Anti-bovine rotavirus recombinant protein vaccine composition and application thereof

Abstract

The invention relates to the technical field related to biological medicine, and particularly provides an anti-bovine rotavirus recombinant protein vaccine composition and application thereof. The recombinant protein vaccine composition for resisting bovine rotavirus comprises a first recombinant protein vaccine which is administrated through nasal drop and a second recombinant protein vaccine which is administrated through subcutaneous injection, the first recombinant protein vaccine comprises VP6 recombinant protein and LTB recombinant protein; the second recombinant protein vaccine comprises VP7 recombinant protein. According to the recombinant protein vaccine disclosed by the invention, the combination of the first recombinant protein vaccine which is administrated through nasal dripping and the second recombinant protein vaccine which is administrated through subcutaneous injection is used, so that the comprehensiveness of preventing the bovine rotavirus is improved.

Description

Technical Field

[0001] The present application relates to the technical field of biomedicine, and specifically to a recombinant protein vaccine composition against bovine rotavirus and its use. Background Art

[0002] Since there are multiple genotypes of bovine rotavirus, conventional recombinant protein vaccines or subunit vaccines can only provide immune protection against some genotypes. Therefore, the comprehensiveness of conventional recombinant protein vaccines or subunit vaccines for the prevention of bovine rotavirus needs to be improved. Summary of the invention

[0003] In view of this, the present application provides a recombinant protein vaccine combination against bovine rotavirus and its use.

[0004] In the first aspect, the present application provides a recombinant protein vaccine combination against bovine rotavirus, wherein the recombinant protein vaccine combination comprises a first recombinant protein vaccine administered by nasal drops and a second recombinant protein vaccine administered by subcutaneous injection; the first recombinant protein vaccine comprises VP6 recombinant protein and LTB recombinant protein; the second recombinant protein vaccine comprises VP7 recombinant protein.

[0005] In some optional embodiments, the VP6 recombinant protein comprises the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.1, or consists of the amino acid sequence shown in SEQ ID NO.1. In some specific embodiments, the VP6 recombinant protein consists of the amino acid sequence shown in SEQ ID NO.1.

[0006] In some optional embodiments, the LTB recombinant protein comprises the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.2, or consists of the amino acid sequence shown in SEQ ID NO.2. In some specific embodiments, the LTB recombinant protein consists of the amino acid sequence shown in SEQ ID NO.2.

[0007] In some optional embodiments, the VP7 recombinant protein comprises the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.3, or consists of the amino acid sequence shown in SEQ ID NO.3. In some specific embodiments, the VP7 recombinant protein consists of the amino acid sequence shown in SEQ ID NO.3.

[0008] In some optional embodiments, the weight ratio of the VP6 recombinant protein to the LTB recombinant protein is (1-9):1, such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1 or 9:1, etc. In other optional embodiments, the weight ratio of the VP6 recombinant protein to the LTB recombinant protein is (1-7):1.

[0009] In some optional embodiments, the total concentration of the VP6 recombinant protein and the LTB recombinant protein in the first recombinant protein vaccine is (500-1500) ug / ml; for example, 500ug / ml, 600ug / ml, 700ug / ml, 800ug / ml, 900ug / ml, 1000ug / ml, 1100ug / ml, 1200ug / ml, 1300ug / ml, 1400ug / ml or 1500ug / ml, etc.

[0010] In some specific embodiments, the concentration of the VP6 recombinant protein in the first recombinant protein vaccine is 500 ug / ml, and the concentration of the LTB recombinant protein in the first recombinant protein vaccine is 100 ug / ml.

[0011] In some optional embodiments, the concentration of the VP7 recombinant protein in the second recombinant protein vaccine is (400-1000) ug / ml; for example, 400ug / ml, 500ug / ml, 600ug / ml, 700ug / ml, 800ug / ml, 900ug / ml or 1000ug / ml, etc.

[0012] In some optional embodiments, the volume of the first recombinant protein vaccine and the second recombinant protein vaccine is 1:(0.5-2); for example, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2, etc.

[0013] In a second aspect, the present application provides a use of the recombinant protein vaccine combination described in the first aspect in the preparation of an anti-bovine rotavirus product.

[0014] In a third aspect, the present application provides an anti-bovine rotavirus product, wherein the anti-bovine rotavirus product comprises the recombinant protein vaccine combination described in the first aspect.

[0015] The present application has the following beneficial effects: the recombinant protein vaccine combination of the present application uses a combination of "a first recombinant protein vaccine administered by nasal drops and a second recombinant protein vaccine administered by subcutaneous injection", thereby improving the comprehensiveness of bovine rotavirus prevention. DETAILED DESCRIPTION

[0016] The present application discloses a recombinant protein vaccine composition against bovine rotavirus and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0017] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.

[0018] Among them, the amino acid sequence information of VP6 recombinant protein, LTB recombinant protein and VP7 recombinant protein in this application is shown in Table 1.

[0019] Table 1 Amino acid sequence information of VP6 recombinant protein, LTB recombinant protein and VP7 recombinant protein in this application:

[0020]

[0021] The VP6 recombinant protein, LTB recombinant protein and VP7 recombinant protein of the present application can be obtained by chemical synthesis (such as liquid phase synthesis, solid phase synthesis) and biosynthesis. Among them, the VP6 recombinant protein, LTB recombinant protein and VP7 recombinant protein of the present application are all obtained by biosynthesis.

[0022] Example 1, Synthesis of coding gene:

[0023] In the present application, the nucleotide sequences of the gene encoding the VP6 recombinant protein, the gene encoding the LTB recombinant protein and the gene encoding the VP7 recombinant protein are shown in Table 2.

[0024] Table 2 Nucleotide sequence information of the gene encoding the VP6 recombinant protein, the gene encoding the LTB recombinant protein and the gene encoding the VP7 recombinant protein in this application:

[0025]

[0026]

[0027] In this preparation example, the coding gene is obtained by phosphoramidite nucleic acid solid phase synthesis. The synthesis method specifically includes the following steps:

[0028] The nucleotide sequence of the gene to be synthesized was introduced into the ABI394 nucleic acid synthesizer, and UniversalCPG / PS was used as a carrier to connect the nucleoside monomers one by one in the direction from the 3' end to the 5' end of the nucleotide sequence. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0029] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0030] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25° C., reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid phase carrier is 5:1.

[0031] The conditions of each coupling reaction are the same. The coupling reaction conditions are: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with a concentration of 0.5M, and the thiolation reagent is an acetonitrile / pyridine mixed solution of hydrogenated xanthanol with a concentration of 0.2mol / L (the volume ratio of acetonitrile and pyridine is 1:1).

[0032] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a volume ratio of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0033] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature of 25°C; reaction time of 3 seconds; oxidation reagent concentration of 0.05M iodine water, the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature of 25°C; reaction time of 360 seconds; thiolation reagent concentration of 0.2M hydrogenated xanthan gum pyridine solution, the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; thiolation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0034] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain single-stranded DNA; wherein:

[0035] Cleavage and deprotection conditions: The synthesized nucleotide sequence connected to the solid phase carrier was added to 25wt% ammonia water, the amount of ammonia water was 0.5ml / μmol, reacted at 55°C for 16 hours, the solvent was removed, and vacuum concentrated to dryness. After ammonia treatment, the product was dissolved with 0.4ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3ml / μmol triethylamine and 0.6ml / μmol triethylamine trihydrofluoride were added to remove the 2'-O-TBDMS protection on the ribose.

[0036] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0037] Detection: After the single-stranded DNA was diluted to a concentration of 0.2 mg / mL using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatogram Hy-Mass Spectrometry, purchased from Waters, model: LCT Premier). The measured value was consistent with the theoretical value, indicating that the target designed coding gene was synthesized.

[0038] Example 2, biosynthesis of recombinant protein:

[0039] (2-1) Cloning and identification of coding genes:

[0040] The synthetic coding gene was connected to the pMD19-T cloning vector in a 16°C constant temperature metal bath overnight. The ligation product was transferred into the DH5a competent cell according to the instruction manual of the DH5a competent cell. The screened positive clones were inoculated into the Amp+ LB liquid culture medium and cultured at 37°C for 10 h. The recombinant plasmid was extracted using a plasmid extraction kit, sequenced and stored at -20°C.

[0041] The recombinant plasmid containing the coding gene and the pET-28a expression vector were double-digested with restriction endonucleases BamHI and XhoI, respectively, and then transferred into TOP10 competent cells after ligation at 16°C for 18 hours using T4 ligase, and cultured overnight at 37°C on a plate containing LB solid medium (kanamycin concentration of 100ug / ml).

[0042] The clone colonies identified as positive after transformation were selected, inoculated into LB liquid culture medium (kanamycin concentration was 100 ug / ml) and cultured overnight at 37°C, and then the recombinant expression plasmid pET28a-coding gene was extracted.

[0043] (2-2) Construction of recombinant engineering bacteria:

[0044] The correctly identified pET28a-edited gene expression plasmid was transfected into Rosetta (DE3), evenly coated on LB solid medium (kanamycin concentration was 50ug / ml), cultured in a 37°C incubator for 16 hours, and then a single colony was picked and inoculated into 4ml of LB liquid medium (kanamycin concentration was 50ug / ml), and shaken in a 37°C incubator for 16 hours. The strain was inoculated into 200ml of LB liquid medium (kanamycin concentration was 50ug / ml) at a ratio of 1:100, cultured at 37°C until the OD was 0.4-0.6, and isopropylthiogalactoside (final concentration was 0.5mM, abbreviated as IPTG, CAS number 367-93-1) was added and induced to express at 25°C. After the expression was completed, the bacterial liquid was collected.

[0045] (2-3) Detection of expression products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE):

[0046] Take an appropriate amount of bacterial solution and centrifuge at 12000rpm for 4min. Resuspend the precipitate with 1×PBS and then take 1ml of the resuspended bacterial solution for ultrasonic disruption. Collect the supernatant and precipitate. Mix equal volumes of supernatant and precipitate with 2×SDS gel loading buffer and boil for 5min. Take 5ul of sample and use empty vector as control for SDS-PAGE electrophoresis. Perform electrophoresis at 80V constant voltage on 12% concentrated gel to 120V constant voltage on 5% separation gel. After the electrophoresis is completed, carefully remove the gel and stain it with Coomassie brilliant blue for 2h, then discard the staining solution and add destaining solution in a shaker overnight. Replace the destaining solution every 6h or so. After the bands on the gel are clearly visible, observe with a gel imaging system.

[0047] (2-4) Purification and identification of recombinant proteins:

[0048] The remaining bacterial resuspension was ultrasonically lysed, and after lysis, it was centrifuged for 10 minutes (12000rpm, 4°C) to collect the precipitate. After washing with buffer to remove impurities, the inclusion bodies were dissolved with 8M urea, and after gradient urea renaturation, they were purified with a Ni-NTA protein purification column with a His tag to obtain the target recombinant protein.

[0049] Example 3: Preparation of the first recombinant protein vaccine:

[0050] In this preparation example, the preparation method of the first recombinant protein vaccine specifically includes the following steps:

[0051] (3-1) VP6 recombinant protein and LTB recombinant protein were mixed in a certain ratio and diluted with a diluent (phosphate buffer at pH = 7.4) to obtain a recombinant protein solution.

[0052] (3-2) The recombinant protein solution and trehalose (CAS No. 99-20-7), gelatin (CAS No. 9000-70-8), ethylenediaminetetraacetic acid (EDTA, CAS No. 60-00-4), and arginine (CAS No. 74-79-3) are mixed uniformly. The amount of trehalose is 5wt% and the amount of gelatin is 2wt% based on the weight of the recombinant protein solution; the amount of ethylenediaminetetraacetic acid is 5mmol and the amount of arginine is 0.4mol per liter of the recombinant protein solution based on the volume of the subunit vaccine.

[0053] (3-3) Adding nano-aluminum adjuvant to the mixed solution obtained in step (3-2) (the volume ratio of the mixed solution to the nano-aluminum adjuvant is 1:2) to obtain the first recombinant protein vaccine.

[0054] Example 4, Preparation of the Second Recombinant Protein Vaccine:

[0055] In this preparation example, the preparation of the second recombinant protein vaccine specifically includes the following steps:

[0056] (4-1) The VP7 recombinant protein was prepared into a VP7 recombinant protein solution using physiological saline, and the VP7 recombinant protein solution was preheated to 37°C.

[0057] (4-2) Adjuvant ISA201 was sterilized by autoclaving at 121°C for 30 minutes and then cooled to 37°C.

[0058] (4-3) The VP7 recombinant protein solution preheated to 37° C. and the adjuvant ISA201 cooled to 37° C. were mixed and emulsified in a volume ratio of 1:1 to obtain a second recombinant protein vaccine.

[0059] Example 5: Preparation of recombinant protein vaccine combination against bovine rotavirus:

[0060] In this preparation example, the recombinant protein vaccine combination against bovine rotavirus includes a first recombinant protein vaccine administered by nasal drops and a second recombinant protein vaccine administered by subcutaneous injection. The ingredients of the recombinant protein vaccine combination against bovine rotavirus are shown in Table 3.

[0061] Table 3 Ingredients of the recombinant protein vaccine combination against bovine rotavirus:

[0062]

[0063] Example 6. Evaluation of recombinant protein vaccine combination against bovine rotavirus:

[0064] Balb / c female mice aged 6 to 8 weeks were randomly divided into 14 groups, with 5 mice in each group. Each female mouse in each group was immunized twice, with the first immunization day being marked as day 0 and the second immunization being performed on day 14. In each immunization, each female mouse in each group was administered 0.2 ml of the first recombinant protein vaccine intranasally and 0.2 ml of the second recombinant protein vaccine subcutaneously. Blood samples were collected from each female mouse on day 28.

[0065] Processing of blood samples: First, place the collected blood samples in a centrifuge tube and let it stand at 37°C for 1 hour; then, centrifuge at 3000rpm at room temperature for 10 minutes, and transfer the supernatant to a clean centrifuge tube; finally, centrifuge at 12000rpm and 4°C for 10 minutes, and transfer the supernatant to 1.5mL centrifuge tubes, with 0.2mL of serum sample in each tube.

[0066] The bovine rotaviruses (TCID 50 For 10 5.5 / 0.1mL) as the attack strain and detect the neutralizing antibody titer of the serum.

[0067] Among them, the neutralizing antibody titer is the highest serum dilution that can protect 50% of cells from developing lesions. In addition to animal protection tests, the neutralizing antibody titer is one of the quantitative indicators that can more objectively and accurately reflect the immune animal's resistance to infection.

[0068] The method for detecting the neutralizing antibody titer of serum is the endpoint neutralization test - fixed virus dilution antiserum method. The specific operation steps are as follows:

[0069] (6-1) The serum to be tested was inactivated at 56°C for 30 min, and then diluted serially in multiples with cell maintenance medium.

[0070] (6-2) Take 1.0 mL of diluted serum of different concentrations and mix with 1.0 mL of standard virus (100 TCID 50 / 0.1 mL) and incubate in a 37°C water bath for 1 h.

[0071] (6-3) Add 0.2 mL of the mixed solution obtained in step (6-2) to 96-well cell culture plates where MA104 cells have grown into a monolayer, and inoculate 4 wells at each dilution; at the same time, set 4 wells of normal MA104 cells as blank controls and set 4 wells of 100 TCID 50 / 0.2mL was used as virus control.

[0072] (6-4) Virus titer control: The standard virus was diluted 10 times serially and added to 96-well cell culture plates where MA104 cells had grown into a monolayer. Four wells were inoculated for each dilution, and 0.1 mL was added to each well.

[0073] (6-5) Place the 96-well cell culture plate at 37°C and 5% CO 2 The cells were incubated in an incubator and the cytopathic effect (CPE) was observed daily.

[0074] (6-6) Neutralizing antibody titer was calculated using the Reed-Muench method based on the degree of cytopathic effect.

[0075] Table 4 Neutralizing antibody titer test results:

[0076]

[0077]

[0078] As can be seen from Table 4, the recombinant protein vaccine combination of the present application uses a "first recombinant protein vaccine administered by nasal drops and a second recombinant protein vaccine administered by subcutaneous injection" to improve the comprehensiveness of bovine rotavirus prevention.

[0079] The above is a detailed introduction to a recombinant protein vaccine combination against bovine rotavirus and its use provided by the present application. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and its core idea of ​​the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A recombinant protein vaccine combination against bovine rotavirus, characterized in that: The recombinant protein vaccine combination comprises a first recombinant protein vaccine administered by nasal drops and a second recombinant protein vaccine administered by subcutaneous injection; the first recombinant protein vaccine comprises VP6 recombinant protein and LTB recombinant protein; the second recombinant protein vaccine comprises VP7 recombinant protein; The VP6 recombinant protein comprises the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.1, or consists of the amino acid sequence shown in SEQ ID NO.1; The LTB recombinant protein comprises the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.2, or consists of the amino acid sequence shown in SEQ ID NO.2; The VP7 recombinant protein comprises the amino acid sequence shown in SEQ ID NO.3, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof relative to SEQ ID NO.3, or consists of the amino acid sequence shown in SEQ ID NO.

3.

2. The recombinant protein vaccine combination according to claim 1, characterized in that: The weight ratio of the VP6 recombinant protein to the LTB recombinant protein is (1-9):

1.

3. The recombinant protein vaccine combination according to claim 2, characterized in that: The weight ratio of the VP6 recombinant protein to the LTB recombinant protein is (1-7):

1.

4. The recombinant protein vaccine combination according to claim 1, characterized in that: The total concentration of the VP6 recombinant protein and the LTB recombinant protein in the first recombinant protein vaccine is (500-1500) ug / ml.

5. The recombinant protein vaccine combination according to claim 1, characterized in that: The concentration of the VP7 recombinant protein in the second recombinant protein vaccine is (400-1000) ug / ml.

6. The recombinant protein vaccine combination according to any one of claims 1 to 5, characterized in that: The volumes of the first recombinant protein vaccine and the second recombinant protein vaccine are 1:(0.5-2).

7. Use of the recombinant protein vaccine combination according to any one of claims 1 to 6 in the preparation of an anti-bovine rotavirus product.

8. An anti-bovine rotavirus product, characterized in that: The anti-bovine rotavirus product comprises the recombinant protein vaccine combination according to any one of claims 1 to 6.

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