codon optimization is carried out on the basis of the coding gene sequence of the subunit F protein shown in SEQ ID NO.8 to obtain the OPTI-F sequence shown in SEQ ID NO.9.

[0021] To achieve the second above-mentioned purpose, the present invention provides a method for preparing the subunit F protein of bovine parainfluenza virus, and the preparation method includes the following steps:

[0022] 1) Construct the coding gene sequence of the subunit F protein of bovine parainfluenza virus as shown in SEQ ID NO.9;

[0023] 2) Clone the coding gene sequence of the subunit F protein constructed in step 1) into a eukaryotic expression vector to obtain a recombinant plasmid containing the coding gene sequence of the subunit F protein;

[0024] 3) Transfect the recombinant plasmid containing the coding gene sequence of the subunit F protein obtained in step 2) into engineered cells of an animal to obtain a cell line;

[0025] 4) Screen out a cell line with high expression from the cell line obtained in step 3); and

[0026] 5) Ferment and culture the cell line with high expression obtained in step 4), and purify to obtain the subunit F protein of bovine parainfluenza virus.

[0027] In the preparation method of the present invention, preferably, in step 2), the eukaryotic expression vector is one of pEE6.4, pEE12.4, pGL4.13 and pcDNA3.1.

[0028] In the preparation method of the present invention, preferably, in step 2), the eukaryotic expression vector is pEE12.4.

[0029] In the preparation method of the present invention, preferably, in step 3), the cell line is one of CHO cell line, HEK293 cell line and 293T / 17 cell line.

[0030] In the preparation method of the present invention, preferably, in step 3), the CHO cell line is one of DG44 cell line, DXB11 cell line, CHO-K1 cell line and CHO-S cell line.

[0031] To achieve the third above-mentioned purpose, the present invention provides an application of the subunit F protein of bovine parainfluenza virus in a subunit vaccine or diagnostic reagent for bovine parainfluenza virus.

[0032] The present invention provides a first subunit F protein, which has excellent immunogenicity and stability of bovine parainfluenza virus F protein, and is convenient for stable and efficient secretion and expression in engineered cell lines. It has high yield and is easy to purify. The purity of the target protein in the cell culture supernatant can reach more than 70%, and the purity of the target protein can reach more than 90% only by one-step affinity chromatography, far meeting the requirements of subunit vaccines and diagnostic reagents. At the same time, it is convenient for large-scale production. Thus, the technical problem of high production cost of the subunit F protein of bovine parainfluenza virus is solved. In addition, since engineered cell lines such as CHO cell line, HEK293 cell line, and 293T / 17 cell line used for production have high controllability, easy quality control, and stable protein production between batches, the amount of other viruses in the subunit F protein of bovine parainfluenza virus produced by the present invention is small, effectively reducing the risk of virus dispersion and having excellent biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It shows a three-dimensional structural model diagram of the pre-fusion subunit F protein;

[0034] Figure 2 It shows the comparison results of the subunit F gene sequence before and after optimization;

[0035] Figure 3 It shows the plasmid map of pEE12.4-OPTI-F;

[0036] Figure 4 It shows the double digestion identification result of pEE12.4-OPTI-F: M is DNA Marker: DL10000 Marker; 1 is the double digestion electrophoresis result of pEE12.4-OPTI-F;

[0037] Figure 5 It shows the SDS-PAGE detection result after purification of the subunit F protein, where 1 is the subunit F protein and M is the Marker;

[0038] Figure 6 It shows the Western blot detection result after purification of the subunit F protein, where 1 is the subunit F protein and M is the Marker;

[0039] Figure 7 It shows the stability detection result after purification of the subunit F protein, where 1 is the Marker and 2 is the subunit F protein after treatment at 4°C;

[0040] Figure 8 It shows the stability detection result after purification of the subunit F protein, where 1 is the Marker and 2 is the subunit F protein after treatment at -20°C. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and do not limit the present invention.

[0042] The strains, plasmids and reagents used in the embodiments of the present invention are all commercially available products.

[0043] The sources of the reagents and drugs of the present invention are listed as follows:

[0044] CHO-K1 cells are derived from the Cell Bank of the Chinese Academy of Sciences' Type Culture Collection Committee, Shanghai Institute of Life Sciences, Chinese Academy of Sciences;

[0045] Cell culture medium and serum are both purchased from Gibco, USA;

[0046] The eukaryotic expression vector pEE12.4 is purchased from Shanghai Linyuan Biotechnology Co., Ltd.;

[0047] Lipofectamine LTX is purchased from Thermo Fisher, USA;

[0048] L-methioninesulfoximine (MSX) is purchased from Sigma;

[0049] BCA Protein Quantification Kit is purchased from Thermo Fisher, USA;

[0050] PLUS TM reagent is purchased from Thermo and is an additive for Lipofectamine LTX transfection reagent;

[0051] CB5 is purchased from Thermo and is a feed for fermentation medium.

[0052] Example 1: 1a: Analyze and optimize the structure of the bovine parainfluenza virus F protein, and construct the coding gene sequence of the subunit F protein of the bovine parainfluenza virus.

[0053] By analyzing the F protein sequence of the bovine parainfluenza virus (GenBank: OR855359.1), it can be known that the genomic sequence 5090-6712 encodes the bovine parainfluenza F protein sequence. Further analysis shows that 1M-18C may be the secretion signal peptide of the F protein, 19Q-493T may be the extracellular region of the F protein, 494I-516F may be the transmembrane region of the F protein, and 517K-540Q may be the intracellular region protein of the F protein. In addition, it is found through analysis that the cleavage site of the F protein is between 109R-110F, that is, 19Q-109R is the F2 protein, and 110F-493T is the F1 protein.

[0054] Combining the experience of previous studies on the expression of viral envelope proteins, the extracellular region of subunit F protein expressed in CHO-K1 cells was selected as the immunogenic protein, namely the amino acid sequence of 1M-481L. Through protein structure prediction and research on the structure of amino acids themselves, we introduced Q162C, L168C, I213C, G230C, A463V, I474Y, and added a trimer motif at the C-terminus to make it easier to form the pre-fusion F trimer. The predicted three-dimensional structure of this amino acid sequence is similar to that of the F proteins of other genera of respiratory viruses. The three-dimensional structure pattern diagram is as shown in Figure 1 shown.

[0055] Among them, based on the amino acid sequence of SEQ ID NO.3, a derivative protein was prepared by substituting, deleting, or adding one or several amino acids, and the homology between this derivative protein and the amino acid sequence of subunit F protein in this example (as shown in SEQ ID NO.4) is as high as 80%-100%, so as to ensure that both have the same immunogenicity. Therefore, this derivative protein also falls within the protection scope of the present invention.

[0056] To facilitate the purification of the subunit F protein, a tag shown in Table 1 can be linked to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.5. In this example, Poly-His is specifically used and linked to the amino terminus of the amino acid sequence shown in SEQ ID NO.5.

[0057] Table 1: Tags and Their Amino Acid Sequences

[0058] Label Sequence Residue Poly-His HHHHHH 6 - 10 (usually 6) FLAG DYKDDDDK 8 c-myc EQKLISEEDL 10 HA YPYDVPDYA 9 Poly-Arg RRRRRR 5 - 6 (usually 6) .

[0059] The coding gene sequence of the amino acid sequence of SEQ ID NO.6 can be as shown in SEQ ID NO.8, or can be obtained by codon optimization of SEQ ID NO.8. In this example, the coding gene sequence of the subunit F protein is codon-optimized based on SEQ ID NO.8 to obtain the OPTI-F sequence, as shown in SEQ ID NO.9, which is the coding gene sequence of the subunit F protein of bovine parainfluenza virus. The artificial synthesis of this gene sequence was entrusted to Nanjing Genscript Biotech Co., Ltd.

[0060] Comparing the codon-optimized sequence with the sequence before codon optimization, the result is as shown in Figure 2 shown, with a total of 386 / 1389 = 27.8% differences.

[0061] 1b: Construction of pEE12.4-OPTI-F recombinant plasmid; 1b.1. PCR amplification of the target fragment OPTI-F; 1b.1.1. PCR reaction

[0062] (1) Primer design and synthesis

[0063] Forward primer:

[0064] 5’-acgaAGCTTGCCGCCACCATGATCACTATC-3’

[0065] Reverse primer:

[0066] 5’- ATTGAATTCtcaATGATGGTGGTGGTGGT-3’

[0067] (2) A 50 μL loading system is shown in Table 2 below.

[0068] Table 2: 50 μL loading system

[0069] Sample Components Volume (μL) Q5 Mix 25 Forward Primer (10 μM) 2.5 Reverse Primer (10 μM) 2.5 OPTI-F 1 <![CDATA[dd H 2 O]]> 19 Total Volume 50 .

[0070] PCR amplification program:

[0071] 95℃ 2 min

[0072] 95℃ 30 s

[0073] 55℃ 45 s

[0074] 72℃ 1 min 30 s

[0075] 72℃ 10 min

[0076] 8℃ forever

[0077] Among them: One cycle is completed in sequence from 95℃ 30 s, 55℃ 45 s, 72℃ 1 min 30 s, and 72℃ 10 min, and this cycle is repeated 30 times.

[0078] 1b.1.2, Gel extraction of PCR products

[0079] (1) Label the sample collection EP tube, adsorption column CB2, and collection tube;

[0080] (2) Weigh the labeled empty EP tube and record the value;

[0081] (3) Carefully cut the single target DNA band from the agarose gel with a scalpel on the gel cutter and put it into a clean 1.5 mL centrifuge tube;

[0082] (4)Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3), place it in a water bath at 50 °C for about 5 min, and gently invert the centrifuge tube up and down continuously during this period to ensure that the gel block is fully dissolved;

[0083] (5)Column equilibration: Add 500 μL of equilibration buffer BL to adsorption column CB2 (adsorption column CB2 is pre-placed in the collection tube), centrifuge at 12000 rpm / min for 1 min, pour out the waste liquid in the collection tube, and place adsorption column CB2 back into the collection tube;

[0084] (6)Add the solution obtained in step (5) to adsorption column CB2, let it stand for 2 min, centrifuge at 10000 rpm / min for 30 s, pour out the waste liquid in the collection tube, and then place adsorption column CB2 into the collection tube;

[0085] (7)Add 600 μL of wash buffer PW buffer to adsorption column CB2, let it stand for 3 min, centrifuge at 10000 rpm / min for 30 s, pour out the waste liquid in the collection tube, and place adsorption column CB2 into the collection tube;

[0086] (8)Repeat step (7);

[0087] (9)Centrifuge the empty adsorption column at 12000 rpm / min for 2 min to remove the wash buffer as much as possible, place adsorption column CB2 at room temperature for 10 min to dry completely;

[0088] (10)Place adsorption column CB2 into the collection tube, suspend and add 50 μL of Elution buffer (preheated at 65 °C) to the middle position of the adsorption membrane, let it stand for 3 min, and centrifuge at 12000 rpm / min for 2 min;

[0089] (11)Take out the centrifuge tube in step (10) from the centrifuge, discard the middle adsorption column CB2, cover the centrifuge tube lid, and retain the DNA sample in the centrifuge tube;

[0090] (12)Store the DNA sample in step 11 at 4 °C and prepare to identify the gel-extracted DNA fragment by agarose gel electrophoresis.

[0091] 1b.2. Double digestion reaction of PCR product and vector

[0092] (1)Label the 1.5 mL EP tubes to be used, add samples and mix them according to Table 3 below in this EP tube. The DNA sample in Table 3 is the DNA fragment finally recovered in step 1b.1.2 (12);

[0093] Table 3: 50 μL reaction system

[0094] Sample Component Name Volume (μL) <![CDATA[dd H 2 0]]> Make up to 50 10× buffer 5 DNA Sample Volume at 2 μg HindⅢ 2.5 EcoRⅠ 2.5

[0095] (2) Place the EP tube in step (1) in a 37°C constant temperature water bath and water bath for 2 - 3 h;

[0096] (3) Gel recovery of the double digestion product: Take out the above double digestion system, perform agarose gel electrophoresis to recover the DNA fragments therein, and the method is the same as the PCR product gel recovery in step 1b.1.2.

[0097] 1b.3. Ligation reaction

[0098] (1) Prepare several clean 1.5 mL EP tubes, make marks, and place them on the EP tube rack for later use;

[0099] (2) Add samples and mix them in the EP tubes in step (1) according to Table 4 below. The target fragment in Table 4 is the DNA fragment finally recovered in step 1b.2 (2);

[0100] Table 4: 10 μL reaction system

[0101] Sample Component Name Experimental Group (μL) Blank Group (μL) <![CDATA[dd H 2 0]]> / 6 10× T4 Ligation buffer 1 1 Target Fragment 6 - Vector 2 2 T4 Ligase 1 1

[0102] (3) After completing the sample addition according to the table in step (2), place each 10 μL reaction system in a 16°C low-temperature coolant circulation machine and water bath for 10 - 16 h;

[0103] (4) Take out the EP tubes in step (3), place them in a 65°C water bath, and water bath for 15 min;

[0104] (5) Take out the EP tubes in step (4), place them at 4°C for storage to obtain the ligation reaction solution.

[0105] 1b.4. Transformation reaction

[0106] (1) Quickly add the 10 μL ligation reaction solution prepared in step 1b.3 (5) to 100 μL of competent cells, pipette and mix well, and ice bath for 30 min;

[0107] (2) Take out the sample tube, place it in a 42°C water bath for 100 s, and then immediately ice bath for 2 min;

[0108] (3) Take out the sample tube, in a laminar flow hood, add 600 μL of liquid LB medium to the sample tube, and then place the sample tube in a 37°C constant temperature shaker at 220 rpm / min for 1 h;

[0109] (4) Plate coating: Take out the sample tube in step (3), centrifuge at 8000 rpm / min at room temperature for 2 min, remove 600 μL of the supernatant, resuspend the cells at the bottom of the tube with the remaining supernatant, place the resuspended bacterial solution in the center of the corresponding transformation plate, and evenly spread the bacterial solution in the center of the transformation plate with a bacterium spreading rod;

[0110] (5) Place the transformation plate obtained in step (4) upright in a biochemical constant temperature incubator, incubate at 37 °C for 1 h, then invert the transformation plate and incubate for 15 h to obtain monoclonal strains.

[0111] 1b.5. Plasmid extraction and double digestion identification; 1b.5.1. Plasmid extraction

[0112] (1) Use a 10 μL pipette tip to pick a monoclonal strain from the transformation plate in step 1b.4(5) into 5 mL of liquid LB medium containing ampicillin resistance, and shake the bacteria overnight at 37 °C and 220 rpm / min;

[0113] (2) Transfer the bacterial solution to a 1.5 mL EP tube, centrifuge at 12000 rpm / min at room temperature for 2 min, and discard the supernatant;

[0114] (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) and thoroughly suspend the cells;

[0115] (4) Add 250 μL of plasmid extraction reagent P2 buffer to the solution in step (3), immediately gently invert the centrifuge tube 5 - 10 times to mix, and let it stand at room temperature for 2 - 4 min;

[0116] (5) Add 350 μL of plasmid extraction reagent P3 buffer to the solution in step (4), immediately gently invert the centrifuge tube 5 - 10 times to mix; let it stand at room temperature for 2 - 4 min;

[0117] (6) Centrifuge the solution in step (5) at 14000 rpm / min at room temperature for 10 min;

[0118] (7) Transfer the supernatant solution in step (6) to the center of the adsorption column, centrifuge at 12000 rpm / min at room temperature for 30 s, and pour out the liquid in the collection tube;

[0119] (8) Add 500 μL of Buffer DW1 to the center of the adsorption column, centrifuge at 12000 rpm / min at room temperature for 30 s, and pour out the liquid in the collection tube;

[0120] (9) Add 500 μL of wash solution to the center of the adsorption column, centrifuge at room temperature at 12,000 rpm for 30 s, pour out the liquid in the collection tube, and repeat once;

[0121] (10) Empty the adsorption column and centrifuge at room temperature at 12,000 rpm for 2 min;

[0122] (11) Place the adsorption column into a clean 1.5 mL centrifuge tube, add 30 μL of Elution buffer to the center of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at room temperature at 12,000 rpm for 2 min, and save the DNA solution in the tube.

[0123] Double digestion identification

[0124] (1) Label the required 1.5 mL EP tubes, add samples according to Table 5 below, where the DNA sample in Table 5 is the DNA solution finally obtained in step 1b.5.1(11);

[0125] Table 5: 20 μL reaction system

[0126] Sample Component Name Volume (μL) <![CDATA[dd H 2 0]]> Make up to 20 μL 10× buffer 2 DNA Sample Volume at 1 μg mass HindⅢ 1 EcoRⅠ 1

[0127] (2) Place the 20 μL reaction system in the EP tube in step (1) in a 37 °C constant temperature water bath for 2 h;

[0128] (3) Perform agarose gel electrophoresis on the double digestion system sample in step (2) to check whether the size of the inserted fragment is correct; the experimental results are shown in Figure 4 : The digestion identification is correctly constructed;

[0129] (4) Select the recombinant plasmid with the correct inserted fragment and send it to a sequencing company for sequencing. In the present invention, this recombinant plasmid was sent to Genewiz Biotechnology Co., Ltd. for determination, and the encoded gene sequence of the subunit F protein was as shown in SEQ ID NO.9.

[0130] 1b.6. Large-scale extraction of endotoxin-free plasmid; 1b.6.1. Extraction of endotoxin-free plasmid

[0131] (1) Inoculate the clone with correct sequencing in step 1b.5.2(4) into 100 mL of medium containing ampicillin resistance, and culture it in a 37 °C constant temperature shaker at 220 rpm for 15 h;

[0132] (2) Transfer the bacterial liquid cultured in step (1) to a 50 mL centrifuge tube, centrifuge at room temperature at 8,000 rpm for 5 min, collect the bacterial cells, and discard the supernatant medium;

[0133] (3) Add 8 mL of plasmid extraction reagent P1 buffer to the centrifuge tube in step (2), and resuspend the bacterial cells thoroughly with a pipette.

[0134] (4) Add 8 mL of plasmid extraction reagent P2 buffer to the centrifuge tube in step (3), immediately invert the centrifuge tube gently 6 - 8 times, and let it stand at room temperature for 5 min.

[0135] (5) Add 8 mL of plasmid extraction reagent P4 buffer to the centrifuge tube in step (4), immediately invert it up and down 6 - 8 times to mix thoroughly until white flocculent precipitates appear in the solution, let it stand at room temperature for about 10 min, and centrifuge at room temperature at 8000 rpm / min for 5 - 10 min to make the white precipitate settle to the bottom of the tube.

[0136] (6) Carefully transfer all the supernatant in step (5) into the filter CS1, slowly push the handle of the filter, and collect the filtrate in a clean 50 mL centrifuge tube.

[0137] (7) Column equilibration: Add 2.5 mL of equilibration buffer BL to the adsorption column CP6 (the adsorption column CP6 is placed in a 50 mL collection tube), centrifuge at room temperature at 8000 rpm / min for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0138] (8) Add isopropanol with a volume 0.3 times that of the filtrate in step (6) to the filtrate, mix it by inverting up and down, and transfer it to the adsorption column CP6. Centrifuge at room temperature at 8000 rpm / min for 2 min, pour out the liquid in the collection tube, and put the adsorption column CP6 back into the same collection tube.

[0139] (9) Add 10 mL of washing buffer PW buffer to the adsorption column CP6 in step (8), centrifuge at room temperature at 8000 rpm / min for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0140] (10) Repeat the operation in step (9) once.

[0141] (11) Add 3 mL of absolute ethanol to the adsorption column CP6 in step (10), centrifuge at room temperature at 8000 rpm / min for 2 min, and pour out the waste liquid.

[0142] (12) Put the adsorption column CP6 in step (11) back into the collection tube, centrifuge at room temperature at 8000 rpm / min for 5 min, open the lid of the adsorption column CP6, and let it stand at room temperature for several minutes to dry.

[0143] (13) Place the adsorption column in a clean 50 mL centrifuge tube, add 1 - 2 mL of TB buffer to the center of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 8000 rpm / min for 2 min at room temperature, transfer all the eluate in the 50 mL centrifuge tube to a clean 1.5 mL centrifuge tube. The eluate is the DNA solution of the pEE12.4 - OPTI - F recombinant plasmid. The map of this recombinant plasmid is as Figure 3 shown. After measuring its concentration, store it at - 20 °C.

[0144] 1c: Establishment of transfection of pEE12.4 - OPTI - F recombinant plasmid into CHO - K1 cells and monoclonal screening; 1c.1, Transfection of CHO - K1 cells

[0145] (1) Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 min; Preheat DMEM / F12 medium (containing 10 wt% serum, 1 wt% double antibody) and PBS buffer in a 37 °C water bath to 37 °C;

[0146] (2) Take out CHO - K1 cells (10 cm cell culture dish) from the 37 °C CO 2 cell culture incubator, discard the supernatant medium, wash the cells once with 8 mL of pre - warmed PBS buffer, and discard the PBS buffer;

[0147] (3) Add 1 - 2 mL of 0.25 wt% trypsin - EDTA to each 10 cm cell culture dish, digest at room temperature for about 2 min, observe under the microscope that the cells shrink and become round and are single cells;

[0148] (4) Add 4 mL of DMEM / F12 medium (containing 10 wt% serum, 1 wt% double antibody) to terminate the digestion reaction, and disperse the cells with a pipette;

[0149] (5) Transfer the digested cells to a 15 mL centrifuge tube, centrifuge at room temperature, 200 g, for 5 min;

[0150] (6) Resuspend the cells with DMEM / F12 medium (containing 10 wt% serum, 1 wt% double antibody) and count;

[0151] (7) Dilute the cells to 2×10 5 cells / mL, take 2 mL of the mixed cells and add them to a six - well plate. Place the six - well plate in a 37 °C, CO 2 CO with a volume percentage of 5% 2 cell culture incubator and incubate overnight;

[0152] (8) Remove the six-well plate from step (7) and observe the cell status: When the cell confluence reaches 80%-90%, transfection can begin. Before transfection, change the medium to DMEM / F12 medium without antibiotics and serum, 2 mL per well;

[0153] (9) Dilute the recombinant plasmid: Dilute the recombinant plasmid with OPTI-MEM medium. Add 2.5 μg of the recombinant plasmid to every 125 μL of OPTI-MEM medium, and then add 2.5 μL of PLUS TM reagent, mix well, and let stand at room temperature for 5 min;

[0154] (10) Dilute Lipofectamine LTX: Add 9 μL of Lipofectamine LTX to 125 μL of OPTI-MEM medium, and then add 2.5 μL of PLUS TM reagent, gently mix well, and let stand at room temperature for 5 min;

[0155] (11) Gently mix the mixtures from steps (10) and (11), let stand at room temperature for 5 min, and then add dropwise to the six-well plate for even distribution;

[0156] (12) Place the six-well plate in a 37 °C, CO 2 cell culture incubator with 5% CO 2 by volume and culture for 4-6 h;

[0157] (13) Change the medium: Discard the supernatant medium, add 2 mL of DMEM / F12 medium (containing 10 wt% serum and 1 wt% double antibody), and place the six-well plate in a 37 °C, CO 2 cell culture incubator with 5% CO 2 by volume for culture.

[0158] 1c.2. Pressure screening

[0159] (1) Start pressure screening 24 h after transfection: Remove the cells in the six-well plate from the CO 2 cell culture incubator in step 1c.1(11), discard the supernatant medium, add 2 mL of DMEM / F12 (containing 10 wt% serum + 25 μM MSX), perform pressure screening for 7 d, and observe the cells in the middle. If there are many dead cells, change the medium.

[0160] 1c.3. Monoclonal screening

[0161] (1) When all the cells in the negative control die after pressure screening in step 1c.2, which takes about 7 d, start monoclonal screening;

[0162] (2)Take out the six-well plate, discard the culture medium, wash once with PBS buffer, then add 300 µL of 0.25 wt% trypsin-EDTA, digest at room temperature for about 2 min, add 2 mL of DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) to terminate the digestion reaction, and disperse the cells with a pipette;

[0163] (3)Transfer the digested cells to a 15 mL centrifuge tube, centrifuge at room temperature, 200 g, for 5 min;

[0164] (4)Resuspend the cells with DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) and count;

[0165] (5)Plating: Dilute the cells to 5 cells / mL, take 200 µL of the mixed cells and add them to a 96-well plate, place it in a 37 °C, 5% CO 2 5% CO by volume 2 cell incubator and incubate for 4 - 6 h;

[0166] (6)Record the wells with single cells;

[0167] (7)When the wells with single cells in the 96-well plate grow up, discard the culture medium, wash once with PBS buffer, add 100 µL of 0.25 wt% trypsin-EDTA, digest at room temperature for about 2 min, add 2 mL of DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) to terminate the digestion reaction, and disperse the cells with a pipette; Transfer the cell suspension to a 12-well plate. When the 12-well plate is full, take the supernatant and detect whether the clone is positive by ELISA. The positive clones with high expression are continuously expanded and cryopreserved.

[0168] 1 d: CHO-K1 cell line is domesticated into suspension culture

[0169] (1)Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 min; Preheat the DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) in a 37 °C water bath to 37 °C;

[0170] (2)Take out the cells obtained in step 3.3 (7) (10 cm cell culture dish), discard the supernatant culture medium, wash the cells once with 8 mL of pre-warmed PBS buffer, and discard the PBS buffer;

[0171] (3)Add 1 - 2 mL of 0.25 wt% trypsin-EDTA to each 10 cm cell culture dish, digest at room temperature for about 2 min, observe under the microscope that the cells shrink and become round and are single cells;

[0172] (4) Add 4 mL of DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) to terminate the digestion reaction, and disperse the cells with a pipette;

[0173] (5) Transfer the digested cells to a 15 mL centrifuge tube, centrifuge at room temperature at 200 g for 5 min;

[0174] (6) Suspend the cells with 100 wt% DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) and count;

[0175] (7) Dilute the cells to 5×10 5 cells / mL, and inoculate 30 mL of the culture medium containing suspended cells obtained in step (6) into a 125 mL shake flask; Place the cell culture flask on an orbital shaker in a 37 °C, 5% CO 2 CO with a volume percentage of 5% 2 cell incubator and incubate overnight at 120 rpm / min;

[0176] (8) Wipe and disinfect the biosafety workbench with 75% alcohol and irradiate with ultraviolet light for 30 min;

[0177] (9) Count the cell density and viability every 24 h;

[0178] (10) When the cell survival rate reaches 94 - 97% after the first generation of cell culture, perform the second-generation culture;

[0179] (11) Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 min; Place 100 wt% DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) and EX-CELL 302 medium in a 5% CO 2 CO with a volume percentage of 5% 2 cell incubator and preheat to 37 °C;

[0180] (12) Take out the cells from the CO 2 cell incubator in step (11) and transfer them to a 50 mL centrifuge tube, centrifuge at room temperature at 200 g for 5 min;

[0181] (13) Mix the DMEM / F12 medium (containing 10 wt% serum + 25 µM MSX) and EX-CELL 302 medium at a ratio of 1:1, resuspend the cells and count;

[0182] (14) Dilute the cells to 5×10 5 cells / mL, and inoculate 30 mL of the culture medium containing suspended cells obtained in step (13) into a 125 mL shake flask; Place the cell culture flask at 37 °C, CO 2CO with a volume concentration of 5% 2 On the orbital shaker in the cell incubator, incubate overnight at 120 rpm / min;

[0183] (15) Wipe and disinfect the biosafety workbench surface with 75 wt% alcohol and irradiate with ultraviolet light for 30 min;

[0184] (16) Count the cell density and viability every 24 h;

[0185] (17) The cell survival rate obtained after culturing the second generation twice is greater than 95%; after culturing the third to sixth generations three times, the cell survival rate is greater than 95%; after 7 weeks, the cells are inoculated and propagated for three generations 3 days later, and the cell density reaches 1×10 6 cells / mL, and at the same time the cell survival rate reaches 95%, and the cells are considered to have adapted to suspension culture; the cell inoculation density is reduced to 3×10 5 cells / mL;

[0186] (18) After domestication, both the 3C2 cell line and the 2B5 cell line in the CHO-K1 cell line meet the requirements, which indicates that both the 3C2 cell line and the 2B5 cell line have been successfully domesticated.

[0187] 1e: Cell shake flask fermentation

[0188] (1) Preparation of the subculture medium: Place 60 wt% of CD-CHO medium + 40 wt% of Ex-cell 302 medium in a water bath at 37 °C and preheat to 37 °C;

[0189] (2) Take out the 3C2 cell line and 2B5 cell line cultured in suspension in step 1d (17) and count them;

[0190] (3) Dilute the 3C2 cell line and 2B5 cell line in step (2) to 2.5×10 5 cells / mL - 3.5×10 5 cells / mL. Inoculate 30 mL of the subculture medium in step (1) into a 125 mL shake flask for each of the two cell lines. Place the cell culture flask at 37 °C, CO 2 CO with a volume concentration of 5% 2 Incubate overnight in a constant temperature shaker at 100 rpm / min;

[0191] (4) Count the cell density and viability every 24 h, measure glucose, and when the blood glucose is lower than 2 g / L, add glucose to 4 g / L; Take 1 mL of the sample every day, and the supernatant is used to detect the protein expression;

[0192] (5) Feeding (about the fourth day): Supplement 70 g / L of CB5, and the addition amount is 10% of the original medium;

[0193] (6) Culture temperature adjustment (the fifth day): Adjust the temperature of the CO 2 incubator to 32 °C;

[0194] (7) Re-feeding (the ninth day): Supplement CB5 at 70 g / L, and the addition amount is 10% of the original culture medium;

[0195] (8) On the twelfth day, harvest the culture media of 3C2 cells and 2B5 cells respectively.

[0196] 1f: Protein purification

[0197] (1) Collect the culture media of 3C2 cells and 2B5 cells harvested in step 1e (8) (about 100 ml per batch), centrifuge at 4 °C and 8000 g for 30 min, take the supernatant, filter through a 0.8 µm filter membrane, load the sample, reserve 80 μL of the sample and add 20 μL of 5×SDS-sample buffer for SDS-PAGE detection to determine the concentration and purity of the sample before purification;

[0198] (2) Column equilibration: Equilibrate with ultrapure water for 2 - 3 CV (column volume), drain the ethanol preservation solution; then equilibrate with Buffer A (50 mM NaH 2 PO 4 (pH = 7.4), 500 mM NaCl) for 2 - 3 CV, and the equilibration rate is 4 - 7 mL / min;

[0199] (3) Loading: Prepare one 5 mL pre-packed column, load the sample at a flow rate of 1 mL / min (adjust the loading flow rate according to the volume of the pre-packed column), with a retention time of 5 min, collect the Flow through (FT), take 80 μL of the sample and add 20 μL of 5×SDS-sample buffer for SDS-PAGE detection to determine the adsorption effect of the target protein on the pre-packed column;

[0200] (4) Washing: Wash the column with 4 wt% 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 to wash away the unbound proteins and weakly bound impurity proteins until the OD value at 280 nm wavelength reaches a stable baseline;

[0201] (5) Elution: Use 50 wt% Buffer B (20 mM NaH 2 PO 4(pH=7.4), 500mM NaCl, 20mM Mimidazole) to elute the target protein until its OD value at 280nm wavelength is equal to the baseline, the elution rate is 2mL / min, the target protein is collected according to the specification of 10mL / tube, 80μL of sample is taken and added to 20μL of 5×SDS-sample buffer for SDS-PAGE detection to determine the elution effect of the target protein;

[0202] (6) Washing: Use 100wt% Buffer B (20mM NaH 2 PO 4 (pH=7.4), 500mM NaCl, 400mM Mimidazole) at a flow rate of 4mL / min for 2-3 CV until the UV baseline is level; then balance with ultrapure water for 2-3 CV, and preserve the HisTrap excel column with 20% ethanol preservation solution for 2-3 CV to obtain the imidazole eluate containing the target protein;

[0203] (7) Dialysis solution exchange: Pour the imidazole eluate containing the target protein obtained in step (6) into a dialysis bag, dialyze 1000 times with 1× PBS buffer to obtain a dialyzed sample, and take 80 μL of the sample for SDS-PAGE detection to determine the concentration and purity of the purified target protein;

[0204] (8) Sterile filtration: In a biological safety cabinet, the dialyzed sample obtained in step (7) is filtered through a low protein binding syringe filter with a pore size of 0.22 μm. In addition, if the dialyzed sample contains too much protein, it can be filtered using a sterilized Nalgene filter with a 0.22 μm filter membrane. The filtered protein solution sample is stored in a -80°C refrigerator.

[0205] (9) Protein concentration determination: The BCA method was used to determine the concentration of the protein after sterilization and filtration. The concentrations of the purified proteins from the 3C2 cell line and the 2B5 cell line were both 2.5 mg / mL to 2.8 mg / ml, and the volumes of both were approximately 40 ml. After calculation (protein yield = protein concentration * protein volume / fermentation supernatant volume), the protein yields of the 3C2 cell line and the 2B5 cell line were both 1 g / L to 1.12 g / L.

[0206] 1g: Identification of subunit F protein; 1g.1, SDS-PAGE detection

[0207] (1) The protein purified in step 1f was subjected to SDS-PAGE detection. The subunit F protein concentration in the sample used was 2 μg / well. The results were as follows: Figure 5 As shown;

[0208] (2)As can be calculated from the figure, the purity of the purified subunit F protein by SDS-PAGE is 90%, and the molecular weight is approximately 52 KD (F 0 ), 40 KD (F 1 ), among which the band at 40 KD is weaker and the band at 52 KD is stronger.

[0209] 1g.2. Western Blot Detection

[0210] (1)The protein purified in step 1f was subjected to Western Blot detection, and the results are as Figure 6 shown. The concentration of subunit F protein (labeled as 1 in the figure) in the sample used was 2 μg / well; the primary antibody used was from the positive serum of cattle immunized with inactivated bovine parainfluenza vaccine, and the dilution ratio was 1:500; the secondary antibody was HRP-labeled donkey anti-bovine IgG secondary antibody, and the dilution ratio was 1:4000;

[0211] (2)As can be seen from the figure, this serum can specifically bind to the subunit F protein of the present invention. Thus, it can be obtained that the subunit F protein prepared by the present invention has excellent immunogenicity.

[0212] 1g.3. ELISA Detection

[0213] (1)Coating: On the enzyme-linked immunosorbent assay (ELISA) plate, the purified subunit F protein was diluted to 0.5 μg / ml with coating buffer (50 mM carbonate buffer, pH = 9.5). Each antigen was coated in 8 wells (4 wells were added with serum samples and 4 wells were added with blocking solution as a control). 100 μl / well of each antigen was added, sealed with a sealing film, and placed in a refrigerator at 4°C overnight;

[0214] (2)Washing: The ELISA plate from step (1) was taken out of the refrigerator and washed 5 times with PBST buffer;

[0215] (3)Blocking: 200 μl of blocking solution (5 wt% skim milk) was added to each well containing subunit F protein, sealed with a sealing film, and incubated at 37°C for 2 h;

[0216] (4)Serum dilution: The positive serum of cattle immunized with inactivated bovine parainfluenza vaccine was diluted 200 times with blocking solution;

[0217] (5)Washing: The same as (2);

[0218] (6)Sample addition: The diluted serum was added, and at the same time, blocking solution was used as a negative control, and incubated at 37°C for 1 h;

[0219] (7)Washing: The same as (2);

[0220] (8) Adding secondary antibody: Add 100 μl of diluted (dilution ratio 1:4000) HRP-labeled donkey anti-bovine IgG secondary antibody to each well and incubate at 37 °C for 0.5 h;

[0221] (9) Washing: The same as (2);

[0222] (10) Color development: Add 100 μl of TMB color development solution to each well under light-proof conditions and incubate at 37 °C for 10 min;

[0223] (11) Termination: Add 50 μl of termination solution (2M H 2 SO 4 ) to each well to terminate the reaction;

[0224] (12) Detection: Measure the OD value of the sample at a wavelength of 450 nm and analyze the data;

[0225] (13) The results are shown in Table 6 below: The well-coated subunit F protein can specifically bind to the serum, and the average value of OD450 is 1.39; there is no specific binding between the well-coated subunit F protein and the blocking solution, and the average value of OD450 is 0.058. This shows that the subunit F protein can be used as an antigen for the ELISA kit, has excellent immunogenicity, and can be developed into a diagnostic kit for detecting bovine parainfluenza infection and immunity after exploring the appropriate coating concentration and serum dilution ratio.

[0226] Table 6: Identification results of subunit F protein by ELISA

[0227] Sample OD450 Value of Coated Subunit F Protein Serum 1.32 Serum 1.43 Serum 1.35 Serum 1.46 Blocking Solution 0.049 Blocking Solution 0.044 Blocking Solution 0.063 Blocking Solution 0.077 .

[0228] 1g.4 Stability verification

[0229] (1) Dilute the purified subunit F protein in step 1f with PBS buffer to 2 mg / ml, divide it into 20 portions, 0.5 ml for each portion; place 10 portions in a refrigerator at 4 °C, take one sample per week for 10 consecutive times;

[0230] (2) Place the other 10 portions in a refrigerator at -20 °C, take one sample per week for 10 consecutive times; measure the protein concentration with BCA after each sampling, and the results are shown in Table 7 below.

[0231] Table 7: Stability of subunit F protein

[0232] Sample Sample Concentration after 4℃ Treatment (mg / ml) Sample Concentration after -20℃ Treatment (mg / ml) First Sampling 2.08 2.03 Second Sampling 1.97 1.98 Third Sampling 1.98 2.02 Fourth Sampling 2.02 2.97 Fifth Sampling 2.03 2.03 Sixth Sampling 1.99 1.97 Seventh Sampling 1.96 2.01 Eighth Sampling 1.97 1.98 Ninth Sampling 1.95 1.96 Tenth Sampling 1.89 1.96 .

[0233] Referring to Table 7, from the perspective of the change in protein concentration, the protein remained basically stable during the two groups of experiments. To further verify whether the treated protein was degraded, we used the samples from the 10th time for SDS-PAGE detection. The specific results are as Figure 7 and Figure 8 shown. Among them, Figure 7 in 1 is the Marker, and 2 is the subunit F protein after treatment at 4°C. The sample loading amount is 2 μg for both; Figure 8 in 1 is the Marker, and 2 is the subunit F protein after treatment at -20°C. The sample loading amount is also 2 μg for both. From Figure 7 and Figure 8 , it can be seen that the treated samples (sampled for the 10th time) are still stable. Thus, it can be obtained that the subunit F protein prepared by the present invention has excellent stability.

[0234] 1h: Vaccine preparation

[0235] (1) Aqueous phase preparation: According to the content of subunit F protein in the vaccine, use PBS buffer (or physiological saline) to dilute the subunit F protein into several portions with different concentration gradients, such as 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, etc. In this example, the subunit F protein is diluted to 50 μg / mL, which is the aqueous phase;

[0236] (2) Oil phase preparation: According to the total amount of the prepared vaccine, measure an appropriate amount of ISA 201 VG adjuvant according to the weight ratio of antigen phase to adjuvant of 1:1 and the volume ratio of 46:54;

[0237] (3) Emulsification: Preheat both the aqueous phase and the oil phase to 33°C. Slowly add the aqueous phase to the oil phase and stir at 200 - 500 rpm / min for 20 - 30 min. Let it stand at 20°C for 1 h and then place it at 4°C overnight;

[0238] (4) Sub-packaging and storage: Sub-package according to needs and store at 4°C for standby after passing the inspection.

[0239] 1i: Vaccine quality inspection

[0240] (1) Physical properties: Observe the appearance (whether it is a milky white emulsion) by visual inspection;

[0241] (2) Use a clean pipette to suck a small amount of the vaccine and drop it into cold water. Observe (except for the first drop), the vaccine should diffuse in a cloud-like manner, and it is judged as a water-in-oil-in-water dosage form;

[0242] (3) Add 10 ml of the vaccine to a centrifuge tube and centrifuge at 3000 r / min for 15 min. The water phase precipitated at the bottom of the tube should be ≤0.5 mL, and it is judged as stable;

[0243] (4)Use a viscometer to detect the viscosity of the vaccine. If the viscosity is between 20 - 50 cp, it is judged as qualified.

[0244] Example 2: The difference between Example 2 and Example 1 is that in this example, the coding amino acid sequence of the subunit F protein is as shown in SEQ ID NO.1.

[0245] Example 3: The difference between Example 3 and Example 1 is that in this example, the coding amino acid sequence of the subunit F protein is as shown in SEQ ID NO.2.

[0246] Example 4: The difference between Example 4 and Example 1 is that in this example, the coding amino acid sequence of the subunit F protein is as shown in SEQ ID NO.3.

[0247] Example 5: The difference between Example 5 and Example 1 is that in this example, the coding amino acid sequence of the subunit F protein is as shown in SEQ ID NO.4.

[0248] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that in this comparative example, the coding gene sequence of the F protein of bovine parainfluenza virus is the genomic sequence from 5144 - 6568 in GenBank: OR855359.

[0249] Example 6: Determination of the expression yield and protein purity of the subunit F protein: Measure the expression yield and protein purity of the subunit F protein in Examples 2 - 5 and the comparative example. The expression results are shown in Table 8 below.

[0250] Table 8: Yield and purity of the subunit F protein in Examples 2 - 5 and the comparative example

[0251] Item Protein Yield (g / L) Protein Purity (%) Example 2 0.1-0.2 60 Example 3 0.3-0.4 65 Example 4 0.4-0.6 75 Example 5 0.5-0.7 80 Comparative Example 0.0-0.02 21 。

[0252] Referring to Table 8, the protein yield of the present invention is between 0.1 g / L - 1.3 g / L, and the protein purity is higher than 60%, which is significantly better than the protein yield of 0.0 g / L - 0.02 g / L and the protein purity of 21% in the comparative example. Thus, it can be obtained that the coding gene of the subunit F protein constructed by the present invention can efficiently secrete and express the subunit F protein in the cell line, and the obtained subunit F protein has a relatively high protein purity.

[0253] The protein yields of Examples 2 to 5 were 0.1 g / L - 0.7 g / L, and the protein purities were 60% - 80%. The protein yield of Example 1 was 1 g / L - 1.12 g / L, and the protein purity reached 90%. Therefore, Example 1 was the preferred example among Examples 1 to 5. It can be obtained that when the amino acid sequence of the subunit F protein was SEQ ID NO.5, its corresponding protein yield and protein purity reached the optimal values.

[0254] In summary, the recombinant plasmid constructed by the present invention can be effectively expressed in an engineered cell line to obtain a subunit F protein of bovine parainfluenza virus with high yield and high purity. The subunit F protein of bovine parainfluenza virus has good specificity and stability, can be mass-produced, effectively reduces the production cost of the subunit F protein of bovine parainfluenza virus, and can also be better applied to subunit vaccines or diagnostic reagents of bovine parainfluenza virus. Thus, the subunit F protein of the present invention has the characteristics of high secretion expression efficiency, high protein purity, easy purification, low production cost, and high safety performance.

[0255] The present invention is illustrated by the above examples. However, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. The purpose of including these specific examples and embodiments here is to help those skilled in the art practice the present invention. Any person skilled in the art can easily make further improvements and refinements without departing from the spirit and scope of the present invention. Therefore, the present invention is only limited by the content and scope of the claims of the present invention, and it is intended to cover all alternative and equivalent solutions included within the spirit and scope of the present invention defined by the appended claims.

Claims

1. A bovine parainfluenza virus subunit F protein mutant, characterized in that: The amino acid sequence of the subunit F protein was mutated by Q162C, L168C, I213C, G230C, A463V, and I474Y to obtain a subunit F protein mutant, and the amino acid sequence of the subunit F protein mutant is shown in SEQ ID NO.

4.

2. A bovine parainfluenza virus subunit F protein mutant, characterized in that: The amino acid sequence of the subunit F protein mutant is shown in SEQ ID NO.

5.

3. The subunit F protein mutant of bovine parainfluenza virus according to claim 2, characterized in that: A tag selected from the group consisting of poly-His, FLAG, c-myc, HA and poly-Arg is connected to the amino terminus or carboxyl terminus of the amino acid sequence of the subunit F protein mutant.

4. The subunit F protein mutant of bovine parainfluenza virus according to claim 3, characterized in that: The tag is poly-His, and its amino acid sequence is shown in SEQ ID NO.

6.

5. A gene encoding a mutant of the subunit F protein of a bovine parainfluenza virus, characterized in that: The coding gene sequence of the subunit F protein mutant is shown in SEQ ID NO.

8. Codon optimization is performed on the basis of the coding gene sequence of the subunit F protein mutant shown in SEQ ID NO.8 to obtain the OPTI-F sequence shown in SEQ ID NO.

9.

6. A method for preparing a mutant of the subunit F protein of bovine parainfluenza virus according to claim 5, characterized in that: The preparation method comprises the following steps: 1) The coding gene sequence of the subunit F protein mutant of bovine parainfluenza virus is shown in SEQ ID NO.9; 2) cloning the coding gene sequence of the subunit F protein mutant constructed in step 1) into a eukaryotic expression vector to obtain a recombinant plasmid containing the coding gene sequence of the subunit F protein mutant; 3) transfecting the recombinant plasmid containing the subunit F protein mutant encoding gene sequence obtained in step 2) into the engineered cells of the animal to obtain a cell line; 4) screening out highly expressed cell lines from the cell lines obtained in step 3); and 5) Fermenting and culturing the highly expressing cell line obtained in step 4), and purifying the subunit F protein mutant of bovine parainfluenza virus.

7. The method for preparing a subunit F protein mutant of bovine parainfluenza virus according to claim 6, characterized in that: In step 2), the eukaryotic expression vector is one of pEE6.4, pEE12.4, pGL4.13 and pcDNA3.

1.

8. The method for preparing a mutant of the subunit F protein of bovine parainfluenza virus according to claim 7, characterized in that: In step 2), the eukaryotic expression vector is pEE12.

4.

9. The method for preparing a mutant of the subunit F protein of bovine parainfluenza virus according to claim 6, characterized in that: In step 3), the cell line is one of a CHO cell line, a HEK293 cell line, and a 293T / 17 cell line.

10. The method for preparing a mutant of the subunit F protein of bovine parainfluenza virus according to claim 9, characterized in that: In step 3), the CHO cell line is one of a DG44 cell line, a DXB11 cell line, a CHO-K1 cell line and a CHO-S cell line.

11. Use of the subunit F protein mutant of bovine parainfluenza virus according to any one of claims 1 to 4 in the preparation of a subunit vaccine or diagnostic reagent for bovine parainfluenza virus.

12. Use of the gene encoding the subunit F protein mutant of bovine parainfluenza virus according to claim 5 in the preparation of a subunit vaccine or diagnostic reagent for bovine parainfluenza virus.

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