A method for preparing and application of a subunit HN protein of bovine parainfluenza virus

Abstract

The present invention relates to a subunit HN protein of bovine parainfluenza virus, a preparation method thereof and an application thereof. 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) a derived amino acid sequence with immunogenicity obtained by substituting, deleting or adding one or several amino acids to SEQ ID NO.1, and the derived amino acid sequence has 80%-100% sequence identity with SEQ ID NO.2. The subunit HN protein is mainly prepared by constructing a recombinant plasmid, transfecting the recombinant plasmid into a cell line, screening a cell line with high expression, and purifying the subunit HN protein of bovine parainfluenza virus. It can be preferably applied to subunit vaccines or diagnostic reagents for bovine parainfluenza virus, and has the characteristics of high secretion expression efficiency, high protein purity, easy purification, reduced production cost, high safety performance, etc.

Description

Technical Field

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

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

[0003] Currently, there is no effective treatment for this disease, and prevention and control mainly rely on vaccines. There is no vaccine on the market in China. There are traditional inactivated vaccines and attenuated live vaccines against 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 members of the Paramyxoviridae family. The virus particles are polymorphic, ranging from spherical to filamentous structures, with a diameter of 150 - 300 nm. The BPIV3 genome encodes 6 major structural proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and large polymerase protein (L) protein. Among them, F protein and HN protein are the main proteins of the virus envelope, which can stimulate the body to produce neutralizing antibodies and are the first glycoproteins of subunit vaccines. The HN protein subunit vaccine does not contain nucleic acid substances and will not produce persistent infection or latent infection after inoculation; the immune response produced can be distinguished from wild virus infection, which is conducive to the control and elimination of the disease. However, the HN subunit vaccine also has obvious defects: the expression yield is relatively low, the production cost is high, and its application is restricted.

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

[0006] Engineered cells are widely used expression cells in current 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. It usually achieves high-density culture in suspension culture, and the culture volume can reach more than 2,000 L, so it can be mass-produced.

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

[0008] Chinese patent application CN202011013669.6 discloses a recombinant antigen of bovine parainfluenza, and its antigen is a heterodimer formed by a truncated F protein and an HN protein, that is, bovine antibody Fc fragments are added to the C-terminal of both the F protein fragment and the HN protein fragment, so that when the two protein fragments are co-expressed in a cell, a more stable heterodimer can be formed. However, only a small fragment of the HN protein is used, and the Fc is not mutated, so a pure heterodimer cannot be obtained, nor can a single HN protein be obtained. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the first object of the present invention is to provide a subunit HN protein of bovine parainfluenza virus, and the subunit HN protein has excellent immunogenicity and stability of the HN protein of bovine parainfluenza virus, and is convenient for stable and efficient secretory expression 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 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 preferably applied to subunit vaccines and diagnostic reagents for bovine parainfluenza virus, thereby facilitating the prevention and control of bovine parainfluenza virus by people.

[0012] 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:

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

[0014] 2) An immunogenic derived amino acid sequence obtained by substituting, deleting or adding one or several amino acids to SEQ ID NO.1.

[0015] 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 HN protein of bovine parainfluenza virus, and its amino acid sequence is shown in SEQ ID NO.2.

[0016] According to the subunit HN protein of the present invention, preferably, the constant region Fc of bovine antibody 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, and a tag selected from poly-His, FLAG, c-myc, HA, poly-Arg.

[0017] According to the subunit HN protein of the present invention, preferably, the amino acid gene sequence of the subunit HN protein is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0018] According to the subunit HN protein of the present invention, preferably, the coding gene sequence of the subunit HN protein with the amino acid gene sequence shown in SEQ ID NO.4 is shown in SEQ ID NO.5.

[0019] To achieve the above second object, the present invention provides a method for preparing a subunit HN protein of bovine parainfluenza virus, and the preparation method includes the following steps:

[0020] 1) Construct the coding gene sequence of the subunit HN protein of bovine parainfluenza virus as shown in SEQ ID NO.5;

[0021] 2) Clone 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;

[0022] 3) Transfect the recombinant plasmid containing the coding gene sequence of the subunit HN protein obtained in step 2) into engineered cells of an animal to obtain a cell line;

[0023] 4) Screen out a cell line with high expression from the cell line obtained in step 3); and

[0024] 5) Ferment and culture the cell line with high expression obtained in step 4), and purify to obtain the subunit HN protein of bovine parainfluenza virus.

[0025] 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.

[0026] In the preparation method of the present invention, preferably, in step 2), the eukaryotic expression vector is pEE12.4.

[0027] In the preparation method of the present invention, preferably, in step 3), the cell line is one of CHO cell line, HEK293 cell line, and 293T / 17 cell line.

[0028] In the preparation method of the present invention, preferably, in step 3), the CHO cell line is one of DG44 cell line, DXB11 cell line, CHO-K1 cell line, and CHO-S cell line.

[0029] In the technical solution of the present invention, preferably, in step ③, the CHO cell line is one of DG44 cell line, DXB11 cell line, CHO-K1 cell line, and CHO-S cell line.

[0030] To achieve the above third object, the present invention provides an application of the subunit HN protein of the bovine parainfluenza virus in a subunit vaccine or diagnostic reagent for bovine parainfluenza virus.

[0031] 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 the HN protein of bovine parainfluenza virus, and is convenient for stable and efficient secretion and expression in engineered cell lines. It has a high yield and is easy to purify. The purity of the target protein in the cell culture supernatant can reach more than 70%, and only one-step affinity chromatography can make the purity of the target protein reach more than 90%, far meeting the requirements of subunit vaccines and diagnostic reagents, and facilitating large-scale production. Thus, the technical problems of low expression yield and high production cost of the HN protein of bovine parainfluenza virus are solved. In addition, since engineered cell lines such as CHO cell lines, HEK293 cell lines, and 293T cell lines used for production have high controllability, easy quality control, and stable protein production among batches during cultivation, the amount of other viruses in the subunit HN protein of bovine parainfluenza virus produced by the present invention is small, effectively reducing the risk of virus dissemination, and having excellent biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It shows a three-dimensional structural model diagram of the predicted subunit HN fusion protein;

[0033] Figure 2 It shows the comparison results of the coding gene sequence of the subunit HN protein before and after optimization, specifically the comparison of the coding gene sequences of SEQ ID NO.6 and SEQ ID NO.7;

[0034] Figure 3 It shows the plasmid map of pEE12.4-OPTI-Fc-HN;

[0035] Figure 4 It shows the double digestion identification result of pEE12.4-OPTI-Fc-HN: M is DNA Marker: DL10000 Marker; 1 is the electrophoresis result of double digestion of pEE12.4-OPTI-Fc-HN with HindIII and EcoRI;

[0036] Figure 5 It shows the SDS-PAGE electrophoresis of the subunit HN protein, where 1 is the electrophoresis detection result of the HN protein in the presence of a reducing agent, 2 is the detection result in the absence of a reducing agent, and M is the Marker;

[0037] Figure 6 It shows the Western Blot detection result after purification of the subunit HN protein, where 1 is the subunit HN protein and 2 is the Marker;

[0038] Figure 7 It shows the stability detection result after purification of the subunit HN protein, where M is the Marker; 1 is the subunit HN protein after treatment at 4°C, and the loading amount is 2 μg;

[0039] Figure 8 It shows the stability detection results of the purified subunit HN protein. Among them, M is the Marker; 1 is the subunit HN protein after treatment at -20°C, and the sample loading amount is 2 μg. Specific implementation manners

[0040] The present invention will be further described below in conjunction with the drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and do not limit the present invention.

[0041] The strains, plasmids and reagents used in the embodiments of the present invention are all commercially available products.

[0042] The sources of the reagents and drugs of the present invention are listed as follows:

[0043] pEE12.4 and CHO-K1 cells are preserved by Zhejiang Hailong Biotechnology Co., Ltd.;

[0044] The cell culture medium and serum are both purchased from Gibco Company, USA;

[0045] Lipofectamine LTX, PLUS TM reagent, CB5, and BCA protein quantification kit are purchased from ThermoFisher Company, USA.

[0046] Example 1: 1a: Analyze and optimize the structure of the bovine parainfluenza virus HN protein, and construct the coding gene sequence of the subunit HN protein of the bovine parainfluenza virus

[0047] By analyzing the bovine parainfluenza virus HN protein sequence (GenBank: OM621819.1), it is found that the genomic sequence is 6818 - 8536 bp, encoding the bovine parainfluenza HN protein sequence. Further analysis shows 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. Combining with the experience of studying the expression of viral envelope proteins, the extracellular region of the HN protein expressed by CHO-K1 cells is selected as the immunogenic protein of the present invention, that is, the amino acid sequence of 57Q - 572S, and this amino acid sequence is a known fragment and can be obtained by querying NBCI.

[0048] However, when expressing the amino acid sequence of 57Q-572S in the CHO-K1 cell line, it was found that the expression level of the fragment protein directly expressed was very low and could not meet the requirements of large-scale production. Further, through protein structure prediction and research on the structure of amino acids themselves, it was found that the HN protein is a homodimer, 57Q-142M is the rod region of the HN protein, and 143T-572S is the head region of the HN protein. The amino acids of 57Q-142M may affect the expression of this protein in CHO-K1 cells, and this polypeptide segment is not the immunogenic site of the HN protein. 143T-572S is the main antigenic epitope region of the HN protein. Further, in order to improve the stability and yield of the HN protein, the bovine antibody constant region sequence was fused with it for expression, and the yield of the HN protein was increased.

[0049] Therefore, the present invention excises the amino acids of 57Q-142M and expresses the amino acid sequence of 143T-572S. The three-dimensional structural pattern diagram predicted for this amino acid sequence is as Figure 1 shown, and 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 was fused with it for expression, and the yield of the HN protein was increased.

[0050] Among them, on the basis of the amino acid sequence SEQ ID NO.3, a derivative protein is prepared by substituting, deleting or adding one or several amino acids, and the homology between this derivative protein and the amino acid sequence of the subunit HN protein in this example (as shown in SEQ ID NO.3) is as high as 80%-100%, so as to ensure that the two have the same immunogenicity, or use the antibody Fc fragment of other species for fusion expression. Therefore, this derivative protein also falls within the protection scope of the present invention.

[0051] In order to facilitate the detection or purification of the subunit HN protein, a tag 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 example, specifically taking Poly-His as an example, it is connected to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.4.

[0052] Table 1: Tags and Their Amino Acid Sequences

[0053] 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) 。

[0054] The coding HN gene sequence part of the amino acid sequence SEQ ID NO.4 can be as shown in SEQ ID NO.6, or can be obtained by codon optimization of SEQ ID NO.7. In this example, the coding gene sequence of the subunit HN protein is codon-optimized based on SEQ ID NO.4 to obtain the OPTI-HN sequence, as shown in SEQ ID NO.5. The artificial synthesis of this gene sequence was commissioned to Nanjing Genscript Biotech Co., Ltd.

[0055] 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), and the result is as Figure 2 shown, with a total of 537 / 2037 = 26.4% differences.

[0056] 1b: Construction of pEE12.4-OPTI-Fc-HN recombinant plasmid; 1b.1. PCR amplification of the target fragment OPTI-Fc-HN; 1b.1.1. PCR reaction

[0057] (1) Primer design and synthesis

[0058] Forward primer:

[0059] 5’-cacgaAGCTTGCCGCCACCATGGCAG-3’

[0060] Reverse primer:

[0061] 5’-AATTGAATTCTCAATGGTGGTGATGGTG-3’

[0062] (2) 50 μL loading system, as shown in Table 2 below.

[0063] Table 2: 50 μL loading system

[0064] Sample components Volume (μL) Q5 Mix 25 Forward primer (10 μM) 2.5 Reverse primer (10 μM) 2.5 OPTI-Fc-HN 1 <![CDATA[dd H2O]]> 19 Total volume 50 .

[0065] PCR amplification program:

[0066] 95℃ 2 min

[0067] 95℃ 30 s

[0068] 58℃ 45 s

[0069] 72℃ 1 min

[0070] 72℃ 10 min

[0071] 10℃ Forever

[0072] Among them: One cycle is completed successively from 95°C for 30 s, 58°C for 45 s, and 72°C for 1 min, and this cycle is repeated 30 times.

[0073] 1b.1.2, Gel extraction of PCR products

[0074] (1) Label the sample collection EP tube, adsorption column CB2, and collection tube;

[0075] (2) Weigh the labeled empty EP tube and record the value;

[0076] (3) Carefully cut the single target DNA band from the agarose gel with a scalpel on a gel cutter and put it into a clean 1.5 mL centrifuge tube;

[0077] (4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3), place it in a 50°C water bath for about 5 min, and gently invert the centrifuge tube up and down continuously during this period to ensure that the gel block is fully dissolved;

[0078] (5) Column equilibration: Add 500 μL of equilibration buffer BL to the adsorption column CB2 (the adsorption column CB2 is pre-placed in the collection tube), centrifuge at 12000 rpm / min for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 back into the collection tube;

[0079] (6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 min, centrifuge at 10000 rpm / min for 30 s, pour out the waste liquid in the collection tube, and then put the adsorption column CB2 into the collection tube;

[0080] (7) Add 600 μL of wash buffer PW buffer to the adsorption column CB2, let it stand for 3 min, centrifuge at 10000 rpm / min for 30 s, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube;

[0081] (8) Repeat step (7);

[0082] (9) Centrifuge the empty adsorption column at 12000 rpm / min for 2 min to remove the wash buffer as much as possible, place the adsorption column CB2 at room temperature for 10 min, and dry it thoroughly;

[0083] (10) Put the adsorption column CB2 into the collection tube, suspend and drop 50 μL of Elutionbuffer (preheated at 65°C) at the middle position of the adsorption membrane, let it stand for 3 min, and centrifuge at 12000 rpm / min for 2 min;

[0084] (11) Take out the centrifuge tube in step (10) from the centrifuge, discard the middle adsorption column CB2, cover the centrifuge tube lid, and retain the DNA sample in the centrifuge tube;

[0085] (12) Place the DNA sample in step 11 at 4 °C for storage, and prepare for agarose gel electrophoresis to identify the gel-extracted DNA fragment.

[0086] 1b.2. Double digestion reaction of PCR product and vector

[0087] (1) Label the required 1.5 mL EP tubes, add samples and mix them in the EP tubes according to Table 3 below. The DNA sample in Table 3 is the DNA fragment finally recovered in step 1b.1.2 (12);

[0088] Table 3: 50 μL reaction system

[0089] Sample component name Volume (μL) <![CDATA[dd H20]]> Make up to 50 10×buffer 5 DNA sample Volume at 2 μg HindⅢ 2.5 EcoRⅠ 2.5

[0090] (2) Place the EP tube in step (1) in a 37 °C constant temperature water bath for 2 - 3 h;

[0091] Gel extraction of double digestion products: Take out the above double digestion system, perform agarose gel electrophoresis to recover the DNA fragment therein, and the method is the same as the gel extraction of PCR products in step 1b.1.2.

[0092] 1b.3. Ligation reaction

[0093] (1) Prepare several clean 1.5 mL EP tubes, make marks, and place them on the EP tube rack for later use;

[0094] (2) Add samples and mix them in the EP tubes in step (1) according to Table 4 below. The target fragment in Table 4 is the DNA fragment finally recovered in step 1b.2 (2);

[0095] Table 4: 10 μL reaction system

[0096] Sample component name Experimental group (μL) Blank group (μL) <![CDATA[dd H20]]> / 6 10× T4 ligation buffer 1 1 Target fragment 6 - Vector 2 2 T4 ligase 1 1

[0097] (3) After completing the sample addition according to the table in step (2), place each 10 μL reaction system in a 16 °C low-temperature coolant circulator for 10 - 16 h;

[0098] (4) Take out the EP tube in step (3) and place it in a 65 °C water bath for 15 min;

[0099] (5) Take out the EP tube in step (4) and place it at 4 °C for storage to obtain the ligation reaction solution.

[0100] 1b.4. Transformation reaction

[0101] (1) Quickly add 10 μL of the ligation reaction solution prepared in step 1b.3(5) to 100 μL of competent cells, pipette and mix well, and incubate on ice for 30 min;

[0102] (2) Take out the sample tube, place it in a 42 °C water bath for 100 s, and then immediately incubate on ice for 2 min;

[0103] (3) Take out the sample tube, in a laminar flow hood, add 600 μL of liquid LB medium to the sample tube, and then place the sample tube in a 37 °C constant temperature shaker at 220 rpm / min for 1 h;

[0104] (4) Plate coating: Take out the sample tube in step (3), centrifuge at room temperature at 8000 rpm / min for 2 min, discard 600 μL of the supernatant, resuspend the cells at the bottom of the tube with the remaining supernatant, place the resuspended bacterial solution in the center of the corresponding transformation plate, and use a bacterium spreading rod to spread the bacterial solution in the center of the transformation plate evenly;

[0105] (5) Place the transformation plate in step (4) upright in a biochemical constant temperature incubator, incubate at 37 °C for 1 h, then invert the transformation plate for incubation for 15 h to obtain monoclonal colonies.

[0106] 1b.5. Plasmid extraction and double digestion identification; 1b.5.1. Plasmid extraction

[0107] (1) Use a 10 μL pipette tip to pick a monoclonal strain from the transformation plate in step 1b.4(5) into 5 mL of liquid LB medium containing ampicillin resistance, and shake the bacteria overnight at 37 °C and 220 rpm / min;

[0108] (2) Transfer the bacterial solution to a 1.5 mL EP tube, centrifuge at room temperature at 12000 rpm / min for 2 min, and discard the supernatant;

[0109] (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to completely suspend the cells;

[0110] (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 well, and let it stand at room temperature for 2 - 4 min;

[0111] (5) Add 350 μL of plasmid extraction reagent P3 buffer to the solution in step (4), immediately gently invert the centrifuge tube 5 - 10 times to mix well; let it stand at room temperature for 2 - 4 min;

[0112] (6) Centrifuge the solution in step (5) at room temperature at 14000 rpm / min for 10 min;

[0113] (7) Transfer the supernatant solution in step (6) to the center of the adsorption column, centrifuge at room temperature at 12,000 rpm / min for 30 s, and pour out the liquid in the collection tube;

[0114] (8) Add 500 μL of Buffer DW1 to the center of the adsorption column, centrifuge at room temperature at 12,000 rpm / min for 30 s, and pour out the liquid in the collection tube;

[0115] (9) Add 500 μL of wash solution to the center of the adsorption column, centrifuge at room temperature at 12,000 rpm / min for 30 s, pour out the liquid in the collection tube, and repeat once;

[0116] (10) Empty the adsorption column and centrifuge at room temperature at 12,000 rpm / min for 2 min;

[0117] (11) Place the adsorption column into a clean 1.5 mL centrifuge tube, add 30 μL of Elution buffer to the center of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at room temperature at 12,000 rpm / min for 2 min, and save the DNA solution in the tube.

[0118] 1b.5.2, Double digestion identification

[0119] (1) Label the required 1.5 mL EP tubes, add samples according to Table 5 below, where the DNA sample in Table 5 is the DNA solution finally obtained in step 1b.5.1 (11);

[0120] Table 5: 20 μL reaction system

[0121] Sample component name Volume (μL) <![CDATA[dd H20]]> Make up to 20 μL 10×buffer 2 DNA sample Volume at 1 μg mass HindⅢ 1 EcoRⅠ 1

[0122] (2) Place the 20 μL reaction system in the EP tube in step (1) in a 37 °C constant temperature water bath for 2 h;

[0123] (3) Perform agarose gel electrophoresis on the double digestion system sample in step (2) to check whether the size of the inserted fragment is correct; the experimental results are shown in Figure 4 : The digestion identification is correctly constructed;

[0124] (4) Select the recombinant plasmid with the correct inserted fragment and send it to a sequencing company for sequencing. In the present invention, this recombinant plasmid was sent to Genewiz Biotechnology Co., Ltd. for determination, and the encoded gene sequence of the subunit Fc-HN protein was as shown in SEQ ID NO.5.

[0125] 1b.6, Large-scale extraction of endotoxin-free plasmid; 1b.6.1, Extraction of endotoxin-free plasmid

[0126] (1) Inoculate the clone with correct sequencing in step 1b.5.2(4) into 100 mL of medium containing ampicillin resistance, and culture it in a constant temperature shaker at 37°C with a rotation speed of 220 rpm / min for 15 h.

[0127] (2) Transfer the bacterial liquid cultured in step (1) into a 50 mL centrifuge tube, centrifuge it at room temperature at 8000 rpm / min for 5 min, collect the bacterial cells, and discard the supernatant medium.

[0128] (3) Add 8 mL of plasmid extraction reagent P1 buffer to the centrifuge tube in step (2), and resuspend the bacterial cells thoroughly with a pipette.

[0129] (4) Add 8 mL of plasmid extraction reagent P2 buffer to the centrifuge tube in step (3), immediately invert the centrifuge tube gently 6 - 8 times, and let it stand at room temperature for 5 min.

[0130] (5) Add 8 mL of plasmid extraction reagent P4 buffer to the centrifuge tube in step (4), immediately invert it up and down 6 - 8 times to mix well until white flocculent precipitate appears in the solution, let it stand at room temperature for about 10 min, and centrifuge it at room temperature at 8000 rpm / min for 5 - 10 min to make the white precipitate settle to the bottom of the tube.

[0131] (6) Carefully transfer all the supernatant in step (5) into the filter CS1, slowly push the handle of the filter, and collect the filtrate in a clean 50 mL centrifuge tube.

[0132] (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 it at room temperature at 8000 rpm / min for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0133] (8) Add isopropanol with a volume 0.3 times that of the filtrate in step (6) to the filtrate, mix it by inverting up and down, and transfer it to the adsorption column CP6. Centrifuge it at room temperature at 8000 rpm / min for 2 min, pour out the liquid in the collection tube, and put the adsorption column CP6 back into the same collection tube.

[0134] (9) Add 10 mL of washing solution PW buffer to the adsorption column CP6 in step (8), centrifuge it at room temperature at 8000 rpm / min for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0135] (10) Repeat the operation in step (9) once.

[0136] (11) Add 3 mL of absolute ethanol to the adsorption column CP6 in step (10), centrifuge at room temperature at 8000 rpm / min for 2 min, and pour off the waste liquid;

[0137] (12) Put the adsorption column CP6 from step (11) back into the collection tube, centrifuge at room temperature at 8000 rpm / min for 5 min, open the lid of the adsorption column CP6, and place it at room temperature for several minutes to dry;

[0138] (13) Put 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 it stand at room temperature for 5 min, centrifuge at room temperature at 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 this recombinant plasmid is as Figure 3 shown. After measuring its concentration, store it at -20 °C.

[0139] 1c: Establishment of transfection of CHO - K1 cells with pEE12.4 - OPTI - Fc - HN recombinant plasmid and monoclonal screening; 1c.1, Transfection of CHO - K1 cells

[0140] (1) Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 min; Preheat the DMEM / F12 medium (containing 10 vol% serum and 1 vol% double antibody) and PBS buffer in a 37 °C water bath to 37 °C;

[0141] (2) Take out the CHO - K1 cells (10 cm cell culture dish) from the 37 °C CO2 cell incubator, discard the supernatant medium, wash the cells once with 8 mL of pre - warmed PBS buffer, and discard the PBS buffer;

[0142] (3) Add 1 - 2 mL of 0.25 vol% trypsin - EDTA to each 10 cm cell culture dish, digest at room temperature for about 2 min, observe under the microscope that the cells shrink and become round and are single cells;

[0143] (4) Add 4 mL of DMEM / F12 medium (containing 10 vol% serum and 1 vol% double antibody) to terminate the digestion reaction, and disperse the cells with a pipette;

[0144] (5) Transfer the digested cells to a 15 mL centrifuge tube, centrifuge at room temperature, 200 g, for 5 min;

[0145] (6) Resuspend the cells with DMEM / F12 medium (containing 10 vol% serum and 1 vol% double antibody) and count;

[0146] (7) Dilute the cells to 2×10 5 cells / mL. Take 2 mL of the well-mixed cells and add them to a six-well plate. Place the six-well plate in a CO2 cell incubator at 37°C with a CO2 volume percentage of 5% and incubate overnight;

[0147] (8) Take out the six-well plate in step (7) and observe the cell state: When the cell confluence reaches 80%-90%, transfection can begin. Before transfection, change the medium to DMEM / F12 medium without antibiotics and serum, 2 mL / well;

[0148] (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;

[0149] (10) Dilute Lipofectamine LTX: Add 9 μL of Lipofectamine LTX to 125 μL of OPTI-MEM medium, and then add 2.5 μL of PLUS TM reagent, gently mix well, and let stand at room temperature for 5 min;

[0150] (11) Gently mix the mixtures in steps (10) and (11), let stand at room temperature for 5 min, and then add dropwise to the six-well plate for even distribution;

[0151] (12) Place the six-well plate in a CO2 cell incubator at 37°C with a CO2 volume percentage of 5% and culture for 4-6 h;

[0152] (13) Change the medium: Discard the supernatant medium, add 2 mL of DMEM / F12 medium (containing 10 vol% serum and 1 vol% double antibody), and place the six-well plate in a CO2 cell incubator at 37°C with a CO2 volume percentage of 5% for culture.

[0153] 1c.2. Pressure screening

[0154] (1) Start pressure screening 24 h after transfection: Take out the cells in the six-well plate from the CO2 cell incubator in step 1c.1 (11), discard the supernatant medium, add 2 mL of DMEM / F12 (containing 10 vol% serum), perform pressure screening for 7 d, observe the cells in the middle, and change the medium if there are many dead cells.

[0155] 1c.3. Monoclonal screening

[0156] (1) When all the cells in the negative control die during the pressure screening in step 1c.2, which is about 7 d, start monoclonal screening;

[0157] (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 medium (containing 10 vol% serum + 25 μM MSX) to terminate the digestion reaction, and disperse the cells with a pipette;

[0158] (3) Transfer the digested cells to a 15 mL centrifuge tube, centrifuge at room temperature, 200 g, for 5 min;

[0159] (4) Resuspend the cells with DMEM / F12 medium (containing 10 vol% serum + 25 μM MSX) and count;

[0160] (5) Plate: Dilute the cells to 5 cells / mL, take 200 μL of the mixed cells and add them to a 96-well plate, place it in a CO2 cell incubator at 37 °C with a CO2 volume percentage of 5% and incubate for 4 - 6 h;

[0161] (6) Record the wells with single cells;

[0162] (7) When the wells with single cells in the 96-well plate grow up, discard the culture medium, wash once with PBS buffer, add 100 μL of 0.25 vol% trypsin-EDTA, digest at room temperature for about 2 min, add 2 mL of DMEM / F12 medium (containing 10 wt% serum + 25 μM MSX) to terminate the digestion reaction, and disperse the cells with a pipette; Transfer the cell suspension to a 12-well plate. When the 12-well plate is full, take the supernatant and detect whether the clone is positive by ELISA. The positive clones with high expression are further expanded and cryopreserved.

[0163] 1d: Domestication of CHO-K1 cell line into suspension culture

[0164] (1) Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 min; Preheat the DMEM / F12 medium (containing 10 vol% serum + 25 μM MSX) in a 37 °C water bath to 37 °C;

[0165] (2) Take out the cells obtained in step 3.3 (7) (10 cm cell culture dish), discard the supernatant culture medium, wash the cells once with 8 mL of pre-warmed PBS buffer, and discard the PBS buffer;

[0166] (3) Add 1 - 2 mL of 0.25 vol% trypsin-EDTA to each 10 cm cell culture dish, digest at room temperature for about 2 min, observe under the microscope that the cells shrink and become round and are single cells;

[0167] (4) Add 4 mL of DMEM / F12 medium (containing 10 vol% serum + 25 µM MSX) to terminate the digestion reaction, and disperse the cells with a pipette;

[0168] (5) Transfer the digested cells to a 15 mL centrifuge tube, centrifuge at room temperature at 200 g for 5 min;

[0169] (6) Suspend the cells with 100 vol% DMEM / F12 medium (containing 10 vol% serum + 25 µM MSX) and count;

[0170] (7) Dilute the cells to 5×10 5 cells / mL, and inoculate 30 mL of the cell suspension obtained in step (6) into a 125 mL shake flask; Place the cell culture flask on an orbital shaker in a CO2 cell incubator at 37 °C with a CO2 volume percentage of 5%, and incubate overnight at 120 rpm / min;

[0171] (8) Wipe and disinfect the biosafety workbench with 75% alcohol and irradiate with ultraviolet light for 30 min;

[0172] (9) Count the cell density and viability every 24 h;

[0173] (10) Perform the second-generation culture when the cell survival rate reaches 94 - 97% after the first-generation cells are cultured once;

[0174] (11) Preparation: Sterilize the biosafety cabinet with ultraviolet light for 30 min; Place 100 vol% DMEM / F12 medium (containing 10 vol% serum + 25 µM MSX) and EX-CELL 302 medium in a CO2 cell incubator with a CO2 volume percentage of 5% and preheat to 37 °C;

[0175] (12) Take out the cells from the CO2 cell incubator in step (11) and transfer them to a 50 mL centrifuge tube, centrifuge at room temperature at 200 g for 5 min;

[0176] (13) Mix the 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;

[0177] (14) Dilute the cells to 5×10 5 cells / mL, and inoculate 30 mL of the cell suspension obtained in step (13) into a 125 mL shake flask; Place the cell culture flask on an orbital shaker in a CO2 cell incubator at 37 °C with a CO2 volume concentration of 5%, and incubate overnight at 120 rpm / min;

[0178] (15)The biosafety workbench surface is wiped and disinfected with 75 wt% alcohol and irradiated with ultraviolet light for 30 min;

[0179] (16)The cell density and viability are counted every 24 h;

[0180] (17)The cell survival rate of the second-generation cells after two passages is greater than 95%; the cell survival rate of the third to sixth-generation cells after three passages is greater than 95%; after 7 weeks, the cells are inoculated and propagated for three generations 3 days later, and the cell density reaches 1×10 6 cells / mL, and at the same time the cell survival rate reaches 95%, and the cells are considered to have adapted to suspension culture; the cell inoculation density is reduced to 1×10 5 - 5×10 5 cells / mL;

[0181] (18)After domestication, both the 7A2 cell line and the 12B8 cell line in the CHO-K1 cell line meet the requirements, indicating that both the 7A2 cell line and the 12B6 cell line have been successfully domesticated.

[0182] 1e: Cell shake flask fermentation

[0183] (1)Preparation of the subculture medium: Place 60 vol% of CD-CHO medium + 40 vol% of Ex-cell 302 medium in a water bath at 37°C and preheat to 37°C;

[0184] (2)Take out the 7A2 cell line and 12B6 cell line in suspension culture in step 1d (17) and count them;

[0185] (3)Dilute the 7A2 cell line and 12B6 cell line in step (2) to 2.5×10 5 cells / mL - 3.5×10 5 cells / mL, and inoculate 30 mL of the subculture medium in step (1) into a 125 mL shake flask for each of the two cell lines. The cell culture flask is placed in a CO2 incubator at 37°C with a CO2 volume concentration of 5% and incubated overnight at 100 rpm / min;

[0186] (4)Count the cell density and viability every 24 h, measure glucose, and add glucose to 4 g / L when the blood glucose is below 2 g / L; take 1 mL of the sample every day, and the supernatant is used to detect the protein expression;

[0187] (5)Feeding (about the fourth day): Supplement CB5 at 70 g / L, and the addition amount is 10% of the original medium;

[0188] (6)Adjust the culture temperature (the fifth day): Adjust the temperature of the CO2 incubator to 32°C;

[0189] (7) Secondary feeding (on the ninth day): Supplement CB5 at 70 g / L, with the addition amount being 10% of the original culture medium;

[0190] (8) On the twelfth day, harvest the culture media of 7A2 cells and 12B6 cells respectively.

[0191] 1f: Protein purification

[0192] (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;

[0193] (2) Column equilibration: Take 20 ml of Protein A packing material and load it into an empty chromatography column. Equilibrate with ultrapure water for 2 - 3 column volumes (CV), drain the ethanol protection solution, and then equilibrate with 1×PBS for 2 - 3 CVs at a flow rate of 5 ml / min;

[0194] (3) Sample loading: Load the filtered supernatant, with a flow rate of 2 ml / min, and collect the flow-through;

[0195] (4) Equilibration: Wash the column with 15 CV of 1×PBS (300 mM NaCl) at a flow rate of 5 ml / min;

[0196] (5) Elution: Elute the target protein with the elution buffer (100 mM Glycine, 300 mM NaCl, pH 3.0) at a flow rate of 5 ml / min, collect the target protein until there is no blue color detected by Coomassie Brilliant Blue G-250, stop collection, and immediately neutralize the eluted target protein with the neutralization solution (1 M Tris, pH 9.0) (v∶v = 1 ml∶0.037 ml);

[0197] (6) Buffer exchange: Place the eluted sample in a dialysis bag and dialyze it with the storage buffer (30 mM Tris, 300 mM NaCl, pH 8.5) at least 1000-fold;

[0198] (7) Sterile filtration: In a biosafety cabinet, filter the dialyzed protein through a 0.22 μm low-protein-binding filter membrane for sterile filtration;

[0199] (8) Protein concentration determination: Use the BCA method to determine the protein concentration. The protein concentration of this batch is 2.942 mg / ml, and the volume of 2.84 mg / ml is approximately 50 ml; After calculation (protein yield = protein concentration × protein volume / volume of the taken culture supernatant), the protein yields of 7A2 cell line and 12B6 cell line are approximately 1.4 g / L - 1.5 g / L.

[0200] 1g: Identification of subunit HN protein; 1g.1, SDS-PAGE detection

[0201] (1)The purified protein from step 1f was detected by SDS-PAGE. The concentration of subunit HN protein in the sample used was 2 μg / well, and the results are as Figure 5 shown;

[0202] (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 absence of a 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.

[0203] 1g.2. Western Blot detection

[0204] (1)The purified protein from step 1f was detected by Western Blot, and the detection results are as Figure 6 shown. The concentration of subunit HN protein (labeled as 1 in the figure) in the sample used was 2 μg / well; the primary antibody 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;

[0205] (2)It can be seen from Figure 6 that this serum can specifically bind to the subunit HN protein of the present invention. Thus, it can be obtained that the subunit HN protein prepared by the present invention has excellent immunogenicity.

[0206] 1g.3. ELISA detection

[0207] (1)Coating: On the enzyme-linked immunosorbent assay (ELISA) plate, the purified subunit HN protein was diluted to 0.5 μg / ml with coating buffer (50 mM carbonate buffer, pH = 9.5). Each antigen was coated in 8 wells (4 wells were added with serum samples, and 4 wells were added with blocking solution as a control). 100 μl / well of each antigen was added, and after sealing with a sealing film, it was placed in a refrigerator at 4 °C overnight;

[0208] (2)Washing: The ELISA plate from step (1) was taken out of the refrigerator and washed 5 times with PBST buffer;

[0209] (3)Blocking: 200 μl of blocking solution (5 wt% skim milk) was added to each well containing subunit HN protein, and after sealing with a sealing film, it was incubated at 37 °C for 2 h;

[0210] (4)Serum dilution: The positive serum of cattle immunized with inactivated bovine parainfluenza vaccine was diluted 200 times with blocking solution;

[0211] (5)Washing: The same as (2);

[0212] (6) Sampling: Add diluted serum, and use the blocking solution as a negative control at the same time. Incubate at 37 °C for 1 h;

[0213] (7) Washing: The same as (2);

[0214] (8) Adding secondary antibody: Add 100 µl of diluted (dilution ratio is 1:6000) HRP-labeled donkey anti-bovine IgG secondary antibody to each well. Incubate at 37 °C for 0.5 h;

[0215] (9) Washing: The same as (2);

[0216] (10) Color development: Add 100 µl of TMB color development solution to each well under light-proof conditions. Incubate at 37 °C for 10 min;

[0217] (11) Termination: Add 50 µl of termination solution (2M H2SO4) to each well to terminate the reaction;

[0218] (12) Detection: Measure the OD value of the sample at a wavelength of 450 nm and analyze the data;

[0219] (13) The results are shown in Table 6 below: The coated subunit HN protein can specifically bind to the serum, and the average value of OD450 is 1.255; neither the coated subunit HN protein nor the blocking solution has specific binding, and the average value of OD450 is 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.

[0220] Table 6: Identification results of subunit HN protein by ELISA

[0221] 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 .

[0222] 1g.4 Stability verification

[0223] (1) Dilute the purified subunit HN protein in step 1f with PBS buffer to 1 mg / ml and divide it into 20 portions, each portion being 0.5 ml; 10 of them are placed in a refrigerator at 4 °C, and one portion is sampled weekly for 10 consecutive times;

[0224] (2) Place the other 10 portions in a refrigerator at -20 °C, and sample one portion weekly for 10 consecutive times; After each sampling, detect the protein concentration with BCA. The results are shown in Table 7 below:

[0225] Table 7: Stability of subunit HN protein

[0226] Sample Sample concentration after 4℃ treatment (mg / ml) Sample concentration after -20℃ treatment (mg / ml) First sampling 1.09 1.08 Second sampling 1.08 1.06 Third sampling 1.08 1.04 Fourth sampling 1.06 1.08 Fifth sampling 0.97 0.99 Sixth sampling 1.03 0.99 Seventh sampling 1,02 1.01 Eighth sampling 0.99 1.00 Ninth sampling 0.95 0.98 Tenth sampling 0.96 0.95 .

[0227] Referring to Table 7, from the perspective of the change in protein concentration, the protein remained basically stable during the two groups of experiments. To further verify whether the treated protein was degraded, we used the samples from the 10th time for SDS-PAGE detection. The specific results are as Figure 7 and Figure 8 shown. Among them, Figure 7 in [Figure / Graph] M is the Marker; 1 is the subunit HN protein after treatment at 4°C, and the sample loading amount is 2 μg; among them, Figure 8 in [Figure / Graph] M is the Marker; 1 is the subunit HN protein after treatment at -20°C, and the sample loading amount is 2 μg. From Figure 7 and Figure 8 it can be seen that the treated samples (sampled for the 10th time) were still stable. Thus, it can be obtained that the subunit HN protein prepared by the present invention has excellent stability.

[0228] 1h: Vaccine Preparation

[0229] (1) Aqueous phase preparation: According to the content of subunit HN protein in the vaccine, use PBS buffer (or physiological saline) to dilute the subunit HN protein into several portions with different concentration gradients, such as 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, etc. In this example, the subunit HN protein was diluted to 50 μg / mL, which is the aqueous phase;

[0230] (2) Oil phase preparation: According to the total amount of the prepared vaccine, measure an appropriate amount of ISA 201 VG adjuvant according to the weight ratio of antigen phase to adjuvant of 1:1 and the volume ratio of 46:54;

[0231] (3) Emulsification: Preheat both the aqueous phase and the oil phase to 33°C, slowly add the aqueous phase to the oil phase, stir at 200 - 500 rpm / min for 20 - 30 min, let it stand at 20°C for 1 h and then place it at 4°C overnight;

[0232] (4) Sub-packaging and storage: Sub-package according to needs, and store at 4°C for standby after passing the inspection.

[0233] 1i. Vaccine Quality Inspection

[0234] (1) Physical properties: Observe the appearance by visual inspection (whether it is a milky white emulsion);

[0235] (2) Use a clean pipette to suck a small amount of the vaccine and drop it into cold water. Observe (except for the first drop), the vaccine should diffuse in a cloud-like manner, and it is judged as a water-in-oil-in-water dosage form;

[0236] (3) Add 10 mL of the vaccine to a centrifuge tube and centrifuge at 3000 r / min for 15 min. The water phase precipitated at the bottom of the tube should be ≤ 0.5 mL, and it is judged as stable;

[0237] (4)Use a viscometer to detect the viscosity of the vaccine, and if it is between 20 - 50 cp, it is judged as qualified.

[0238] Example 2: The difference from Example 1 is that in this example, the encoded amino acid sequence of the subunit HN protein is the extracellular region of the HN protein, 57Q - 572S.

[0239] Example 3: The difference from Example 1 is that in this example, the encoded gene sequence of the subunit HN protein is the extracellular region of the HN protein, 143T - 572S.

[0240] Example 4: The difference from Example 1 is that in this example, for the subunit HN protein, a bovine Fc sequence is fused based on HN shown in SEQ ID NO.1.

[0241] Example 5: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pEE6.4, and the cell line is the DG44 cell line in the CHO cell line.

[0242] Example 6: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pGL4.13, and the cell line is the CHO - K1 cell line in the CHO cell line.

[0243] Example 7: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pcDNA3.1, and the cell line is the CHO - S cell line in the CHO cell line.

[0244] Example 8: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pEE12.4, and the cell line is the CHO - S cell line in the CHO cell line.

[0245] Example 9: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pEE12.4, the cell line is the HEK293 cell line, and the subunit HN protein does not require the domestication operation in step 1d during the preparation process.

[0246] Example 10: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pEE12.4, the cell line is the 293T / 17 cell line, and the subunit HN protein does not require the operation in step 1d during the preparation process.

[0247] Example 11: The difference from Example 1 is that in this example, the eukaryotic expression vector used is pCEP4, and the cell line is the DG44 cell line in the CHO cell line.

[0248] Comparative Example: The difference from Example 1 is that in this comparative example, the coding gene sequence of the HN protein of bovine parainfluenza virus is the genomic sequence of 6986 - 8536 bp in GenBank: OM621819.

[0249] Example 12: Determination of the expression yield and protein purity of the subunit HN protein

[0250] The expression yields and protein purities of the subunit HN proteins in Examples 2 - 11 and the comparative example were measured, and the expression results are shown in Table 8 below.

[0251] Table 8: Yield and purity of the subunit HN protein in Examples 2 - 11 and the comparative example

[0252] Item 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 Example 11 1.2-1.4 80 Comparative example 0.0-0.02 39 。

[0253] 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. Thus, it can be obtained that the subunit HN protein - coding gene constructed in the present invention can efficiently secrete and express the subunit HN protein in the cell line, and the protein purity of the obtained subunit HN protein is relatively high.

[0254] The protein yields in Examples 2 - 4 are 0.1 g / L - 0.9 g / L, and the protein purities are 60% - 80%. The protein yield in Example 1 is 1.4 g / L - 1.5 g / L, and the protein purity reaches 90%. Therefore, Example 1 is the preferred example among Examples 1 to 4. Thus, when the coding gene sequence of the subunit HN protein is SEQ ID NO.5, its corresponding protein yield and protein purity reach the optimal values.

[0255] The protein yields in Examples 5 - 8 are 1.1 g / L - 1.4 g / L, and the protein purities are 85% - 88%, which are lower than the detection results of Example 1. The protein yields in Examples 9 - 11 are 1.2 g / L - 1.6 g / L, and the protein purities are 80% - 85%. Thus, it can be obtained that when the eukaryotic expression vector is selected from pEE12.4 and the cell line is selected from one of the CHO cell line, HEK293 cell line, and 293T / 17 cell line, the expression yield and protein purity of the subunit HN protein are relatively high; especially when the eukaryotic expression vector is pEE12.4 and the cell line is CHO - K1 cell line, the expression level and protein purity of the subunit HN protein reach the optimal values.

[0256] In summary, the recombinant plasmid constructed by the present invention can be effectively expressed in engineered cell lines to obtain a subunit HN protein of bovine parainfluenza virus with high yield and high purity. The subunit HN protein of bovine parainfluenza virus has good specificity and stability, can be mass-produced, effectively reduces the production cost of the subunit HN protein of bovine parainfluenza virus, and can also be better applied to subunit vaccines or diagnostic reagents for bovine parainfluenza virus. Therefore, the subunit HN protein of the present invention has the characteristics of high secretion expression efficiency, high protein purity, easy purification, low production cost, high safety performance, etc.

[0257] The present invention is illustrated by the above examples. However, it should be understood that the present invention is not limited to the specific examples and embodiments described herein. The purpose of including these specific examples and embodiments here is to help those skilled in the art practice the present invention. Any person skilled in the art can easily make further improvements and refinements without departing from the spirit and scope of the present invention. Therefore, the present invention is only limited by the content and scope of the claims of the present invention, and it is intended to cover all alternative and equivalent solutions included within the spirit and scope of the present invention defined by the appended claims.

Claims

1. A subunit HN protein of a bovine parainfluenza virus, characterized in that The subunit HN protein includes a truncated protein of the extracellular region of the bovine parainfluenza virus HN protein and a bovine antibody constant region Fc, the amino acid sequence of the truncated protein of the extracellular region is shown in SEQ ID NO.2, the bovine antibody constant region Fc is connected to the amino terminus of the amino acid sequence shown in SEQ ID NO.2, and the amino acid sequence of the bovine antibody constant region Fc is shown in SEQ ID NO.8; the subunit HN protein is a homodimer.

2. The subunit HN protein according to claim 1, characterized in that The subunit HN protein is also connected to a tag selected from the group consisting of poly-His, FLAG, c-myc, HA, and poly-Arg.

3. The subunit HN protein of bovine parainfluenza virus according to claim 2, characterized in that The tag connected to the subunit HN protein is poly-His, and the amino acid sequence of the obtained subunit HN protein is shown in SEQ ID NO.

4.

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

5.

5. The method for preparing the subunit HN protein of bovine parainfluenza virus according to claim 4, characterized in that: The preparation method comprises the following steps: 1) constructing the gene sequence encoding the subunit HN protein of bovine parainfluenza virus 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 strains from the cell strains 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.

6. The method for preparing the subunit HN protein of bovine parainfluenza virus according to claim 5, characterized in that: In step 2), the eukaryotic expression vector is one of pEE6.4, pEE12.4, pGL4.13 and pcDNA3.

1.

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 pEE12.

4.

8. The method for preparing the subunit HN protein of bovine parainfluenza virus according to claim 5, 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.

9. The method for preparing the subunit HN protein of bovine parainfluenza virus according to claim 8, characterized in that: 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.

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

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