Preparation method and application of subunit HN protein of bovine parainfluenza virus

Abstract

The invention relates to a subunit HN protein of a bovine parainfluenza virus and a preparation method and application thereof, the subunit HN protein is an extracellular region of the bovine parainfluenza virus HN protein, and the amino acid sequence of the subunit HN protein is as follows: 1) an amino acid sequence as shown in SEQ ID NO.1; 2) a derived amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the SEQ ID NO.1 and has immunogenicity, wherein the derived amino acid sequence has 80%-100% sequence identity with a sequence SEQ ID NO.2. The subunit HN protein is mainly prepared by constructing a recombinant plasmid, transfecting a cell strain with the recombinant plasmid, screening a high-expression cell strain and purifying the subunit HN protein of the bovine parainfluenza virus, and can be better suitable for a subunit vaccine or a diagnostic reagent of the bovine parainfluenza virus. The method has the characteristics of high secretory expression efficiency, high protein purity, easiness in purification, low production cost, high safety performance and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of animal vaccines and veterinary biological products, and particularly relates to a subunit HN protein of a bovine parainfluenza virus and a preparation method and application thereof. Background Art

[0002] 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.

[0003] 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.

[0004] 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 virus envelope, which can stimulate the body to produce neutralizing antibodies and are the first glycoproteins of subunit vaccines. HN 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, HN subunit vaccine also has obvious defects: the expression yield is relatively low, the production cost is high, and the application is limited.

[0005] The cost of subunit vaccines is mainly in the production of subunit proteins. The inventors have noticed that HN protein is a type II transmembrane glycoprotein that plays an important role in virus entry and release. At the same time, HN protein is also a multifunctional protein that has neuraminidase activity, hemagglutinin activity and sialic acid binding on the cell surface. HN protein is generally 572 amino acids long and consists of three domains: N-terminal cytoplasmic tail, transmembrane domain and C-terminal extracellular domain. The C-terminal domain of the outer membrane is further divided into a globular head region and a neck region. The C-terminal extracellular domain is responsible for separating sialic acid from glycoproteins on the surface of infected cells, which is necessary for the release of viral particles from cells. At the same time, HN protein is also a sialic acid receptor on the cell surface, mediating the adsorption process of viruses and cells. Therefore, HN protein is an important virulence protein and protective antigen of bovine parainfluenza virus. As a glycosylated modified protein of the envelope, HN protein must be achieved in animal cells to ensure that the expressed protein can be glycosylated and form a complete and active HN protein.

[0006] Engineered cells are currently the most 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, post-transcriptional modification and other biological functions. They are usually cultured in suspension to achieve high density, and the culture volume can reach more than 2,000 L, so they can be produced on a large scale.

[0007] However, when using engineered cells to express HN protein, the expression yield of the C-terminal extracellular region of HN protein is very low, making it difficult to obtain HN protein with excellent immunogenicity and stability for the prevention and control of bovine parainfluenza virus. Therefore, when using engineered cells to express HN protein, structural analysis and optimization of the coding gene sequence of HN protein is a necessary process.

[0008] 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. However, a small fragment of the HN protein is used, and the Fc is not mutated, so a pure heterodimer cannot be obtained, and no separate HN protein is obtained. Summary of the invention

[0009] In view of the shortcomings of the prior art, the first object of the present invention is to provide a subunit HN protein of bovine parainfluenza virus, which has the excellent immunogenicity and stability of the HN protein of bovine parainfluenza virus and is easy to be stably and efficiently secreted and expressed in CHO cell lines.

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

[0011] The third object of the present invention is to provide an application of a subunit HN 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.

[0012] In order to achieve the above first object, the present invention provides a subunit HN protein of bovine parainfluenza virus, wherein the subunit HN protein is the extracellular region of the HN protein of bovine parainfluenza virus, and its amino acid sequence is: 1) The amino acid sequence shown in SEQ ID NO.1; 2) An immunogenic derivative amino acid sequence obtained by substituting, deleting or adding one or more amino acids from SEQ ID NO.1.

[0013] According to the subunit HN protein of the present invention, preferably, the subunit HN protein is a truncated protein of the extracellular region of the bovine parainfluenza virus HN protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0014] According to the subunit HN protein of the present invention, preferably, a bovine antibody constant region Fc is connected to the amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.8, as well as a tag selected from poly-His, FLAG, c-myc, HA, and poly-Arg.

[0015] According to the subunit HN protein of the present invention, preferably, the amino acid gene sequence of the subunit HN protein is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0016] According to the subunit HN protein of the present invention, preferably, the coding gene sequence of the subunit HN protein of the amino acid gene sequence shown in SEQ ID NO.4 is shown in SEQ ID NO.5.

[0017] In order to achieve the above second object, the present invention provides a method for preparing bovine parainfluenza virus HN protein, the preparation method comprising the following steps: 1) The gene sequence encoding the subunit HN protein of bovine parainfluenza virus is constructed as shown in SEQ ID NO.5; 2) cloning the coding gene sequence of the subunit HN protein constructed in step 1) into a eukaryotic expression vector to obtain a recombinant plasmid containing the coding gene sequence of the subunit HN protein; 3) transfecting the recombinant plasmid containing the coding gene sequence of the subunit HN protein 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 HN protein of bovine parainfluenza virus.

[0018] 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.

[0019] In the preparation method of the present invention, preferably, in step 2), the eukaryotic expression vector is pEE12.4.

[0020] 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.

[0021] 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.

[0022] In the technical solution of the present invention, preferably, in step ③, the CHO cell strain is one of the DG44 cell strain, the DXB11 cell strain, the CHO-K1 cell strain and the CHO-S cell strain.

[0023] In order to achieve the third objective, the present invention provides a use of the subunit HN protein of bovine parainfluenza virus in a subunit vaccine or diagnostic reagent for bovine parainfluenza virus.

[0024] In summary, the present invention has the following beneficial effects: the present invention provides a subunit HN protein, which has excellent immunogenicity and stability of bovine parainfluenza virus HN 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, so as to facilitate large-scale production, thereby solving the technical problems of low expression yield and high production cost of bovine parainfluenza virus HN protein. In addition, since the engineered cell strains such as CHO cell strains, HEK293 cell strains, and 293T cell strains used for production have high controllability during culture, easy quality control, and stable production batches of proteins, the amount of other viruses in the bovine parainfluenza virus subunit HN protein 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

[0025] Figure 1 A schematic diagram showing the predicted three-dimensional structure of the subunit HN fusion protein; Figure 2 It represents the comparison result of the coding gene sequence of the subunit HN protein before and after optimization, specifically the comparison of the coding gene sequence of SEQ ID NO.6 and SEQ ID NO.7; Figure 3 represents the plasmid map of pEE12.4-OPTI-Fc-HN; Figure 4 Indicates the results of double enzyme digestion of pEE12.4-OPTI-Fc-HN: M is DNA Marker: DL10000 Marker; 1 is the result of double enzyme digestion electrophoresis of pEE12.4-OPTI-Fc-HN HindIII and EcoRI; Figure 5 It represents the SDS-PAGE electrophoresis of subunit HN protein, where 1 is the electrophoresis detection result of HN protein in the presence of reducing agent, 2 is the detection result in the presence of non-reducing agent, and M is Marker; Figure 6 It represents the Western Blot test results after purification of subunit HN protein, where 1 is subunit HN protein and 2 is marker; Figure 7 It represents the stability test results of the purified subunit HN protein, where M is the marker; 1 is the subunit HN protein treated at 4°C, and the loading amount is 2 μg; Figure 8 It represents the stability test result of the purified subunit HN protein, wherein M is the marker; 1 is the subunit HN protein after -20℃ treatment, and the loading amount is 2μg. DETAILED DESCRIPTION

[0026] 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.

[0027] The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

[0028] The sources of the reagents and drugs of the present invention are listed as follows: pEE12.4 and CHO-K1 cells were stored by Zhejiang Hilong Biotechnology Co., Ltd.; Cell culture medium and serum were purchased from Gibco, USA; Lipofectamine LTX, PLUS TM Reagent, CB5, and BCA protein quantification kit were purchased from ThermoFisher, USA.

[0029] Example 1: 1a: Structural analysis and optimization of the HN protein of bovine parainfluenza virus to construct the coding gene sequence of the subunit HN protein of bovine parainfluenza virus By analyzing the bovine parainfluenza virus HN protein sequence (GenBank: OM621819.1), it was found that the genome sequence 6818-8536bp encodes the bovine parainfluenza HN protein sequence. Further analysis showed that 1M-33M is the intracellular region of the HN protein, 34S-56I is the transmembrane region of the HN protein, and 57Q-572S is the extracellular region of the HN protein. Combined with the experience of expressing viral envelope protein research, the extracellular region of the HN protein expressed by CHO-K1 cells was selected as the immunogenic protein of the present invention, that is, the amino acid sequence of 57Q-572S, and the amino acid sequence is a known fragment that can be obtained through NBCI query.

[0030] However, by expressing the amino acid sequence of 57Q-572S through the CHO-K1 cell line, it was found that the expression level of the protein fragment directly expressed was very low and could not meet the needs of large-scale production. Further, through protein structure prediction and the study of the amino acid structure itself, we found that the HN protein is a homodimer, 57Q-142M is the stem region of the HN protein, and 143T-572S is the head region of the HN protein. The 57Q-142M amino acids may affect the expression of the protein in CHO-K1 cells, and this segment of the polypeptide is not the immunogenic site of the HN protein. 143T-572S is the main antigenic epitope region of the HN protein. In order to further improve the stability and yield of the HN protein, the bovine antibody constant region sequence was fused with it for expression, which increased the yield of the HN protein.

[0031] Therefore, the present invention removes the amino acids 57Q-142M and expresses the amino acid sequence 143T-572S. The three-dimensional structure model predicted by this amino acid sequence is shown in the figure Figure 1 As shown, it can be efficiently secreted and expressed in CHO-K1 cells to obtain the corresponding subunit HN protein. In order to improve the stability and yield of the HN protein, the bovine antibody constant region sequence is fused with it for expression, thereby increasing the yield of the HN protein.

[0032] 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 HN protein in this embodiment (as shown in SEQ ID NO.3), thereby ensuring that the two have the same immunogenicity, or the antibody Fc fragment of other species is used for fusion expression, so the derived protein also falls within the protection scope of the present invention.

[0033] In order to facilitate the detection or purification of the subunit HN 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.3. In this embodiment, Poly-His is taken as an example and connected to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.4.

[0034] 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)

[0035] The portion of the HN gene sequence encoding the amino acid sequence SEQ ID NO.4 may be as shown in SEQ ID NO.6, or may be obtained by codon optimization of SEQ ID NO.7. The gene sequence encoding the subunit HN protein in this embodiment is codon optimized on the basis of SEQ ID NO.4 to obtain the OPTI-HN sequence, as shown in SEQ ID NO.5. The artificial synthesis of the gene sequence was entrusted to Nanjing GenScript Biotechnology Co., Ltd.

[0036] The sequence of HN after codon optimization (as shown in SEQ ID NO.7) was compared with the sequence of HN before codon optimization (as shown in SEQ ID NO.6). Figure 2 As shown, a total of 537 / 2037=26.4% are different.

[0037] 1b: Construction of pEE12.4-OPTI-Fc-HN recombinant plasmid; 1b.1, PCR amplification of target fragment OPTI-Fc-HN; 1b.1.1, PCR reaction (1) Primer design and synthesis Upstream primer: 5'-cacgaAGCTTGCCCGCCACCATGGCAG-3' Downstream primer: 5'-AATTGAATTCTCAATGGTGGTGATGGTG-3' (2) 50 μL sample loading system, as shown in Table 2 below.

[0038] 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-Fc-HN 1 <![CDATA[dd H2O]]> 19 Total volume 50

[0039] PCR amplification procedure: 95℃ 2 min 95℃ 30 s 58℃ 45 s 72℃ 1 min 72℃ 10 min 10℃ Forever Among them: one cycle was completed from 95℃ 30 s, 58℃ 45 s, 72℃ 1 min, and this cycle was repeated 30 times.

[0040] 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 into 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.

[0041] 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) <![CDATA[dd H20]]> 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; Gel recovery of double-enzyme digestion products: Take out the above double-enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein, using the same method as the gel recovery of PCR products in step 1b.1.2.

[0042] 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) <![CDATA[dd H20]]> / 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.

[0043] 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 single clone colony.

[0044] 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.

[0045] 1b.5.2. 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) <![CDATA[dd H20]]> 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) Perform agarose gel electrophoresis on the double enzyme digestion system sample in step (2) to check whether the inserted fragment size is correct; the experimental results are shown in Figure 4 :Enzyme digestion confirmed the construction was 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 Fc-HN protein encoding gene was measured as shown in SEQ ID NO.5.

[0046] 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-HN recombinant plasmid. The map of the recombinant plasmid is shown in Figure 3 The concentration was measured as shown and then stored at -20°C.

[0047] 1c: Transfection of CHO-K1 cells with pEE12.4-OPTI-Fc-HN 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 vol% serum, 1 vol% 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 vol% 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 vol% serum and 1 vol% 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 10 vol% serum and 1 vol% double antibody) and count; (7) Dilute cells to 2×10 5 / mL, take 2mL of the mixed cells and add them to a six-well plate, and place the six-well plate in a CO2 cell culture incubator at 37°C and 5% CO2 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 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; (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, 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 vol% serum and 1 vol% double antibody), and place the six-well plate in a CO2 cell culture incubator at 37°C and 5% CO2 volume percentage.

[0048] 1c.2, Pressurized screening (1) Start pressurization 24 h 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 vol% serum), and pressurize for 7 days. Observe the cells in the middle and replace the medium frequently if there are dead cells.

[0049] 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 vol% trypsin-EDTA, digest at room temperature for about 2 min, add 2 mL of DMEM / F12 culture medium (containing 10 vol% serum + 25 µM MSX) to terminate the digestion reaction, and blow off the cells 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 10 vol% serum + 25 µM 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 vol% 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 away the cells; 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.

[0050] 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 vol% 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 vol% 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 vol% 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) Resuspend the cells in 100 vol% DMEM / F12 medium (containing 10 vol% serum + 25 µM MSX) and count; (7) Dilute cells to 5×10 5 / mL, inoculate 30mL of the culture medium containing suspended cells obtained in step (6) into a 125mL shake flask; place the cell culture flask in a CO2 cell culture incubator at 37°C and 5% CO2 volume percentage on an orbital shaker, and incubate at 120rpm / 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: Sterilize in a biosafety cabinet with UV for 30 min; 100 vol% DMEM / F12 medium (containing 10 vol% serum + 25 µM MSX) and EX-CELL 302 medium were 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 vol% serum + 25 µM MSX) and EX-CELL 302 medium at a ratio of 1:1, resuspend the cells, and count them; (14) Dilute cells to 5×10 5 / mL, 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 on an orbital shaker in a CO2 cell culture incubator at 37°C and a CO2 volume concentration of 5%, and incubate 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) The cell survival rate after two second-generation cultures was greater than 95%; the cell survival rate after three third- to sixth-generation cultures was greater than 95%; after 7 weeks, the cells were inoculated and propagated for three generations, and the cell density reached 1×10 6 / mL, and the cell viability reached 95%, the cells were considered to have adapted to suspension culture; the cell seeding density was reduced to 1×10 5 ~5×10 5 Pieces / mL; (18) After domestication, the 7A2 cell line and the 12B8 cell line in the CHO-K1 cell line both met the requirements, which indicated that the 7A2 cell line and the 12B6 cell line were successfully domesticated.

[0051] 1e: Shake flask fermentation (1) Preparation of subculture medium: Preheat 60 vol% CD-CHO medium + 40 vol% Ex-cell 302 medium to 37°C in a 37°C water bath; (2) Take out the 7A2 cell line and 12B6 cell line cultured in suspension in step 1d (17) and count them; (3) Dilute the 7A2 cell line and 12B6 cell line in step (2) to 2.5×10 5 / mL~3.5×10 5 Each of the two cell lines was inoculated with 30 mL of the subculture medium in step (1) into a 125 mL shake flask. The cell culture flask was placed in a CO2 constant temperature shaker at 37°C and a CO2 volume concentration of 5%, and incubated 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 (about 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 7A2 cells and the culture medium of 12B6 cells were harvested respectively.

[0052] 1f: Protein purification (1) Culture medium treatment: Take 100 ml of cell culture medium, centrifuge at 8000 g for 30 minutes at 4°C, and filter the supernatant through a 0.8 μm filter membrane as the sample; (2) Column equilibration: Load 20 ml of Protein A filler into an empty chromatography column. Equilibrate with ultrapure water for 2 to 3 column volumes (CV), drain the ethanol protective solution, and then equilibrate with 1× PBS for 2 to 3 CV at a flow rate of 5 ml / min. (3) Load the supernatant after filtration at a flow rate of 2 ml / min and collect the flow-through; (4) Equilibrate the column with 15 CV of 1× PBS (300 mM NaCl) at a flow rate of 5 ml / min. (5) Elution: Elute the target protein with elution buffer (100 mM Glycine, 300 mM NaCl, pH 3.0) at a flow rate of 5 ml / min. Collect the target protein until no blue color appears in the Coomassie Brilliant Blue G-250 test. Stop collecting the target protein and immediately neutralize it with neutralization buffer (1 M Tris, pH 9.0) (v:v = 1 ml: 0.037 ml). (6) Change the solution: Place the eluted sample in a dialysis bag and dialyze it at least 1000 times with storage solution (30 mM Tris, 300 mM NaCl, pH 8.5); (7) Sterilization by filtration: In a biological safety cabinet, sterilize the dialyzed protein using a 0.22 μm low protein binding filter membrane; (8) Protein concentration determination The BCA method was used to determine the protein concentration. The protein concentration of this batch was 2.942 mg / ml. The volume of 2.84 mg / ml was approximately 50 ml. After calculation (protein yield = protein concentration × protein volume / volume of culture supernatant), the protein yield of 7A2 cell line and 12B6 cell line was approximately 1.4 g / L to 1.5 g / L.

[0053] 1g: Identification of subunit HN protein; 1g.1, SDS-PAGE detection (1) The protein purified in step 1f was subjected to SDS-PAGE detection. The subunit HN protein concentration in the sample used was 2 μg / well. The results were as follows: Figure 5 As shown; (2) It can be calculated from the figure that the SDS-PAGE purity of the purified subunit HN protein is 95%. It exists in the form of a dimer in the presence of a non-reducing agent with a molecular weight of about 180 kD. It exists in the form of a monomer in the presence of a reducing agent with a molecular weight of about 90 kD.

[0054] 1g.2. Western Blot Detection (1) The protein purified in step 1f was subjected to Western Blot detection. The detection results were as follows: Figure 6 As shown. The concentration of subunit HN protein (marked as 1 in the figure) in the sample used was 2 μg / well; the primary antibody used was derived from the serum of cattle immunized with inactivated bovine parainfluenza vaccine, and the dilution ratio was 1:200; the secondary antibody was HRP-labeled donkey anti-bovine IgG secondary antibody, and the dilution ratio was 1:6000; (2) From Figure 6 It can be seen that the serum can specifically bind to the subunit HN protein of the present invention, thus, it can be concluded that the subunit HN protein prepared by the present invention has excellent immunogenicity.

[0055] 1g.3. ELISA test (1) Coating: On the ELISA plate, dilute the purified subunit HN 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 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 HN 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 1:6000) 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 H2SO4) 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 HN protein can specifically bind to the serum, with an average OD450 of 1.255; the coated subunit HN protein has no specific binding to the blocking solution, with an average OD450 of 0.055. This shows that the subunit HN protein can be used as an antigen for the ELISA kit and has good immunogenicity. After exploring the appropriate coating concentration and serum dilution ratio, a diagnostic kit for detecting bovine parainfluenza infection and immunity can be developed.

[0056] Table 6: Results of ELISA assay for identification of subunit HN protein sample OD450 value of coated HN protein serum 1.26 serum 1.25 serum 1.28 serum 1.23 Blocking solution 0.058 Blocking solution 0.047 Blocking solution 0.050 Blocking solution 0.063

[0057] 1g.4 Stability Verification (1) Dilute the purified subunit HN protein in step 1f to 1 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 by BCA after each sampling. The results are shown in Table 7 below: Table 7: Stability of subunit HN proteins sample Sample concentration after treatment at 4℃ (mg / ml) Sample concentration after -20℃ treatment (mg / ml) First sampling 1.09 1.08 Second sampling 1.08 1.06 The third sampling 1.08 1.04 The fourth sampling 1.06 1.08 The fifth sampling 0.97 0.99 The sixth sampling 1.03 0.99 The seventh sampling 1,02 1.01 The eighth sampling 0.99 1.00 The ninth sampling 0.95 0.98 The tenth sampling 0.96 0.95

[0058] See 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 was degraded, we used the 10th sample for SDS-PAGE detection. The specific results are as follows: Figure 7 and Figure 8 As shown. Figure 7 M is the marker; 1 is the subunit HN protein after treatment at 4℃, and the loading amount is 2μg; Figure 8 M is the marker; 1 is the subunit HN protein after -20℃ treatment, and the loading amount is 2μg. Figure 7 and Figure 8 It can be seen that the treated sample (the 10th sampling) is still stable, which shows that the subunit HN protein prepared by the present invention has excellent stability.

[0059] 1h: Vaccine preparation (1) Preparation of aqueous phase: according to the content of subunit HN protein in the vaccine, the subunit HN 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 HN 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.

[0060] 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 20-50 cp to be considered acceptable.

[0061] Example 2: The difference from Example 1 is that the amino acid sequence encoded by the subunit HN protein in this example is the extracellular region of the HN protein, 57Q-572S.

[0062] Example 3: The difference from Example 1 is that the coding gene sequence of the subunit HN protein in this example is the extracellular region of the HN protein, 143T-572S.

[0063] Example 4: The difference from Example 1 is that in this example, the subunit HN protein is fused with a bovine Fc sequence based on the HN shown in SEQ ID NO.1.

[0064] Example 5: The difference from Example 1 is that the eukaryotic expression vector used in this example is pEE6.4, and the cell line is the DG44 cell line among the CHO cell lines.

[0065] Example 6: The difference from Example 1 is that the eukaryotic expression vector used in this example is pGL4.13, and the cell line is the CHO-K1 cell line among the CHO cell lines.

[0066] Example 7: The difference from Example 1 is that the eukaryotic expression vector used in this example is pcDNA3.1, and the cell line is the CHO-S cell line among the CHO cell lines.

[0067] Example 8: The difference from Example 1 is that the eukaryotic expression vector used in this example is pEE12.4, and the cell line is the CHO-S cell line among the CHO cell lines.

[0068] Example 9: The difference from Example 1 is that the eukaryotic expression vector used in this example is pEE12.4, the cell line is HEK293 cell line, and the subunit HN protein does not require the acclimation operation in step 1d during the preparation process.

[0069] Example 10: The difference from Example 1 is that the eukaryotic expression vector used in this example is pEE12.4, the cell line is 293T / 17 cell line, and the subunit HN protein does not require the acclimation operation in step 1d during the preparation process.

[0070] Example 11: The difference from Example 1 is that the eukaryotic expression vector used in this example is pCEP4, and the cell line is the DG44 cell line among the CHO cell lines.

[0071] Comparative Example: The difference from Example 1 is that the coding gene sequence of the HN protein of the bovine parainfluenza virus in this comparative example is the genome sequence of 6986-8536 bp in GenBank: OM621819.

[0072] Example 12: Determination of the expression yield and protein purity of subunit HN protein The expression yield and protein purity of the subunit HN protein in Examples 2 to 11 and the comparative example were measured, and the expression results are shown in Table 8 below.

[0073] Table 8: Yield and purity of subunit HN protein of Examples 2-11 and Comparative Examples project Protein yield (g / L) Protein purity (%) Example 2 0.1-0.2 60 Example 3 0.5-0.7 70 Example 4 0.7-0.9 80 Example 5 1.3-1.4 88 Example 6 1.2-1.4 86 Example 7 1.1-1.3 85 Example 8 1.2-1.4 85 Example 9 1.5-1.6 82 Example 10 1.4-1.6 85 Embodiment 11 1.2-1.4 80 Comparative Example 0.0-0.02 39

[0074] Referring to Table 8, the protein yield and protein purity of the present invention are significantly better than the protein yield of 0.0 g / L-0.02 g / L and the protein purity of 39% in the comparative example. It can be concluded that the subunit HN protein encoding gene constructed by the present invention can efficiently secrete and express the subunit HN protein in the cell line, and the obtained subunit HN protein has a high protein purity.

[0075] The protein yields of Examples 2 to 4 are between 0.1 g / L and 0.9 g / L, and the protein purity is between 60% and 80%, while the protein yield of Example 1 is between 1.4 g / L and 1.5 g / L, and the protein purity is 90%. Therefore, Example 1 is a preferred embodiment among Examples 1 to 4. It can be concluded that when the coding gene sequence of the subunit HN protein is SEQ ID NO.5, the corresponding protein yield and protein purity reach the optimal values.

[0076] The protein yields of Examples 5 to 8 were 1.1 g / L-1.4 g / L, and the protein purity was 85%-88%, which were lower than the test results of Example 1. The protein yields of Examples 9 to 11 were 1.2 g / L-1.6 g / L, and the protein purity was 80%-85%. It can be concluded that when the eukaryotic expression vector selected pEE12.4 and the cell line selected one of the CHO cell line, the HEK293 cell line, and the 293T / 17 cell line, the expression yield and protein purity of the subunit HN protein were both high; especially when the eukaryotic expression vector selected pEE12.4 and the cell line selected CHO-K1 cell line, the expression amount and protein purity of the subunit HN protein reached the optimal value.

[0077] 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 HN 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 HN 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 HN protein of the present invention has the characteristics of efficient secretion expression, high protein purity, easy purification, low production cost, high safety performance, etc.

[0078] 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

1. A subunit HN protein of a bovine parainfluenza virus, characterized in that The subunit HN protein is the extracellular region of the bovine parainfluenza virus HN protein, and its amino acid sequence is: 1) The amino acid sequence shown in SEQ ID NO.1; 2) An immunogenic derivative amino acid sequence obtained by substituting, deleting or adding one or more amino acids from SEQ ID NO.

1.

2. The subunit HN protein according to claim 1, characterized in that The subunit HN protein is a truncated protein of the extracellular region of the bovine parainfluenza virus HN protein, and its amino acid sequence is shown in SEQ ID NO.

2.

3. The subunit HN protein according to claim 1 or 2, characterized in that The amino terminus or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.2 is connected to a bovine antibody constant region Fc, whose amino acid sequence is shown in SEQ ID NO.8, and a tag selected from poly-His, FLAG, c-myc, HA, and poly-Arg.

4. The subunit HN protein of bovine parainfluenza virus according to claim 3, characterized in that The amino acid gene sequence of the subunit HN protein is shown in SEQ ID NO.3 or SEQ ID NO.

4.

5. The subunit HN protein of bovine parainfluenza virus according to claim 4, characterized in that The coding gene sequence of the subunit HN protein of the amino acid gene sequence shown in SEQ ID NO.4 is shown in SEQ ID NO.5 or SEQ ID NO.

6.

6. The method for preparing the subunit HN protein of bovine parainfluenza virus according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: 1) The coding gene sequence of the subunit HN protein of bovine parainfluenza virus is constructed as shown in SEQ ID NO.5; 2) cloning the coding gene sequence of the subunit HN protein constructed in step 1) into a eukaryotic expression vector to obtain a recombinant plasmid containing the coding gene sequence of the subunit HN protein; 3) transfecting the recombinant plasmid containing the coding gene sequence of the subunit HN protein 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 HN protein of bovine parainfluenza virus.

7. The method for preparing the subunit HN protein 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 the subunit HN 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 the subunit HN 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 the subunit HN 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 HN protein of bovine parainfluenza virus according to any one of claims 1 to 5 in a subunit vaccine or diagnostic reagent for bovine parainfluenza virus.

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