Artificially synthesized bovine rotavirus VP7-M protein mutant and vaccine and application thereof

Abstract

The invention relates to the technical field of veterinary products, in particular to an artificially synthesized bovine rotavirus VP7-M protein mutant, a vaccine and application. According to the designed bovine rotavirus VP7-M mutant, disulfide bonds and hydrophobic hole filling mutation are introduced, five amino acid mutations are designed in an original protein amino acid sequence, and then a CHO cell line capable of stably expressing the bovine rotavirus VP7 protein mutant is constructed; the cell line can efficiently secrete and express VP7-M mutant protein, the conformation of the VP7-M mutant protein is similar to that of natural protein, the secreted and expressed VP7-M mutant protein is subjected to one-step affinity chromatography purification and prepared into the subunit vaccine, the immunogenicity of the VP7-M mutant protein is evaluated by immunizing a mouse, and animal experiment results show that the VP7-M subunit vaccine is good in safety and has good application prospects. The bovine rotavirus subunit vaccine has good immunogenicity and has a protection effect similar to or even better than that of a traditional inactivated vaccine, so that a foundation is laid for further preparation of the bovine rotavirus subunit vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and in particular relates to an artificially synthesized bovine rotavirus VP7-M protein mutant, a vaccine and an application thereof. Background Art

[0002] Calf diarrhea is a major concern for the cattle industry, and bovine rotavirus is one of the main pathogens that cause calf diarrhea in large-scale cattle farms. Rotavirus infection in calves and mixed infection with other intestinal pathogens can lead to aggravated diarrhea symptoms, and in severe cases, death.

[0003] Bovine rotavirus is an icosahedral symmetrical double-stranded RNA virus with a diameter of about 70nm. It has no envelope and a structure similar to a wheel. The complete and infectious virus particle has a three-layer capsid structure. The structural proteins include core proteins (VP1, VP2 and VP3), inner capsid (VP6) and outer capsid layer (VP4 and VP7). The size of the rotavirus genome is about 18555bp, encoding 6 structural proteins (VP1-VP4, VP6, VP7) and 5 or 6 non-structural proteins (NSP1~NSP5 or NSP6). Among them, VP4 and VP7 proteins can make animals produce neutralizing antibodies; VP6 protein is a group-specific antigen shared by all rotaviruses; VP1, VP2 and VP3 and other non-structural proteins (NSP) play a key role in viral transcription. Bovine rotavirus is currently classified into G type and P type according to the outer capsid protein VP7 and VP4. According to the survey report, bovine rotavirus is distributed globally, including 12 G types and 11 P types, among which G6, G8 and G10, P[1], P[5] and P

[11] are the most common serotypes, and G10 mainly appears in dairy cow herds.

[0004] There are many methods for detecting bovine rotavirus infection, but there is no direct treatment method or specific drug. As for the symptoms of rotavirus infection, the course of the disease is short and the onset is acute. It is difficult to effectively cure it with only supportive treatment. Therefore, the development of bovine rotavirus vaccine has practical application value and important theoretical significance. The in vitro pure culture amplification effect of bovine rotavirus is poor, the genetic variability is strong, and there are many genotypes. Therefore, the subunit vaccine that can combine the advantages of inactivated vaccines and attenuated vaccines has become the preferred solution for the development of rotavirus vaccines. Subunit vaccines can not only induce humoral immunity, but also induce cellular immunity in the body.

[0005] The VP7 protein of bovine rotavirus is a trimer structure and an N-oligomannosyl glycoprotein. It constitutes the smooth part of the outer capsid on the surface of bovine rotavirus, accounting for about 30% of the total protein of bovine rotavirus, and is an important neutralizing antigen. At present, the expression systems of VP7 protein include Escherichia coli, insect baculovirus, yeast, etc., but these expression systems are not very ideal for the expression effect and immunogenicity of VP7 protein. This may be because VP7 is a glycoprotein, and the neutralization epitope is highly dependent on the conformation. These recombinantly expressed VP7 lack post-translational modification and cannot form the correct natural conformation, and cannot stimulate the body to produce high-titer neutralizing antibodies. Summary of the invention

[0006] Based on the above situation, the present invention analyzes and designs the bovine rotavirus VP7 protein, and constructs a CHO cell line that stably expresses the bovine rotavirus VP7 protein mutant through genetic engineering technology. The system has accurate post-translational modification function, and the expressed protein is closest to the natural state in any aspect; in addition, the system has the ability to efficiently amplify and express recombinant genes; CHO belongs to fibroblasts, and rarely secretes endogenous proteins, which is conducive to the separation and purification of target proteins. The cell line can efficiently secrete and express VP7 mutant proteins, purify the secreted VP7 mutant proteins and prepare them into subunit vaccines, and evaluate their immunogenicity by immunizing mice, indicating that the VP7 subunit vaccine has good safety and good immunogenicity, thus laying the foundation for the preparation of the bovine rotavirus subunit vaccine in the next step.

[0007] In order to achieve the above-mentioned purpose, the present invention proposes an artificially synthesized and efficiently expressed bovine rotavirus VP7-M protein, wherein the bovine rotavirus VP7-M protein is a protein obtained by subjecting the following amino acid mutations to the natural VP7 protein of bovine rotavirus G10 type: the valine at position 68 is mutated to glutamic acid, the valine at position 129 is mutated to glutamine, the asparagine at position 157 is mutated to aspartic acid, the serine at position 273 is mutated to cysteine, and the glutamine at position 308 is mutated to cysteine.

[0008] The amino acid sequence of the bovine rotavirus VP7-M protein is shown in SEQ ID No. 2. The bovine rotavirus VP7-M protein can be used to prepare a bovine rotavirus vaccine.

[0009] The present invention also provides a nucleotide sequence encoding the bovine rotavirus VP7-M protein.

[0010] The present invention also provides a recombinant plasmid, wherein the recombinant plasmid is connected with a nucleotide sequence encoding the bovine rotavirus VP7-M protein.

[0011] Furthermore, the plasmid is pXC17.4.

[0012] The invention also provides a high-efficiency expression system containing the recombinant plasmid, and the receptor cells of the expression system include CHO cell lines.

[0013] Finally, the present invention proposes an application of the bovine rotavirus VP7-M protein secreted by the above-mentioned high-efficiency expression system in the preparation of a bovine rotavirus vaccine.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The invention constructs a pXC17.4-VP7-M eukaryotic expression plasmid, and utilizes a CHO eukaryotic expression system to construct a cell line stably expressing a bovine rotavirus VP7 protein mutant. The cell line can efficiently secrete and express the VP7-M protein, and the expression amount of the VP7-M protein is much higher than that of the wild-type VP7 protein. The secreted and expressed VP7-M protein is in the culture medium supernatant, and the supernatant can be harvested by simple centrifugation without breaking the cells, and then purified by a step of affinity chromatography to obtain the pure VP7-M protein, and the purity of the VP7-M protein can reach more than 90%. The VP7-M protein expressed and purified by the CHO eukaryotic expression system has post-translational modification, can form a stable trimer, is closer to the natural conformation than other expression systems, and is endotoxin-free. Preliminary mouse immunization experiments and suckling mouse virus challenge experiments were conducted. By comparing the antibody levels of mice before and after immunization, it was found that the immunized group could induce the body to produce high-titer neutralizing antibodies, which was significantly different from the neutralizing antibody levels of mice in the control group, indicating that the VP7-M protein has good immunogenicity; the suckling mouse virus challenge protection experiment showed that the neutralizing antibodies produced by the VP7-M protein subunit vaccine can be transferred to the offspring through the passive immunization process and provide protection for suckling mice. The present invention has conducted a preliminary study on the immunogenicity of bovine rotavirus VP7-M. The experiment showed that the VP7-M protein subunit vaccine and the traditional inactivated vaccine have similar protective effects, and even better immune effects than the inactivated vaccine; in addition, the VP7-M protein is secreted and expressed by suspended CHO cells, and the purification step is simple. It can be produced by large-scale high-density fermentation in the later stage, which is simpler and safer than the traditional inactivated vaccine production, and the quality of the prepared vaccine batches is more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The PCR identification results of the recombinant expression plasmid in Example 2, wherein M: DNA marker; 1: VP7-M fragment positive control result; 2: negative control amplification result; 3-7: 5 monoclonal strain PCR amplification results.

[0017] Figure 2 This is the map of the pXC17.4-VP7-M eukaryotic expression plasmid in Example 2.

[0018] Figure 3The expression detection of VP7-M cell line, A is the protein expression detection by SDS-PAGE electrophoresis, and B is the immunogenicity detection of the expressed VP7-M by Western Blot, where M: protein marker; 1: CHO-K1 blank cell expression supernatant; 2: VP7-M cell line expression supernatant; 3: VP7 wild-type cell line expression supernatant.

[0019] Figure 4 This is the cooling expression detection of VP7-M cell line, where M: protein marker; 1: protein expression supernatant before cooling; 2-9: protein expression supernatant after cooling for 1 to 8 days.

[0020] Figure 5 These are the results of VP7-M protein purification, M: protein marker; 1: VP7-M purified sample reduced sample; 2: VP7-M purified sample reduced sample diluted one-fold; 3: 1 mg / ml BSA standard; 4: 0.5 mg / ml BSA standard; 5: 0.25 mg / ml BSA standard; 7: 2 μg VP7-M non-reduced sample; 8: 4 μg VP7-M non-reduced sample. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0022] Example 1 Gene synthesis of bovine rotavirus VP7 protein mutants

[0023] In this example, bovine rotavirus was selected as the virus isolated from the MA-104 cell line.

[0024] This embodiment is based on the bovine rotavirus G10-HB01 strain (deposit number CCTCCNO: V202436) isolated and preserved in the laboratory of our company, which has been published in patent CN202410423353.6. The VP7 amino acid sequence is shown in SEQ ID No. 3. By introducing disulfide bonds and hydrophobic cavity filling mutations, 5 amino acid mutated VP7 protein mutants were designed, and the 68th valine was mutated to glutamic acid, the 129th valine was mutated to glutamine, the 157th asparagine was mutated to aspartic acid, the 273rd serine was mutated to cysteine, and the 308th glutamine was mutated to cysteine. At the same time, the Kozak sequence, secretion signal peptide and 8 histidine tags were added to the N-terminus of the mutant protein. The gene sequence was synthesized by chemical synthesis, which contained 882 nucleotides and encoded a VP7 mutant of 291 amino acids, named VP7-M protein. Among them, positions 1-10 are Kozak sequences, positions 10-69 are secretion signal peptides, positions 70-93 are 8 histidine tag sequences, and positions 94-882 are VP7 proteins. Specifically, the nucleotide sequence of VP7-M protein is shown in SEQ ID No. 1, and the amino acid sequence of VP7-M protein is shown in SEQ ID No. 2. The size of the artificially synthesized VP7-M protein gene is 882bp.

[0025] Example 2 Construction of CHO expression vector

[0026] Using the artificially synthesized VP7-M protein gene in Example 1 as a template, PCR amplification was performed using primers.

[0027] The upstream primer VP7-F sequence is: 5'-CGC AAGCTT GCCACCATGGAAACTGATACTCTACT-3', introducing restriction endonuclease Hind III site and protective bases;

[0028] The downstream primer VP7-R sequence is: 5'-CCG GAATTC TCACACTCTGTAGTAGAAGGCGGCGG-3', introducing restriction endonuclease EcoR I site and protective bases.

[0029] The PCR reaction system was: PrimeSTAR Max Premix (2X), 25 μl; VP7-F primer, 2 μl; VP7-R primer, 2 μl; VP7-M gene template, 1 μl; sterile water was added to 50 μl. The PCR reaction conditions were: 98°C pre-denaturation for 3 min; 98°C denaturation for 30 s, 55°C annealing for 15 s, 72°C extension for 1 min, for a total of 30 cycles; and finally 72°C loop extension for 5 min. The PCR product was detected and analyzed by 1% agarose gel electrophoresis and recovered and purified.

[0030] The target DNA band amplified in the above step was recovered, digested with Hind III / EcoR I, and ligated with the pXC17.4 vector digested with the same enzymes to obtain a synthesized plasmid.

[0031] The ligated plasmid was transformed into DH5α competent cells, and 5 single clones were picked and placed in LB liquid culture medium containing ampicillin. The cells were cultured with shaking at 37°C overnight, and the VP7-M gene insertion in the cultured bacterial liquid was identified by PCR.

[0032] PCR identification results Figure 1 As shown: strains 1, 2, and 5 ( Figure 1 The VP7-M positive gene was amplified (lanes 3, 4, and 7 in the middle), and the positive clone strain was selected to extract the plasmid for sequencing verification, and the recombinant expression plasmid pXC17.4-VP7-M with the VP7-M gene inserted was obtained. Figure 2 shown.

[0033] Example 3 Construction of a CHO cell line that efficiently expresses VP7-M protein

[0034] Step 1: After linearization of the pXC17.4-VP7-M plasmid constructed in Example 2 with restriction endonuclease PvuI, 10 μg of the linear plasmid was electroporated into suspended CHO-K1 cells with a voltage of 280 V, 3 pulses, a pulse time of 5 ms, and a pulse interval of 0.784 s.

[0035] Step 2: After culturing the cells in step 1 in a shaker at 37°C for 48 h, the culture medium was removed by centrifugation, and the selection medium containing 50 μM MSX (methionine sulfonyl fluoride) of CD-CHO-AGT was added to continue culturing. The cell density and activity were observed every two days. When the cell density increased to 1x10 6 cells / ml, the cells were passaged to a cell density of approximately 1x10 cells / ml using CD-CHO-AGT selection medium containing 50 μM MSX (methionine sulfonyl fluoride). 5 cells / ml and continue culturing.

[0036] Step 3. After three times of serial dilution and subculture screening, dilute the cells with CHO cloning medium and spread them on 96-well plates. When the single cells in the 96-well plates are full, take the supernatant and use Elisa to detect whether the monoclonal cells are VP7-M positive. Select the positive monoclonal cells and transfer them to CHO-2 suspension culture medium for expanded production. After adaptation, freeze the seed cells and name them CHO-VP7-M. Continue to culture the remaining cells in suspension.

[0037] The supernatant was taken and SDS-PAGE electrophoresis was used to detect the protein expression, and the immunogenicity of the expressed VP7-M was detected by Western Blot. The detection results of VP7-M protein expression are shown in Figure 3 As shown, VP7-M cell line ( Figure 3 A) showed an obvious expression band, which was consistent with the expected size, and the expression level of VP7-M was much higher than that of VP7 wild-type cell line ( Figure 3 Lane 3 of A), Western Blot results showed that the expressed VP7-M protein could be recognized by bovine rotavirus positive serum just like the VP7 wild type.

[0038] The above results indicate that the VP7-M cell line constructed in this example can effectively secrete and express VP7-M protein and has good immunogenicity.

[0039] Example 4 Expression and purification of VP7-M protein

[0040] 1. Expression of VP7-M protein

[0041] Step 1: CHO-VP7-M cells revived at 37°C in step 3 of Example 3 were cultured at a cell density of 0.7 x 10 6 cells / ml were inoculated into a 1L cell culture flask containing 200ml CHO-2 medium and cultured continuously.

[0042] Step 2: After 72 hours of culture, take samples to count cell density, activity, pH value, and monitor glucose concentration. When the sugar concentration is greater than 3 g / L, add feed medium at a rate of 5%; when the sugar concentration is less than 3 g / L, use glucose concentrate to make the final sugar concentration reach 6 g / L, and add feed medium at a rate of 5%; continue to culture, and when the cell density reaches 10x10 6 cells / ml, the culture temperature was lowered to 33° C. Thereafter, 5% volume of feed was added every other day, and glucose was added every day to maintain its concentration not less than 3 g / L.

[0043] Step 3: Monitor cell density, diameter, and activity. When all cells become larger and the activity is below 80%, harvest the supernatant. During the culture period, take the culture supernatant every day for SDS-PAGE electrophoresis to detect protein expression.

[0044] Test results such as Figure 4 As shown, obvious expression bands appeared after cooling culture, and the expression level of VP7-M protein gradually increased with the cooling time.

[0045] 2. Purification of VP7-M Protein

[0046] Step 1: Centrifuge the fermentation broth obtained in step 3 above at 5000 rpm for 10 min to harvest the culture supernatant, filter it with a 0.45 μm membrane, and dilute the filtered supernatant by one half with PBS buffer for later use.

[0047] Step 2: Purify by Ni-NTA affinity chromatography. Load 5 ml of Ni-NTA filler into the chromatography column. Rinse the column with 5 times the column volume of pure water to remove ethanol at a flow rate of 1.5 ml / min. Equilibrate the column with 5 times the column volume of PBS buffer at a flow rate of 1.5 ml / min. Pass the supernatant protein filtered and diluted in step 1 through the column at a flow rate of 1 ml / min. Wash the column with 5 times the column volume of PBS buffer containing 30 mM imidazole to remove impurities at a flow rate of 2 ml / min. Elute VP7-M protein with 3 times the column volume of PBS buffer containing 500 mM imidazole at a flow rate of 2 ml / min. Desalt and filter the protein elution sample and store it for later use.

[0048] The purification results were detected by reducing and non-reducing SDS-PAGE. Figure 5 As shown, after one-step purification by Ni-NTA, the purity of VP7-M protein can reach more than 90% and the protein concentration can reach 3 mg / ml by grayscale analysis of ImageJ software. In addition, the molecular weight of the purified VP7-M protein is about 35 kDa in a monomeric state under reducing conditions, and about 100 kDa in a trimer form under non-reducing conditions.

[0049] Example 5 Mouse Immunization Test

[0050] In this experiment, ISA201 was selected as the adjuvant, and was diluted and emulsified with the bovine rotavirus VP7-M protein obtained in Example 5 to prepare a VP7-M subunit vaccine, wherein the volume ratio of protein to adjuvant was 1:1, the final concentration of the high-concentration vaccine group protein was 300 μg / ml, and the concentration of the low-concentration vaccine group protein was 100 μg / ml. Female BALB / c mice were immunized, and the immunogenicity of the VP7-M subunit vaccine was evaluated by a serum neutralization test.

[0051] SPF-grade 6-week-old Balb / c female mice were used for immunization. Blood was collected from the eye sockets of all mice before the first immunization, and the serum was separated as the basic serum. The concentration of the prepared VP7-M protein was detected using the BCA protein concentration determination kit. The immunization test procedure is shown in Table 1. Immunization was performed once every 14 days, for a total of three immunizations. Blood was collected 14 days after the three immunizations to separate serum and detect the titer of neutralizing antibodies in the serum of immunized female mice. The immunization grouping was set up as a PBS control group, a G10 inactivated vaccine group, and a test group with different doses of bovine rotavirus VP7-M protein. The G10 inactivated vaccine was prepared by concentrating and inactivating the bovine rotavirus G10-HB01 strain virus liquid and emulsifying it with ISA201 adjuvant at a volume ratio of 1:1. The virus titer in the finished product was 10 7.5 TCID50 / ml.

[0052] Neutralization titer test was performed as follows: the collected mouse serum was inactivated in a 56°C water bath for 30 min, and the serum was serially diluted in a 96-well plate, with the first well being diluted 1:8, followed by 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024 dilutions, for a total of 8 dilutions. Four wells were prepared for each dilution and each well was incubated with 100 μL of 200 TCID 50 The trypsin-treated bovine rotavirus G10-HB01 strain virus liquid was mixed and incubated for 1 hour; then 100 μL of the serum-virus mixture was added to MA-104 cells plated in a 96-well plate and infected at 37°C for 14 hours; the control group was mixed with the treated virus using the diluent, and then infected with MA-104 cells at 37°C for 14 hours; after incubation, indirect immunofluorescence detection was performed using the laboratory-prepared VP6 monoclonal antibody 5B11.

[0053] The test results are shown in Table 2. The average neutralizing titers of the G10 inactivated vaccine group, VP7-MA group, and VP7-MB group after 14 days of immunization were 409.6, 358.4, and 563.2, respectively. The neutralizing antibody titers of the three vaccine immunization groups were significantly higher than those of the PBS control group. Among them, the neutralizing antibody titer of the low-dose VP7-MA group was equivalent to that of the G10 inactivated vaccine, and the neutralizing antibody titer of the high-dose VP7-MB group was higher than that of the low-dose VP7-MA group and the G10 inactivated vaccine group, indicating that the VP7-M protein has good immunogenicity, and the subunit vaccine prepared with it can induce the body to produce high-titer neutralizing antibodies.

[0054] Table 1 Mouse immunization program

[0055] Group Immunization dose / each Injection volume Adjuvant Number of animals Immune pathway Control group PBS 100μl ISA201 5 Subcutaneous injection G10 inactivated vaccine <![CDATA[10 6.5 TCID 50 ]]> 100μl ISA201 5 Subcutaneous injection VP7-MA 10 μg 100μl ISA201 5 Subcutaneous injection VP7-MB 30 μg 100μl ISA201 5 Subcutaneous injection

[0056] Table 2 Results of mouse neutralizing antibody detection

[0057]

[0058]

[0059] Example 6 Sucking mouse challenge protection test

[0060] All female mice 2 weeks after the third immunization in Example 5 were mated and separated into cages to give birth. The 7-day-old suckling mice were challenged with G10 rotavirus by oral administration at a dose of 5×10 6 TCID50 / mouse, observe the clinical symptoms of the suckling mice, such as diarrhea or death, body hair, mental activity, etc. every day for 7 consecutive days. The results are shown in Table 3. Under the same challenge dose, all suckling mice in the PBS control group developed diarrhea on the second day after the challenge, and the diarrhea lasted for 3 to 4 days. Two of the pups born to the female mice in the VP7-MA immunization group and the G10 inactivated vaccine immunization group began to have diarrhea on the 3rd to 4th day after the virus attack, which lasted for 2 to 3 days. The diarrhea rates were 11.1% and 10%, respectively, which were significantly lower than the 100% diarrhea rate in the PBS control group. The diarrhea symptoms were milder and the duration of the symptoms was significantly shortened. The virus attack protection rate of the pups born to the high-dose VP7-MB immunization group was 100%, which was slightly higher than that of the VP7-MA group and the G10 inactivated vaccine group. The pups' protection results were consistent with the neutralizing antibody level test results. The results showed that the neutralizing antibodies produced in the immunized female mice can be transmitted to the offspring through the passive immunization process and provide protection for the pups. The subunit vaccine prepared with VP7-M protein has similar protective effects to the traditional inactivated vaccine, and is even better than the inactivated vaccine.

[0061] Table 3 Results of the challenge protection test on suckling mice

[0062]

[0063]

Claims

1. An artificially synthesized and highly expressed bovine rotavirus VP7-M protein, characterized in that: The bovine rotavirus VP7-M protein is a protein obtained by subjecting the following amino acid mutations to the natural VP7 protein of bovine rotavirus G10 type: the 68th valine is mutated to glutamic acid, the 129th valine is mutated to glutamine, the 157th asparagine is mutated to aspartic acid, the 273rd serine is mutated to cysteine, and the 308th glutamine is mutated to cysteine.

2. The bovine rotavirus VP7-M protein according to claim 1, characterized in that The amino acid sequence of the bovine rotavirus VP7-M protein is shown in SEQ ID No.

2.

3. Use of the bovine rotavirus VP7-M protein as claimed in claim 1 in the preparation of a bovine rotavirus vaccine.

4. A nucleotide sequence comprising a nucleotide sequence encoding the bovine rotavirus VP7-M protein according to any one of claims 1 to 2.

5. A recombinant plasmid, characterized in that: The recombinant plasmid contains the nucleotide sequence of claim 4.

6. The recombinant plasmid according to claim 5, characterized in that The plasmid is pXC17.

4.

7. A high-efficiency expression system comprising the recombinant plasmid according to any one of claims 5 to 6, characterized in that: The recipient cells of the expression system include CHO cell lines.

8. Use of the bovine rotavirus VP7-M protein secreted by the expression system according to claim 7 in the preparation of a bovine rotavirus vaccine.

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