阅读说明：本技术 一种狂犬病毒疫苗及其制备方法 (Rabies virus vaccine and preparation method thereof ) 是由 董春升 熊思东 梁明龙 于 2021-08-09 设计创作，主要内容包括：本发明公开了一种狂犬病毒疫苗及其制备方法。本发明的狂犬病毒疫苗是将水疱性口炎病毒的囊膜蛋白G替换为狂犬病毒囊膜蛋白RABV-(G)。本发明通过分子生物学技术,在编码水疱性口炎病毒基因组质粒上,将VSV囊膜蛋白替换为狂犬病毒囊膜蛋白RABV-(G),利用VSV反向遗传学系统在293T细胞中包装成表达RABV-(G)的以VSV为载体的疫苗,将上述疫苗滴鼻免疫小鼠,证实可有效诱导小鼠针对狂犬病毒的中和抗体,显著提高免疫小鼠抵御病毒感染的能力,具备作为新型狂犬疫苗的应用价值和潜力。(The invention discloses a rabies virus vaccine and a preparation method thereof. The rabies virus vaccine replaces the envelope protein G of the vesicular stomatitis virus with the envelope protein RABV of the rabies virus G . The invention replaces VSV envelope protein with rabies virus envelope protein RABV on a genome plasmid for coding vesicular stomatitis virus by a molecular biology technology G Packaging into expression RABV in 293T cells using VSV reverse genetics system G The vaccine using VSV as a vector is used for nasal drip immunization of mice, and proves that the neutralizing antibody of the mice against rabies virus can be effectively induced, and the effect is obviousRemarkably improves the capability of immune mice to resist virus infection and has application value and potential as a novel rabies vaccine.)

1. A rabies virus vaccine is characterized in that the rabies virus vaccine is prepared by replacing envelope protein G of vesicular stomatitis virus with rabies virus envelope protein RABV_G。

2. The rabies virus vaccine according to claim 1, wherein the envelope protein RABV_GComprises the following steps: envelope protein RABV with GenBank AAA47218.1_GOr the envelope protein RABV with the number GenBank: AAA47218.1_GOn the basis of the expression vector, the envelope protein RABV is obtained after one or more amino acid insertions, deletions or mutations_GAn active protein.

3. A method for preparing a rabies virus vaccine according to any one of claims 1 or 2, comprising the steps of:

s1, construction of plasmid pXN2-G_RABV: the gene sequence of the envelope protein G on the pXN2 vector is cut off and replaced by rabies virus envelope protein RABV_GTo obtain plasmid pXN2-G_RABV；

S2, expression recombinant VSV-RABV_G: the first cells to be infected were infected with poxvirus with T7 RNA transcriptase and the infected cells were treated with pXN2-G_RABVAnd cotransfecting the pP, pL and pN plasmids, collecting the supernatant of the pathological cell after transfection, infecting a second cell to be infected by adopting the supernatant, collecting the cell culture supernatant infected by the virus after virus amplification, and obtaining the rabies virus vaccine.

4. The method according to claim 3, wherein the first cell to be infected is a BHK cell, 293T cell, BSRT7 cell or Vero cell.

5. The method according to claim 3, wherein the second cell to be infected is a Vero cell, a BHK cell, a 293T cell or a BSRT7 cell.

6. The method according to claim 4 or 5, wherein the BHK cells are BHK-21 cells.

7. The method according to claim 3, wherein pXN2-G_RABVpP, pL and pN plasmids, pXN2-G_RABVThe ratio of pP, pL and pN plasmids is 10: 5: 4: 2.

8. the method according to claim 3, wherein the poxvirus is infected for 1 to 3 hours.

9. The method according to claim 3, wherein the supernatant of the diseased cells is collected 2 to 4 days after cotransfection.

10. A neutralizing antibody induced by the rabies vaccine according to any one of claims 1 or 2.

Technical Field

The invention relates to a rabies virus vaccine and a preparation method thereof, belonging to the technical field of biological medicines.

Background

Rabies virus (rabi virus, RABV) belongs to the rhabdoviridae, is a typical neurotropic virus, and virions are bullet-shaped, single-stranded negative-strand RNA viruses with capsular membrane segments, can invade the brain of a host in a reverse direction through the central nervous system, cause encephalomyelitis of warm-blooded animals, and once the disease occurs, the death rate is 100%. At present, no effective treatment method exists, and rabies vaccine inoculation before or after exposure is the only effective prevention means.

At present, due to the change of life style and concept of residents in China, the market scale of the pet industry in China market is continuously enlarged, the number of cats and dogs in the pet industry in 2019 is increased to 2024 yuan, the number of the cats and the dogs in the pet industry is increased by 10 years, and the increase of the demand of rabies vaccines for people is promoted. In the first half of 2020, 4795 thousands of rabies vaccines are distributed in batches for human beings, and the ratio is increased by 36%. Wherein the adult organism accounts for 39%, Ningbo Rong and Ansheng biopharmaceutical industry Co., Ltd accounts for 30%, and Duxing medicine controlled thigh Co., Ltd., Dalian Delivery biopharmaceutical Co., Ltd., accounts for 11%. The vaccine sold on the spot is mainly an inactivated virus vaccine, the virus inactivation usually adopts beta-propiolactone as an inactivating agent, and the inactivated vaccine can be divided into Vero cell vaccine, Human Diploid Cell Vaccine (HDCV), Primary Hamster Kidney (PHK) cell vaccine and chick embryo cell vaccine from cultured cell sources. The yield of the Vero cell crazy seedlings is large, the quality is controllable, the Vero cell crazy seedlings are main products of the crazy seedlings in China, about 90 percent of the crazy seedling batch issuance is occupied every year, all enterprises occupying the market of the former four are enterprises producing the Vero cell crazy seedlings, and at present, only one adult Ducheng Kanghua enterprise produces HDCV. Therefore, the rabies vaccine for human or veterinary use is rapidly increased every year, and the market demand is very large.

Vesicular Stomatitis Virus (VSV) can be used as a vaccine vector, an Ebola vaccine using VSV as the vaccine vector is also granted to the market by the U.S. FDA and European drug administration EMA in 2019, and the safety of VSV in human bodies can be fully ensured. However, VSV has limitations as a viral vector, and firstly, the capacity of the viral vector is limited, the size of the inserted foreign gene is usually about 1-2K, and the antibody response of the specificity induced by the VSV as a vaccine vector to the body is also different, so that the myocarditis vaccine of coxsackie virus B3 using VSV as a vector can induce an effective immune protection response, but the significant antibody protection response cannot be induced by VSV as a vector using a novel coronavirus receptor binding region RBD as an antigen. Since the difference between the envelope protein of other viruses inserted from outside and the original envelope protein of the VSV virus exists, the recombinant VSV replacing the envelope protein of the VSV can not be replicated, so that the rescue of the recombinant VSV of the envelope replacement type is a great technical challenge. There has been no report of using vesicular stomatitis virus as a rabies virus vaccine vector.

Disclosure of Invention

In order to solve the vacancy of rabies vaccines in the prior art, the invention uses a VSV reverse genetic system to replace the envelope protein G of the vesicular stomatitis virus with the envelope protein RABV of the rabies virus_GPackaging and preparing the recombinant VSV-RABV expressing the rabies virus envelope protein_GViruses and their immune response and protective effect were tested in mouse models.

The first purpose of the invention is to provide a rabies virus vaccine, which is obtained by replacing envelope protein G of vesicular stomatitis virus with envelope protein RABV of rabies virus_G。

Further, the envelope protein RABV_GComprises the following steps: envelope protein RABV with GenBank AAA47218.1_GOr the envelope protein RABV with the number GenBank: AAA47218.1_GOn the basis of the expression vector, the envelope protein RABV is obtained after one or more amino acid insertions, deletions or mutations_GAn active protein.

In the present invention, the rabies virus base envelope protein RABV_GWithout limitation, the envelope protein RABV with GenBank accession AAA47218.1_GAlso comprises similar rabies virus basal envelope protein RABV of other plant types_G。

The second purpose of the invention is to provide a preparation method of the rabies virus vaccine, which comprises the following steps:

s1, construction of plasmid pXN2-G_RABV: the gene sequence of the envelope protein G on the pXN2 vector is cut off and replaced by rabies virus envelope protein RABV_GTo obtain plasmid pXN2-G_RABV；

S2, expression recombinant VSV-RABV_G: the first cells to be infected were affected by pox with T7 RNA transcriptaseToxic infection, pXN2-G for infected cells_RABVAnd cotransfecting the pP, pL and pN plasmids, collecting the supernatant of the pathological cell after transfection, infecting a second cell to be infected by adopting the supernatant, collecting the cell culture supernatant infected by the virus after virus amplification, and obtaining the rabies virus vaccine.

Further, the first cell to be infected is a BHK cell, 293T cell, BSRT7 cell or Vero cell.

Further, the second cell to be infected is a Vero cell, a BHK cell, a 293T cell or a BSRT7 cell.

Further, the BHK cell is a BHK-21 cell.

Further, pXN2-G_RABVpP, pL and pN plasmids, pXN2-G_RABVThe ratio of pP, pL and pN plasmids is 10: 5: 4: 2.

further, the time of pox virus infection is 1-3 hours.

Further, the supernatant of the diseased cells was collected 2-4 days after cotransfection.

In the present invention, the reverse genetic recombination system of vesicular stomatitis virus is based on pXN2, pP, pL and pN. pXN2 contains the full-length cDNA sequence of the virus, the T7 promoter, and the delta hepatitis ribozyme, and is primarily responsible for providing the full-length RNA genome of the virus without redundant sequences. The pP, pN and pL plasmids are capable of providing P, N and L proteins to make up the RNA polymerase core. The T7 promoter was used on all four plasmids, as described in 10: 5: 4: 2, to co-transfect BHK-21 cells. T7 RNA polymerase is not present in eukaryotic cells, and therefore prior to co-transfection of the four plasmids, the cells are infected with a recombinant poxvirus comprising the T7 RNA polymerase gene to provide the T7 RNA polymerase protein.

The third purpose of the invention is to provide a neutralizing antibody, wherein the neutralizing antibody is induced and produced by the rabies virus vaccine.

The invention has the beneficial effects that:

the invention replaces VSV envelope protein with rabies virus envelope on a genome plasmid for coding vesicular stomatitis virus by a molecular biology technologyProtein RABV_GPackaging into expression RABV in 293T cells using VSV reverse genetics system_GThe vaccine using the VSV as the vector is used for nasal drip immunization of a mouse, so that the neutralizing antibody of the mouse against the rabies virus can be effectively induced, the capability of the immunized mouse in resisting virus infection is obviously improved, and the vaccine has application value and potential as a novel rabies vaccine.

Description of the drawings:

FIG. 1 is VSV-RABV_GSchematic diagram of construction of recombinant virus reverse genetic recombinant plasmid.

FIG. 2 is a recombinant VSV-RABV_GWestern Blot identification of RABV GP 24 hours after infection of 293T cells with VSV-GFP, Control was uninfected 293T cells.

FIG. 3 shows VSV-RABV_GVaccine nasal drip immunization induces RABV G specific serum IgG produced by mice.

FIG. 4 is VSV-RABV_GAnd (3) detecting the survival rate of the vaccine after the vaccine is subjected to nasal drop immunization and induced mice to be attacked by the virus.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

The plasmids, strains, cells, animals and reagents used in the examples were as follows:

plasmids pXN2, pP, pL, pN and pVSVG were stored in the laboratory as host bacteria DH5 α, stable 2 (Shanghai Weidi Biotech Co., Ltd.). BHK21 and Vero E6 were maintained by the laboratory. Gene synthesis entrusted Suzhou Jinwei Zhi Co. The goat anti-mouse IgG polyclonal antibody was labeled with HRP, and the goat anti-mouse IgA polyclonal antibody was labeled with HRP (southern Biotech Co.). Restriction endonucleases XhoI and NheI (TaKaRa Co.), T4 DNA ligase (TaKaRa); taq DNA polymerase (TaKaRa Co.); dntp (promega); LB medium (OXOID, UK), agar powder, agarose, EB (Shanghai chemical reagent procurement supply station), Tris (USB), agarose gel recovery kit (Shunhun, Shanghai), bulk plasmid extraction kit (Qiagen), lactate dehydrogenase LDH kit was purchased from Roche. Male BALB/c mice at 6-8 weeks of age were purchased from Shanghai Slek laboratory animal center and housed in clean-grade. Animal feeding and operation meet the relevant national regulations.

Example 1: recombinant VSV-RABV_GConstruction of vaccine and in vitro expression function identification

1.1) plasmid pXN2-G_RABVConstruction: the G gene sequence is contained between the enzyme cutting sites of the pXN2 vector xbaI and Mlu I, so we entrust Jinwei to synthesize the XbaI-Mlu I fragment, the VSV G gene is replaced by the RABV epidemic strain G gene, and the xbaI and Mlu I enzyme cutting sites are connected to the pXN2 vector to obtain pXN2-G_RABVPlasmid, sequencing verification, figure 1 is a schematic construction diagram.

1.2) recombinant VSV-RABV_GPreparation and expression detection of (1): BHK-21 cells were first infected with poxvirus harboring T7 RNA transcriptase, and 2 hours after infection cells were treated with pXN2-G_RABVpP, pL and pN plasmids were used in a ratio of 10: 5: 4: 2 co-transfection. Cytopathic effect was observed 3 days after transfection, and cell supernatants were collected to re-infect Vero cells, amplifying the virus. We collected the virus-infected cell culture supernatant and detected VSV-RABV by Western blotting_GMiddle RABV envelope protein G_RABVAs shown in FIG. 2.

Example 2: recombinant VSV-RABV_GVaccine-induced specific antibody responses

2.1) mouse nose drop immunization method and serum collection

BALB/c male mice at 6-8 weeks were divided into 3 groups, and 2 groups of nasal drops were immunized: 10⁶PFU VSV-GFP (negative control), 10⁶PFU VSV-RABV_GGroup 1 intramuscular injection 10⁶PFU LBNSE (attenuated vaccine), 6 mice per group. The nasal drop immunization procedure was as follows: mice were injected intraperitoneally with 80-120 μ l of 0.75% sodium pentobarbital and lightly anesthetized. Dripping PBS virus vaccine solution containing VSV-GFP into nasal cavity on two sides of mouse drop by drop_GThe same method of operation is taken by the group. Blood was collected via the posterior angular vein of the eye socket on days 10, 20 and 30 after immunization, and the blood was allowed to stand at 37 ℃ for 30min, centrifuged at 6000rpm for 10min, and serum was collected and frozen at-80 ℃.

2.2) ELISA detection of RABV G-specific serum IgG:

the ELISA method comprises the following steps: coating with 20ug/ml RABV G holoprotein and coating solution (0.1M Na)₂CO₃pH9.6, 0.1% BSA)4 ℃ plate wrapping; blocking with 5% mil-PBS; sequentially adding 1: diluting serum, HRP-goat anti-mouse IgG, IgA and OPD at 40 deg.C, stopping color development, and measuring OD_450nm。

VSV-RABV_GThe immune group induces higher level of specific IgG after 10 days of immunization, and the OD value reaches about 1.0 at day 30, which is lower than the antibody level (OD) generated by the live attenuated vaccine LBNSE_450nm1.5). However, the empty virus VSV-GFP alone cannot induce serum IgG, OD_450nmThe value is about 0.15, significantly lower than the former. And (4) prompting by a result: VSV-RABV_GAnd LBNSE immunized mice, both induced a strong neutralizing antibody immune response in serum (FIG. 3).

Example 3: recombinant VSV-RABV in a mouse infection model_GVaccine protection response assay

BALB/c male mice at 6-8 weeks were divided into 3 groups of 10 mice each, and the mice were immunized as described in 2.1.

At week 13 after immunization, we infected mice with intracerebral injection and observed survival of mice within 20. We found that the VSV-GFP immunised group began to die 8 days post infection, and by 13 days mice all died; while the last survival rate of LBNSE group mice was 60% (6/10), VSV-RABV_GAlthough the mice in the group were lower in antibody level than in the LBNSE group, they all survived the challenge experiment (n 10), possibly inducing more neutralizing antibodies (fig. 4). Since the challenge experiment in mice was at 13 weeks post-immunization, our results also suggest VSV-RABV_GCan provide a long-term protective immune response.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.