CN119708169B - Bovine parainfluenza virus subunit F protein and preparation method and application thereof - Google Patents

Abstract

The present invention relates to a subunit F protein of bovine parainfluenza virus and a preparation method and application thereof, wherein the amino acid sequence of the subunit F protein is: 1) an amino acid sequence as shown in SEQ ID NO.2; 2) an immunogenic derived amino acid sequence obtained by replacing, deleting or adding one or more amino acids from SEQ ID NO.2. The subunit F protein of the present invention is mainly prepared by constructing a recombinant plasmid, transfecting a cell strain with the recombinant plasmid, screening a highly expressed cell strain, and purifying the subunit F protein of the bovine parainfluenza virus, and can be well applied to the subunit vaccine or diagnostic reagent of the bovine parainfluenza virus, and has the characteristics of efficient secretion expression, high protein purity, easy purification, reduced production cost, high safety performance, etc.

Description

A subunit F protein of bovine parainfluenza virus and its preparation method and application

Technical Field

The invention belongs to the technical field of animal vaccines and veterinary biological products, and particularly relates to a subunit F protein of a bovine parainfluenza virus and a preparation method and application thereof.

Background Art

Bovine parainfluenza virus type 3 (BPIV3) is a highly contagious viral disease. It can cause fever, coughing, runny nose, decreased spirit and appetite in cattle, leading to decreased productivity. It is often mixed with bovine respiratory syncytial infection, bovine viral diarrhea, bovine infectious rhinotracheitis, bovine Pasteurella multocida, etc., causing calf respiratory syndrome. The disease is prevalent worldwide, bringing huge economic burdens and production losses to the cattle industry, seriously affecting the development of the cattle industry.

Currently, there is no effective treatment for the disease, and prevention and control mainly rely on vaccines. There is no vaccine available in China, but there are traditional inactivated and live attenuated 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, it is necessary to develop new safe and effective vaccines.

BPIV3 virus belongs to the Paramyxoviridae family. The virus particles are polymorphic, ranging from spherical to filamentous structures, with a diameter of 150-300nm. The BPIV3 genome encodes 6 main structural proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large polyadenylation protein (L). Among them, F protein and HN protein are the main proteins of the viral envelope, which can stimulate the body to produce neutralizing antibodies and are the first glycoproteins of subunit vaccines. The F protein subunit vaccine does not contain nucleic acid substances and will not cause persistent infection or latent infection after vaccination; the immune response generated 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 the application is limited.

The cost of subunit vaccines is mainly in the production of subunit proteins. F protein is a glycosylated trimer protein of the envelope necessary for bovine parainfluenza virus to enter cells. It is generally composed of 540 amino acids and is called protein F0 before cleavage. In order to obtain a subunit F protein with biological activity, F0 needs to be hydrolyzed by cell proteases into a polypeptide F1 and F2 connected by a disulfide bond. Therefore, in order to ensure that the expressed protein can be glycosylated and form a complete and active F protein, it must be achieved in animal cells.

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 molecular structure, physicochemical properties, and biological functions such as post-transcriptional modification. They can also be cultured at high density in suspension culture, and the culture volume can reach more than 2,000 L, which allows for large-scale production.

Studies have 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 F protein, if the coding gene sequence of the F protein is not optimized by mutation modification, the engineered cells basically express low amounts of F protein, and most of the obtained proteins are post-fusion proteins, making it difficult to obtain F proteins with excellent immunogenicity and stability for the prevention and control of bovine parainfluenza virus. Therefore, when using engineered cells to express F protein, it is a necessary process to optimize the coding gene mutation sequence of the F protein.

Chinese patent application CN202011013669.6 discloses a recombinant bovine parainfluenza antigen, which is a heterodimer formed by a truncated F protein and an HN protein, that is, a bovine antibody Fc fragment is added to the C-terminus of the F protein fragment and the HN protein fragment, so that the two protein fragments can form a more stable heterodimer when expressed simultaneously in a cell, but the F protein uses a very small fragment and does not have a pre-fusion conformation, and a separate F protein is not obtained.

Summary of the invention

In view of the shortcomings of the prior art, the first object of the present invention is to provide a subunit F protein of bovine parainfluenza virus, which has the excellent immunogenicity and stability of bovine parainfluenza virus F protein on the basis of the original F protein, and is easy to be stably and efficiently secreted and expressed in CHO cell lines.

The second object of the present invention is to provide a method for preparing a 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.

The third object of the present invention is to provide an application of a subunit F protein of bovine parainfluenza virus, which can be well applied to subunit vaccines and diagnostic reagents of bovine parainfluenza virus, thereby facilitating people's prevention and control of bovine parainfluenza virus.

In order to achieve the above first object, the present invention provides a subunit F protein of bovine parainfluenza virus, wherein the amino acid sequence of the subunit F protein is:

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 more amino acids from SEQ ID NO. 2.

According to the subunit F protein of the bovine parainfluenza virus of the present invention, preferably, a trimer motif GCN4 protein is further connected to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.2, and the amino acid sequence of the trimer motif GCN4 protein is shown in SEQ ID NO.7, so as to obtain a fusion protein I of the subunit F protein and the trimer motif GCN4 protein, and the amino acid sequence of the fusion protein I is shown in SEQ ID NO.3.

According to the subunit F protein of the bovine parainfluenza virus of the present invention, preferably, the amino acid sequence of the subunit F protein is 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.

According to the subunit F protein of the bovine parainfluenza virus of the present invention, preferably, a trimer motif GCN4 protein is further connected to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.4, and the amino acid sequence of the trimer motif GCN4 protein is shown in SEQ ID NO.7, so as to obtain a fusion protein II of the subunit F protein mutant and the trimer motif GCN4 protein, and the amino acid sequence of the fusion protein II is shown in SEQ ID NO.5.

According to the subunit F protein of bovine parainfluenza virus of the present invention, preferably, a tag selected from 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 fusion protein II.

According to the subunit F protein of bovine parainfluenza virus of the present invention, preferably, the tag is poly-His, and the subunit F protein of bovine parainfluenza virus is obtained, and its amino acid sequence is shown in SEQ ID NO.6.

According to the subunit F protein of the bovine parainfluenza virus of the present invention, preferably, the coding gene sequence of the subunit F protein is as shown in SEQ ID NO.8, and codon optimization is performed 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.

In order to achieve the above second object, the present invention provides a method for preparing a subunit F protein of a bovine parainfluenza virus, the preparation method comprising the following steps:

1) The coding gene sequence of the subunit F protein of bovine parainfluenza virus is constructed as shown in SEQ ID NO.9;

2) cloning the subunit F protein encoding gene sequence constructed in step 1) into a eukaryotic expression vector to obtain a recombinant plasmid containing the subunit F protein encoding gene sequence;

3) transfecting the recombinant plasmid containing the subunit F protein 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 of bovine parainfluenza virus.

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.

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

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

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

In order to achieve the third objective, the present invention provides a use of a subunit F protein of a bovine parainfluenza virus in a subunit vaccine or a diagnostic reagent for a bovine parainfluenza virus.

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 secretory expression in engineered cell lines, with high yield and easy purification, and the purity of the target protein in the cell culture supernatant can reach more than 70%, and only one-step affinity chromatography is required to make the purity of the target protein reach more than 90%, which far meets the needs of subunit vaccines and diagnostic reagents, and is convenient for large-scale production, thereby solving the technical problem of high production cost of subunit F protein of bovine parainfluenza virus. In addition, since the engineered cell strains such as CHO cell strains, HEK293 cell strains, 293T/17 cell strains used for production have high controllability during cultivation, easy quality control, and stable production between batches of protein, the amount of other viruses in the subunit F protein of bovine parainfluenza virus produced by the present invention is small, which effectively reduces the risk of spreading the virus and has excellent biosafety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a schematic diagram of the predicted three-dimensional structure of the subunit F protein before fusion;

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

Figure 3 shows the plasmid map of pEE12.4-OPTI-F;

Figure 4 shows the results of double enzyme digestion of pEE12.4-OPTI-F: M is DNA Marker: DL10000 Marker; 1 is the result of double enzyme digestion electrophoresis of pEE12.4-OPTI-F;

FIG5 shows the results of SDS-PAGE detection after purification of subunit F protein, wherein 1 is subunit F protein, and M is Marker;

FIG6 shows the Western blot detection results after purification of subunit F protein, wherein 1 is subunit F protein, and M is Marker;

FIG7 shows the stability test results of the purified subunit F protein, wherein 1 is the marker, and 2 is the subunit F protein after treatment at 4° C.;

FIG8 shows the stability test results of the purified subunit F protein, wherein 1 is the marker and 2 is the subunit F protein after being treated at -20°C.

DETAILED DESCRIPTION

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, but are not intended to limit 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 reagents and drugs of the present invention are listed as follows:

CHO-K1 cells were obtained from the Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences and the Cell Bank of Shanghai Institute of Life Sciences of Chinese Academy of Sciences;

Cell culture medium and serum were purchased from Gibco, USA;

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

Lipofectamine LTX was purchased from Thermo Fisher, USA;

Methionine sulfoximine (MSX) was purchased from Sigma;

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

PLUS ^TM reagent was purchased from Thermo Company and is an additive to Lipofectamine LTX transfection reagent;

CB5 was purchased from Thermo Company and used as feed for fermentation medium.

Example 1: 1a: The structure of the bovine parainfluenza virus F protein was analyzed and optimized to construct the coding gene sequence of the subunit F protein of the bovine parainfluenza virus.

By analyzing the F protein sequence of bovine parainfluenza virus (GenBank: OR855359.1), it can be known that the genome sequence 5090-6712 encodes the F protein sequence of bovine parainfluenza. Further analysis showed 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, analysis found that the restriction site of the F protein is between 109R-110F, that is, 19Q-109R is the F2 protein, and 110F-493T is the F1 protein.

Combined with the experience of previous studies on the expression of viral envelope proteins, the extracellular region of the subunit F protein expressed by CHO-K1 cells was selected as the immunogenic protein, namely the amino acid sequence of 1M-481L. Through protein structure prediction and the study of the amino acid structure itself, we added a trimer motif to Q162C, L168C, I213C, G230C, A463V, I474Y, and the C-terminus to make it easier to form the pre-fusion F trimer. The three-dimensional structure predicted by this amino acid sequence is similar to that of other respiratory virus F proteins, and its three-dimensional structure model is shown in Figure 1.

Among them, on the basis of the amino acid sequence SEQ ID NO.3, a derivative protein is prepared by replacing, deleting or adding one or several amino acids, and the derived protein has a homology of up to 80%-100% with the amino acid sequence of the subunit F protein in this embodiment (as shown in SEQ ID NO.4), thereby ensuring that the two have the same immunogenicity, and therefore the derived protein also falls within the scope of protection of the present invention.

In order to facilitate the purification of the subunit F protein, a tag as shown in Table 1 can be connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.5. In this embodiment, Poly-His is taken as an example and connected to the amino terminus of the amino acid sequence shown in SEQ ID NO.5.

Table 1: Tags and their amino acid sequences

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)

.

The coding gene sequence of the amino acid sequence 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. The coding gene sequence of the subunit F protein in this embodiment is codon optimized on the basis of 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 the gene sequence was entrusted to Nanjing GenScript Biotechnology Co., Ltd.

The sequence after codon optimization was compared with the sequence before codon optimization, and the results are shown in Figure 2, with a total of 386/1389=27.8% differences.

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

(1) Primer design and synthesis

Upstream primer:

5’-acgaAGCTTGCCCGCCACCATGATCACTATC-3’

Downstream primer:

5’-ATTGAATTCtcaATGATGGTGGTGGTGGT-3’

(2) 50 μL sample loading system, as shown in Table 2 below.

Table 2: 50μL injection system

Added ingredients Volume (μL) Q5 Mix 25 Upstream primer (10 μM) 2.5 Downstream primer (10 μM) 2.5 OPTI-F 1 dd _H2O 19 Total volume 50

.

PCR amplification procedure:

95℃ 2 min

95℃ 30 s

55℃ 45 s

72℃ 1 min30s

72℃ 10 min

8℃ forever

Among them: one cycle was completed from 95℃ 30 s, 55℃ 45 s, 72℃ 1 min 30 s, 72℃ 10 min, and this cycle was repeated 30 times.

1b.1.2. PCR product gel recovery

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

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

(3) Carefully cut the single target DNA band from the agarose gel using a scalpel and place it into a clean 1.5 mL centrifuge tube;

(4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3) and place in a 50°C water bath for about 5 min, gently turning the centrifuge tube upside down to ensure that the gel block is fully dissolved;

(5) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column CB2 is placed in the collection tube in advance), centrifuge at 12000 rpm/min for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 back into the collection tube;

(6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 min, centrifuge at 10,000 rpm/min for 30 s, pour out the waste liquid in the collection tube, and then place the adsorption column CB2 in the collection tube;

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

(8) Repeat step (7);

(9) Centrifuge the empty adsorption column at 12000 rpm/min for 2 min to remove as much rinse solution as possible. Place the adsorption column CB2 at room temperature for 10 min to dry thoroughly.

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

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

(12) Store the DNA sample in step 11 at 4°C and prepare agarose gel electrophoresis to identify the DNA fragments.

1b.2. Double restriction enzyme digestion reaction of PCR product and vector

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

Table 3: 50 μL reaction system

Sample ingredient name Volume (μL) dd H ₂ 0 Refill to 50 10×buffer 5 DNA samples Volume at 2 μg HindⅢ 2.5 EcoRⅠ 2.5

(2) Place the EP tube in step (1) in a constant temperature water bath at 37°C for 2-3 hours;

(3) Gel recovery of double-enzyme digestion products: Take out the double-enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein. The method is the same as the gel recovery of PCR products in step 1b.1.2.

1b.3. Ligation reaction

(1) Prepare several clean 1.5 mL EP tubes, label them, and place them in an EP tube rack for later use;

(2) Add samples to the EP tube in step (1) and mix them according to Table 4 below, where the target fragment in Table 4 is the DNA fragment finally recovered in step 1b.2 (2);

Table 4: 10 μL reaction system

Sample ingredient name Experimental group (μL) Blank group (μL) dd H ₂ 0 / 6 10× T4 connection buffer 1 1 Purpose fragment 6 - Carrier 2 2 T4 ligase 1 1

(3) After adding samples according to the table in step (2), place each 10 μL reaction system in a 16°C low-temperature cooling liquid circulation machine and water bath for 10-16 h;

(4) Take out the EP tube in step (3) and place it in a 65°C water bath for 15 min;

(5) Take out the EP tube in step (4) and store it at 4°C to obtain the ligation reaction solution.

1b.4. Conversion reaction

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

(2) Take out the sample tube, place it in a 42°C water bath for 100 seconds, and then immediately place it in an ice bath for 2 minutes;

(3) Take out the sample tube, add 600 μL of liquid LB medium to the sample tube in the clean bench, and then place the sample tube in a 37°C constant temperature shaker at 220 rpm/min for 1 h.

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

(5) Place the transformation plate prepared in step (4) upright in a biochemical constant temperature incubator and culture at 37°C for 1 hour. Then, invert the transformation plate and culture it for 15 hours to obtain a monoclonal strain.

1b.5. Plasmid extraction and double enzyme digestion identification; 1b.5.1. Plasmid extraction

(1) Use a 10 μL pipette tip to pick a single clone from the transformation plate in step 1b.4 (5) and transfer it to 5 mL of liquid LB medium containing ampicillin resistance. Incubate the culture overnight at 37°C and 220 rpm/min.

(2) Transfer the bacterial solution to a 1.5 mL EP tube, centrifuge at room temperature and 12,000 rpm for 2 min, and discard the supernatant;

(3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to thoroughly suspend the bacteria;

(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 stand at room temperature for 2-4 minutes;

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

(6) Centrifuge the solution from step (5) at room temperature and 14,000 rpm for 10 min;

(7) Transfer the supernatant solution from step (6) to the center of the adsorption column, centrifuge at room temperature and 12000 rpm/min for 30 seconds, and discard the liquid in the collection tube;

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

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

(10) Centrifuge the empty adsorption column at room temperature and 12000 rpm for 2 min.

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

Double enzyme digestion identification

(1) Label the 1.5 mL EP tubes to be used and add samples according to Table 5 below. The DNA sample in Table 5 is the DNA solution obtained in step 1b.5.1 (11);

Table 5: 20 μL reaction system

Sample ingredient name Volume (μL) dd H ₂ 0 Add to 20 μL 10×buffer 2 DNA samples Volume when mass is 1 μg HindⅢ 1 EcoRⅠ 1

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

(3) The double enzyme digestion system sample in step (2) was subjected to agarose gel electrophoresis to check whether the insert fragment size is correct; the experimental results are shown in Figure 4: enzyme digestion identification shows that the construction is correct;

(4) The recombinant plasmid with the correct insertion fragment was selected and sent to a sequencing company for sequencing. In the present invention, the recombinant plasmid was sent to Genewise Biotechnology Co., Ltd. for sequencing. The sequence of the subunit F protein encoding gene was measured as shown in SEQ ID NO.9.

1b.6, Endotoxin-free plasmid extraction; 1b.6.1, Endotoxin-free plasmid extraction

(1) Inoculate the clone sequenced correctly in step 1b.5.2 (4) into 100 mL of culture medium containing ampicillin resistance and culture in a constant temperature shaker at 37°C, 220 rpm/min, for 15 h;

(2) Transfer the bacterial culture in step (1) to a 50 mL centrifuge tube, centrifuge at room temperature and 8000 rpm/min for 5 min, collect the bacteria, and discard the supernatant culture medium;

(3) Add 8 mL of plasmid extraction reagent P1 buffer to the centrifuge tube in step (2) and fully resuspend the bacteria using a pipette;

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

(5) Add 8 mL of plasmid extraction reagent P4 buffer to the centrifuge tube in step (4), immediately invert the tube 6-8 times, mix thoroughly until white flocculent precipitate appears in the solution, leave it at room temperature for about 10 min, and centrifuge it at room temperature and 8000 rpm/min for 5-10 min to allow the white precipitate to settle to the bottom of the tube;

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

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

(8) Add 0.3 times the volume of isopropanol to the filtrate from step (6), mix thoroughly by inverting, and transfer to adsorption column CP6. Centrifuge at room temperature and 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;

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

(10) Repeat step (9) once;

(11) Add 3 mL of anhydrous ethanol to the adsorption column CP6 in step (10), centrifuge at room temperature and 8000 rpm/min for 2 min, and discard the waste liquid;

(12) Place the adsorption column CP6 from step (11) back into the collection tube, centrifuge at room temperature and 8000 rpm/min for 5 min, open the cover of the adsorption column CP6, and leave it at room temperature for several minutes to dry;

(13) Place the adsorption column in step (12) into a clean 50 mL centrifuge tube, add 1-2 mL of TB buffer to the center of the adsorption membrane, let stand at room temperature for 5 min, centrifuge at room temperature and 8000 rpm/min for 2 min, and transfer all the eluate in the 50 mL centrifuge tube into a clean 1.5 mL centrifuge tube. The eluate is the DNA solution of the pEE12.4-OPTI-F recombinant plasmid. The map of the recombinant plasmid is shown in Figure 3. After measuring the concentration, store it at -20°C.

1c: Transfection of CHO-K1 cells with pEE12.4-OPTI-F recombinant plasmid and establishment of monoclonal screening; 1c.1, CHO-K1 cell transfection

(1) Preparation: UV sterilize in a biosafety cabinet for 30 min; DMEM/F12 medium (containing 10 wt% serum, 1 wt% double antibody) and PBS buffer were placed in a 37 ℃ water bath and preheated to 37 ℃;

(2) Take out CHO-K1 cells (10 cm cell culture dish) from the 37°C _CO2 cell culture incubator, discard the supernatant culture medium, wash the cells once with 8 mL of pre-warmed PBS buffer, and discard the PBS buffer;

(3) Add 1-2 mL of 0.25 wt% trypsin-EDTA to each 10 cm cell culture dish and digest for about 2 min at room temperature. Observe the cells under a microscope to see that they shrink and become round and appear as single cells.

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

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

(6) Resuspend the cells in DMEM/F12 medium (containing 10wt% serum and 1wt% double antibody) and count;

(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 CO ₂ cell culture incubator at 37°C and 5% CO ₂ volume percentage and incubate overnight.

(8) Take out the six-well plate in step (7) and observe the cell status: when the cell confluence reaches 80%-90%, transfection can be started. Before transfection, replace the culture medium with antibiotic-free and serum-free DMEM/F12 culture medium, 2 mL/well;

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

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

(11) Gently mix the mixture of step (10) and step (11), let it stand at room temperature for 5 min, and then add it dropwise into a six-well plate and distribute it evenly;

(12) Place the six-well plate in a _CO2 cell culture incubator at 37°C and 5% _CO2 for 4-6 hours;

(13) Medium change: discard the supernatant culture medium, add 2 mL of DMEM/F12 culture medium (containing 10 wt% serum and 1 wt% double antibody), and place the six-well plate in a _CO2 cell culture incubator at 37°C and 5% _CO2 volume percentage.

1c.2. Pressurized screening

(1) Start pressurization 24 hours after transfection: Remove the six-well plate cells from the _CO2 cell culture incubator in step 1c.1 (11), discard the supernatant culture medium, add 2 mL of DMEM/F12 (containing 10 wt% serum + 25 µM MSX), and pressurize for 7 days. Observe the cells in the middle and replace the medium frequently if there are dead cells.

1c.3. Monoclonal screening

(1) When all negative control cells in step 1c.2 are dead, about 7 days later, start monoclonal screening;

(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 culture medium (containing 10 wt% serum + 25 µM MSX) to terminate the digestion reaction, and blow the cells apart with a pipette;

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

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

(5) Plating: Dilute cells to 5 cells/mL, take 200 µL of the mixed cells and add them to a 96-well plate. Place the plate in a _CO2 cell culture incubator at 37°C and 5% _CO2 for 4-6 hours.

(6) Recording the pores of a single cell;

(7) When a single cell grows in the wells of the 96-well plate, 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 culture medium (containing 10 wt% serum + 25 µM MSX) to terminate the digestion reaction, and use a pipette to blow the cells apart; transfer the cell fluid to a 12-well plate, and when the 12-well plate is full, take the supernatant, and detect whether the clone is positive by ELISA. The positive clones with high-efficiency expression continue to be expanded and cultured and frozen.

1d: CHO-K1 cell line is adapted to suspension culture

(1) Preparation: UV sterilize in a biosafety cabinet for 30 min; preheat DMEM/F12 medium (containing 10 wt% serum + 25 µM MSX) to 37°C in a 37°C water bath;

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

(3) Add 1-2 mL of 0.25 wt% trypsin-EDTA to each 10 cm cell culture dish and digest for about 2 min at room temperature. Observe the cells under a microscope to see that they shrink and become round and appear as single cells.

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

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

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

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

(8) The biosafety cabinet surface was wiped and disinfected with 75% alcohol and then exposed to ultraviolet light for 30 minutes;

(9) Count cell density and viability every 24 h;

(10) After the first generation of cells is cultured once, when the cell survival rate reaches 94-97%, the second generation of cells is cultured;

(11) Preparation: UV sterilize in a biosafety cabinet for 30 min; 100 wt% DMEM/F12 medium (containing 10 wt% serum + 25 µM MSX) and EX-CELL 302 medium are placed in a _CO2 cell culture incubator with a CO2 volume percentage of ₅ % and preheated to 37°C;

(12) Remove the cells from the _CO2 cell culture incubator in step (11) and transfer them to a 50 mL centrifuge tube. Centrifuge at 200 g for 5 min at room temperature.

(13) Mix 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 them;

(14) Dilute the cells to 5 × 10 ⁵ cells/mL and inoculate 30 mL of the culture medium containing the suspended cells obtained in step (13) into a 125 mL shake flask; place the cell culture flask in a CO ₂ cell culture incubator at 37°C and 5% CO ₂ on an orbital shaker at 120 rpm/min overnight;

(15) The biosafety cabinet surface was wiped and disinfected with 75wt% alcohol and exposed to ultraviolet light for 30 minutes;

(16) Count cell density and viability every 24 h;

(17) After two second-generation cultures, the cell survival rate is greater than 95%; after three third- to sixth-generation cultures, the cell survival rate is greater than 95%; after 7 weeks, the cells are propagated for three generations 3 days after inoculation, the cell density reaches 1×10 ⁶ cells/mL, and 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;

(18) After domestication, the 3C2 cell line and the 2B5 cell line in the CHO-K1 cell line both met the requirements, which indicated that the 3C2 cell line and the 2B5 cell line were successfully domesticated.

1e: Shake flask fermentation

(1) Preparation of subculture medium: Preheat 60wt% CD-CHO medium + 40wt% Ex-cell 302 medium to 37℃ in a 37℃ water bath;

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

(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, respectively, and inoculate 30 mL of the subculture medium in step (1) into a 125 mL shake flask. Place the cell culture flask in a CO ₂ constant temperature shaker at 37°C and a CO ₂ volume concentration of 5%, and incubate at 100 rpm/min overnight;

(4) Count cell density and viability every 24 hours, measure glucose, and add glucose to 4 g/L when blood glucose is lower than 2 g/L; take 1 mL of sample every day, and use the supernatant to detect protein expression;

(5) Feed (around the fourth day): Supplement with 70 g/L CB5, which is 10% of the original culture medium;

(6) Culture temperature adjustment (day 5): adjust the temperature of the _CO2 incubator to 32°C;

(7) Re-feeding (day 9): Supplement with 70 g/L CB5, which is 10% of the original culture medium;

(8) On the twelfth day, the culture medium of 3C2 cells and the culture medium of 2B5 cells were harvested respectively.

1f: Protein purification

(1) Collect the 3C2 cell culture medium and 2B5 cell culture medium harvested in step 1e (8) (about 100 ml for each batch), centrifuge at 4°C and 8000 g for 30 min, take the supernatant, filter through a 0.8 µm filter, load the sample, and 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;

(2) Column equilibration: Use ultrapure water _to equilibrate for 2-3CV (column volume), drain the ethanol storage solution; then use Buffer A (50mM _NaH2PO4 (pH=7.4), 500mM NaCl) to equilibrate for 2-3CV, with an equilibration speed of 4-7mL/min;

(3) Sample loading: prepare a 5 mL pre-packed column, load the sample at a flow rate of 1 mL/min (adjust the sample loading flow rate according to the volume of the pre-packed column), retain for 5 min, collect the flow through (FT), take 80 μL of 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;

(4) Washing: Wash the column with 4 wt% Buffer B (20 mM NaH ₂ PO ₄ (pH = 7.4), 500 mM NaCl, 20 mM Mimidazole) at a flow rate of 4 mL/min to wash away the proteins that are not bound to the column and the impurities with weak binding ability until the OD baseline at a wavelength of 280 nm is stable;

(5) Elution: Use 50wt% Buffer B (20mM _NaH2PO4 (pH=7.4), 500mM NaCl, 20mMimidazole) _to elute the target protein until its OD value at 280nm wavelength reaches baseline. The elution rate is 2mL/min. Collect the target protein at a specification of 10mL/tube. Take 80μL of sample and add 20μL of 5×SDS-sample buffer for SDS-PAGE detection to determine the elution effect of the target protein.

(6) Washing: Wash with 100wt% Buffer B (20mM _NaH2PO4 (pH=7.4), 500mM NaCl, 400mMimidazole) 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. The HisTrap excel column can be balanced with 20% ethanol storage solution for 2-3 CV to obtain the imidazole eluate containing the target protein;

(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;

(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.

(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.

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

(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 are shown in FIG5 ;

(2) From the figure, it can be calculated that the SDS-PAGE purity of the purified subunit F protein is 90%, and the molecular weight is approximately 52 KD (F ₀ ) and 40 KD (F ₁ ). The band at 40 KD is weaker, and the band at 52 KD is stronger.

1g.2、Western Blot detection

(1) The protein purified in step 1f was subjected to Western Blot detection, and the results are shown in Figure 6. The concentration of subunit F protein (marked as 1 in the figure) in the sample used was 2 μg/well; the primary antibody used was derived from the positive serum of cattle immunized with inactivated bovine parainfluenza virus 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;

(2) As can be seen from the figure, the serum can specifically bind to the subunit F protein of the present invention, which shows that the subunit F protein prepared by the present invention has excellent immunogenicity.

1g.3. ELISA test

(1) Coating: On the ELISA plate, dilute the purified subunit F protein to 0.5 µg/ml with coating solution (50 mM carbonate buffer, pH = 9.5). Coat 8 wells with each antigen (4 wells with serum samples and 4 wells with blocking solution as a control). Add 100 µl/well of each antigen, seal with sealing film and place in a refrigerator at 4°C overnight.

(2) Washing: Take out the ELISA plate from step (1) from the refrigerator and wash the plate 5 times with PBST buffer;

(3) Blocking: Add 200 µl of blocking solution (5 wt% skim milk) to each well containing subunit F protein, seal with sealing film and incubate at 37°C for 2 h;

(4) Serum dilution: dilute the positive serum of cattle immunized with inactivated bovine parainfluenza vaccine 200 times with blocking solution;

(5) Washing: same as (2);

(6) Sample addition: Add diluted serum and use blocking solution as a negative control, and incubate at 37°C for 1 h.

(7) Washing: same as (2);

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

(9) Washing: same as (2);

(10) Color development: Add 100 µl of TMB color development solution to each well in the dark and incubate at 37°C for 10 min.

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

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

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

Table 6: Identification results of subunit F protein by ELISA

sample OD450 value of coating 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

.

1g.4 Stability Verification

(1) Dilute the purified subunit F protein in step 1f to 2 mg/ml with PBS buffer and divide into 20 portions, each with 0.5 ml; 10 portions are placed in a 4°C refrigerator and one portion is sampled every week for 10 consecutive times;

(2) The other 10 samples were placed in a -20°C refrigerator and one sample was taken every week for 10 consecutive times. The protein concentration was tested using BCA after each sampling. The results are shown in Table 7 below.

Table 7: Stability of subunit F protein

sample Sample concentration after treatment at 4℃ (mg/ml) Sample concentration after -20℃ treatment (mg/ml) First sampling 2.08 2.03 Second sampling 1.97 1.98 The third sampling 1.98 2.02 The fourth sampling 2.02 2.97 The fifth sampling 2.03 2.03 The sixth sampling 1.99 1.97 The seventh sampling 1.96 2.01 The eighth sampling 1.97 1.98 The ninth sampling 1.95 1.96 The tenth sampling 1.89 1.96

.

Referring to Table 7, from the changes in protein concentration, the protein remained basically stable during the two groups of experiments. In order to further verify whether the treated protein is degraded, we used the 10th sample for SDS-PAGE detection, and the specific results are shown in Figures 7 and 8. Among them, 1 in Figure 7 is Marker, 2 is the subunit F protein after treatment at 4°C, and the sample amount is 2μg; 1 in Figure 8 is Marker, 2 is the subunit F protein after treatment at -20°C, and the sample amount is also 2μg. It can be seen from Figures 7 and 8 that the treated sample (the 10th sampling) is still stable, which shows that the subunit F protein prepared by the present invention has excellent stability.

1h: Vaccine preparation

(1) Preparation of aqueous phase: According to the content of subunit F protein in the vaccine, the subunit F protein is diluted into several portions with different concentration gradients using PBS buffer (or normal saline), such as 50 μg/mL, 100 μg/mL, 200 μg/mL, 400 μg/mL, etc. In this embodiment, the subunit F protein is diluted to 50 μg/mL, which is the aqueous phase;

(2) Oil phase preparation: according to the total amount of vaccine to be prepared, with the weight ratio of antigen phase to adjuvant being 1:1 and the volume ratio being 46:54, take an appropriate amount of ISA 201 VG adjuvant;

(3) Emulsification: Preheat both the water phase and the oil phase to 33°C, slowly add the water phase to the oil phase, stir at 200-500 rpm/min for 20-30 min, let stand at 20°C for 1 sec, and then place at 4°C overnight;

(4) Packaging and storage: Pack the product according to needs and store it at 4°C for future use after passing inspection.

1i: Vaccine quality inspection

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

(2) Use a clean pipette to draw a small amount of vaccine and drop it into cold water. Observe (except the first drop). The vaccine should diffuse in a cloud-like manner, which indicates that it is a water-in-oil-in-water dosage form.

(3) Add 10 ml of the vaccine into a centrifuge tube and centrifuge at 3000 r/min for 15 min. If the amount of water precipitated at the bottom of the tube is ≤ 0.5 mL, it is considered stable.

(4) Use a viscometer to test the viscosity of the vaccine, which should be between 20-50cp to be considered qualified.

Example 2: The difference between Example 2 and Example 1 is that the amino acid sequence encoding the subunit F protein in this example is shown in SEQ ID NO.1.

Example 3: The difference between Example 3 and Example 1 is that the amino acid sequence encoding the subunit F protein in this example is shown in SEQ ID NO.2.

Example 4: The difference between Example 4 and Example 1 is that the amino acid sequence encoding the subunit F protein in this example is shown in SEQ ID NO.3.

Example 5: The difference between Example 5 and Example 1 is that the amino acid sequence encoding the subunit F protein in this example is shown in SEQ ID NO.4.

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

Example 6: Determination of the expression yield and protein purity of the subunit F protein: The expression yield and protein purity of the subunit F protein in Examples 2 to 5 and the comparative example were determined, and the expression results are shown in Table 8 below.

Table 8: Yield and purity of subunit F protein of Examples 2-5 and Comparative Examples

project 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 twenty one

.

Referring to Table 8, the protein yield of the present invention is 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. It can be concluded that the subunit F protein encoding gene 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 high protein purity.

The protein yields of Examples 2 to 5 are between 0.1 g/L and 0.7 g/L, and the protein purity is between 60% and 80%, while the protein yield of Example 1 is between 1 g/L and 1.12 g/L, and the protein purity is 90%. Therefore, Example 1 is a preferred embodiment among Examples 1 to 5. It can be concluded that when the amino acid sequence of the subunit F protein is SEQ ID NO.5, the corresponding protein yield and protein purity reach the optimal values.

In summary, the recombinant plasmid constructed by the present invention can be effectively expressed in an engineered cell line to obtain a high-yield, high-purity subunit F protein of a bovine parainfluenza virus, which has good specificity and stability, can be mass-produced, and effectively reduces the production cost of the subunit F protein of the bovine parainfluenza virus, and can also be well applied to the application of the subunit vaccine or diagnostic reagent of the bovine parainfluenza virus. Therefore, the subunit F protein of the present invention has the characteristics of efficient secretion expression, high protein purity, easy purification, low production cost, high safety performance, etc.

The present invention is illustrated by the above examples, but 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 to practice the present invention. Any person skilled in the art is easy to make further improvements and perfections without departing from the spirit and scope of the present invention, so 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 alternatives and equivalents included in the spirit and scope of the present invention defined by the appendix claims.

Claims (12)

1. The subunit F protein mutant of bovine parainfluenza virus is characterized in that the amino acid sequence of the subunit F protein is mutated by Q162C, L168C, I213C, G230C, A463V and I474Y to obtain the subunit F protein mutant, and the amino acid sequence of the subunit F protein mutant is shown as SEQ ID NO. 4.

2. The subunit F protein mutant of bovine parainfluenza virus is characterized in that the amino acid sequence of the subunit F protein mutant is shown as SEQ ID NO. 5.

3. The mutant bovine parainfluenza virus subunit F protein of claim 2, wherein a tag of poly-His, FLAG, c-myc, HA, poly-Arg is attached to the amino terminus or the carboxy terminus of the amino acid sequence of the mutant subunit F protein.

4. A mutant bovine parainfluenza virus subunit F protein according to claim 3 wherein the tag is poly-His and the amino acid sequence is as shown in SEQ ID No. 6.

5. The coding gene of the subunit F protein mutant of bovine parainfluenza virus is characterized in that the coding gene sequence of the subunit F protein mutant is shown as SEQ ID NO.8, and codon optimization is performed on the basis of the coding gene sequence of the subunit F protein mutant shown as SEQ ID NO.8 to obtain an OPTI-F sequence shown as SEQ ID NO. 9.

6. A method of preparing a mutant bovine parainfluenza virus subunit F protein according to claim 5, comprising the steps of:

1) The coding gene sequence of the subunit F protein mutant of the bovine parainfluenza virus is shown as SEQ ID NO. 9;

2) Cloning the coding gene sequence of the subunit F protein mutant constructed in the 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 coding gene sequence obtained in the step 2) into an engineering cell of an animal to obtain a cell strain;

4) Screening out highly expressed cell lines from the cell lines obtained in step 3), and

5) And (3) fermenting and culturing the cell strain with high expression obtained in the step (4), and purifying to obtain the subunit F protein mutant of bovine parainfluenza virus.

7. The method for producing a mutant bovine parainfluenza virus subunit F protein according to claim 6, wherein in step 2), the eukaryotic expression vector is one of pEE6.4, pEE12.4, pGL4.13 and pcDNA3.1.

8. The method for producing a mutant bovine parainfluenza virus subunit F protein of claim 7, wherein in step 2) the eukaryotic expression vector is pee12.4.

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

10. The method for producing a mutant bovine parainfluenza virus subunit F protein according to claim 9, wherein 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.

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

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