CN114807195A - Fusion gene for improving heat-resistant stability and immune effect of rabies virus, recombinant rabies virus and application of fusion gene - Google Patents
Fusion gene for improving heat-resistant stability and immune effect of rabies virus, recombinant rabies virus and application of fusion gene Download PDFInfo
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Abstract
The invention provides a fusion gene for improving heat-resistant stability and immune effect of rabies virus, recombinant rabies virus and application thereof, belonging to the technical field of virus. The rabies virus SAD B19 strain is used as a framework, a fusion gene formed by a mineralization gene W6 and a mineralization gene G is inserted between a genome N and a genome P, recombinant full-length plasmids pBNSP-RV-GW6 and pBNSP-RV-W6G are constructed, and a recombinant rabies virus containing the mineralization gene and double G genes is rescued by utilizing a rabies reverse genetic operation system. The recombinant rabies virus is stably proliferated on cells, has the heat-resistant stability characteristic, can induce and generate a higher rabies virus specific antibody by immunizing a mouse, can resist the attack of a lethal dose of rabies virus, has the heat-resistant stability and high-efficiency immune effect, can be directly used as a vaccine for application, and has a higher application prospect.
Description
Technical Field
The invention belongs to the technical field of viruses, and particularly relates to a fusion gene for improving heat-resistant stability and immune effect of rabies virus, recombinant rabies virus and application thereof.
Background
Rabies (Rabies) is an ancient zoonosis caused by Rabies Virus (RV) infection, no specific treatment medicine exists so far, and vaccination still remains the most important measure for preventing and controlling the disease. Once rabies occurs, the fatality rate reaches almost 100%. Rabies virus infects almost all warm-blooded animals, including domestic animals such as horses, cattle, sheep, pigs, dogs, cats and the like, and wild animals including mainly bat, fox, wolf, jackal, raccoon, badger and the like. Rabies is transmitted between animals and animals or humans mainly through saliva when the animals bite, and additionally scratches, vaccination with incompletely inactivated vaccines or transplantation of toxic organs, etc. of the animals may also cause infection with viruses. In developed countries in Europe and America, because of implementing strict immunization policy on domestic animals, the infection of the domestic animals rabies to people is basically controlled, wild animals are the main infection sources of rabies viruses, and therefore, implementing large-scale oral immunization on the wild animals is the main measure for controlling rabies in developed countries.
At present, the traditional rabies vaccine must rely on strict cold chain transport, and the deviation from the cold chain transport causes the reduction of the immune efficacy of the vaccine and further influences the immune effect. Therefore, the development of a novel heat-resistant and efficient rabies vaccine is very necessary for preventing and controlling rabies and has important practical significance.
Disclosure of Invention
In view of the above, the present invention aims to provide a fusion gene for improving heat resistance stability and immune effect of rabies virus, so that the prepared recombinant rabies virus has the characteristics of heat resistance and high-efficiency immune effect, and has a wide application prospect for preparing a novel rabies vaccine.
The invention provides a fusion gene for improving heat-resistant stability and immune effect of rabies virus, which comprises a mineralization gene W6 and a G gene;
the nucleotide sequence of the mineralization gene W6 is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the G gene is shown as SEQ ID NO: 2, respectively.
Preferably, the fusion gene comprises W6G and GW 6;
the nucleotide sequence of W6G is shown in SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of GW6 is shown as SEQ ID NO: 4, respectively.
The invention provides a recombinant rabies virus carrying mineralized genes, which comprises the fusion genes;
the fusion gene is inserted between the N and P genes of the backbone rabies virus genome.
Preferably, the fusion gene is inserted between N and P of the backbone rabies virus genome via bsiwi i and Nhe i cleavage sites.
Preferably, the strain of the rabies virus is the rabies virus SAD-B19 strain.
Preferably, the recombinant rabies virus comprises rBNSP-RV-GW6 and rBNSP-RV-W6G.
The invention provides a construction method of the recombinant rabies virus, which comprises the following steps:
1) inserting the fusion gene between the N gene and the P gene of the full-length cDNA plasmid of the rabies virus to obtain a recombinant plasmid;
2) and rescuing the recombinant plasmid by a reverse genetic operation system to obtain the recombinant rabies virus.
Preferably, an in vitro mineralization treatment is also performed after said rescue;
the method for the in vitro mineralization treatment comprises the steps of treating the recombinant rabies viruses by using a mineralization A liquid, and then adding a mineralization B liquid for treatment;
the treatment time of the mineralized liquid A is 50-70 min, and the temperature is 3-5 ℃; the mineralized A liquid is Na-containing 2 HPO4 0.36g/L、NaH 2 PO 4 0.30g/L of aqueous solution with the pH value of 8;
the treatment time of the mineralized B liquid is 50-70 min, and the temperature is 3-5 ℃; the mineralized B solution is 3.33g/L CaCL with the pH value of 8 2 An aqueous solution.
The invention provides application of the recombinant rabies virus or the recombinant rabies virus obtained by the construction method in preparing a vaccine for preventing and controlling rabies.
Preferably, the transport temperature of the rabies prevention and control vaccine is not more than 38 ℃.
The invention provides a fusion gene for improving heat-resistant stability and immune effect of rabies virus, which comprises a mineralization gene W6 and a G gene; the nucleotide sequence of the mineralization gene is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the G gene is shown as SEQ ID NO: 2, respectively. The fusion gene is introduced into the rabies virus, so that the content of G protein in the virus is increased, a high-level anti-rabies virus antibody is induced and generated, and a good immune effect is achieved; meanwhile, the mineralization gene improves the heat-resistant stability of the virus, and is convenient for the storage and transportation of subsequently prepared vaccines.
Furthermore, the invention specifically defines the connection relationship between the mineralized gene W6 and the G gene, wherein the sequence of the G gene sequentially comprises a signal peptide, an extracellular region, a transmembrane region and an intracellular region, GW6 indicates that the mineralized gene W6 is located behind the extracellular region and before the transmembrane region of the G gene, and W6G indicates that the mineralized gene W6 is located behind the signal peptide of the G gene and before the extracellular region. Experiments show that the connection relation of the mineralization gene W6 and the mineralization gene G is a factor influencing the heat-resistant stability of the recombinant rabies virus, and the GW6 fusion gene is favorable for improving the heat-resistant stability of the recombinant rabies virus.
The invention provides a recombinant rabies virus carrying mineralized genes, which comprises the fusion genes; the coding sequence of the fusion gene is inserted between the N and P genes of the rabies virus genome. In order to evaluate the effect of the recombinant rabies virus as a novel rabies vaccine, the biological characteristics such as a growth curve, pathogenicity, heat resistance, immunogenicity, toxicity attack protection, genetic stability and the like of the recombinant rabies virus are researched, the growth characteristics of the recombinant rabies virus are basically consistent compared with those of a parent strain, and the titer of the recombinant rabies virus on BSR cells is slightly lower than that of the parent strain BNSP-333; the pathogenicity of the vaccine as an attenuated vaccine is low, and the vaccine cannot die adult mice; because the recombinant rabies virus carries double G genes, the prepared mineralized vaccine has good immunogenicity, and can induce organisms to generate rabies neutralizing antibodies with protective efficacy; meanwhile, the recombinant rabies virus has heat-resistant stability and is convenient to store and transport.
Drawings
FIG. 1 is a gene recombination strategy for constructing recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G, wherein A is an insertion site of a fusion gene, B is a sequence structure schematic diagram of GW6, and C is a sequence structure schematic diagram of W6G;
FIG. 2 is a diagram of the identification electrophoresis of pBNSP-RV-GW6 and pBNSP-RV-W6G plasmids by BsiWI and NheI double digestion; m is gamma-EcoRT 14digest, 1, 2 is pBNSP-RV-GW6 plasmid, 3, 4 is pBNSP-RV-W6G plasmid;
FIG. 3 shows the immunofluorescence identification results of recombinant rabies viruses pBNSP-RV-GW6 and pBNSP-RV-W6G after rescue; blue is DAPI nuclear staining, and green fluorescence is rabies N protein antibody;
FIG. 4 shows the result of Western blotting detection of target protein, where M is protein Marker, 1: recombinant virus rBNSP-RV-GW 6; 2 is blank control; 3 is recombinant virus rBNSP-RV-W6G;
fig. 5 shows the results of RT-PCR detection of genetic stability of recombinant rabies virus rBNSP-RV-GW6 and rBNSP-RV-W6G genes, and identification of genetic stability of a. bnsp-GW6, wherein M: trans 2K Marker; 1: generation 5; 2: generation 10; 3: generation 15; bnsp-W6G genetic stability identification results, wherein M: trans 2K Marker; 1: generation 5; 2: generation 10; 3: generation 15
FIG. 6 shows the growth curves of recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G of chimeric GW6 and W6G genes and the constructed recombinant viruses of chimeric GFP genes and RV-G genes on Vero;
FIG. 7 shows the rabies antibody levels of antigens prepared by recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G of chimeric GW6 and W6G genes in Balb/c mice, and simultaneously establishes the antibody levels of the vaccine prepared with the GEL adjuvant and the negative control group of PBS at different periods after immunization;
FIG. 8 shows the survival rate of immunized mice 28 days after immunization of the antigens prepared from recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G and the negative control of vaccine prepared with GEL adjuvant and PBS;
FIG. 9 shows the results of the thermostability experiments for different virus strains after the maniation treatment.
Detailed Description
The invention provides a fusion gene for improving heat-resistant stability and immune effect of rabies virus, which comprises a mineralization gene W6 and a G gene; the nucleotide sequence of the mineralization gene W6 is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the G gene is shown as SEQ ID NO: 2, respectively.
In the present invention, the fusion gene includes a G gene. The G gene is the only protein of the rabies virus which can generate a neutralizing antibody, and the recombinant rabies virus contains double G genes by limiting the G gene in the fusion gene, so that the content of immunogen is improved, and the induction of the antibody with higher level is facilitated, and the prepared recombinant rabies virus is more suitable for preparing vaccine candidate strains.
In the present invention, the fusion gene includes the mineralization gene W6. The mineralization gene W6 has the characteristic of heat stability and is convenient to store and transport.
In the present invention, the fusion gene preferably includes W6G and GW6, depending on the connection relationship of the mineralization gene W6 and the G gene; the W6G is mineralized gene W6 located behind the signal peptide of the G gene and in front of the extracellular region. The nucleotide sequence of the W6G is shown as SEQ ID NO: 3, wherein the 7 th to 122 th sites are signal peptide sequences, the 123 th to 191 th sites are W6 sequences, the 192 th to 1278bp extracellular region sequences, the 1279 th to 1464 th sites are transmembrane region sequences, and the 1280 th to 1650 th intracellular region sequences; the 5 'end and the 3' end are BsiW1 and Nhe1 restriction sites respectively. The GW6 is mineralized gene W6 located behind the extracellular region of the G gene and in front of the transmembrane region. The nucleotide sequence of GW6 is shown as SEQ ID NO: 4, wherein the 7 th to 122 th sites are signal peptide sequences, the 123 th to 1210 th extracellular region sequences, the 1210 th to 1278 th sites are W6 sequences, the 1279 th to 1464 th sites are transmembrane region sequences, the 1465 th to 1650 th intracellular region sequences, and the 5 'and 3' ends are BsiW1 and Nhe1 enzyme cutting sites respectively. Experiments show that the GW6 fusion gene is more favorable for improving the heat-resistant stability of the recombinant rabies virus than the W6G fusion gene.
The invention provides a recombinant rabies virus carrying mineralized genes, which comprises the fusion genes; the coding sequence of the fusion gene is inserted between the N and P genes of the rabies virus genome.
In the present invention, the fusion gene is preferably inserted between the N gene and the P gene of the rabies virus genome through bsiwi and Nhe i cleavage sites. The framework strain of the rabies virus is preferably the rabies virus SAD-B19 strain. The rabies virus strain SAD-B19 is a known virus strain reported in the prior art, see in particular the prior art (Genetic stability (in vivo) of the attached anode viruses vaccine SAD B19.Beckert A, Geue L, Vos A, Neubert A, free C, Muller T. Beckert A, et al. Microbiol Immunol.2009Jan; 53(1) 16-21.).
In the present invention, the recombinant rabies virus preferably comprises rBNSP-RV-GW6 and rBNSP-RV-W6G. After inserting GW6 fusion gene into SAD-B19 strain as basic strain, rBNSP-RV-GW6 is obtained. After inserting the W6G fusion gene into SAD-B19 strain as basic strain, rBNSP-RV-W6G is obtained.
The invention provides a construction method of the recombinant rabies virus, which comprises the following steps:
1) inserting the fusion gene between N and P of the rabies virus full-length cDNA plasmid to obtain a recombinant plasmid;
2) and rescuing the recombinant plasmid by a reverse genetic operating system to obtain the recombinant rabies virus.
In the invention, the insertion method preferably introduces sequences of insertion enzyme cutting sites at two ends when artificially synthesizing the fusion gene, and realizes the insertion of the fusion gene through restriction enzyme cutting and connection. The restriction enzymes with restriction sites ligated at both ends are preferably BsiWI and Nhe I. The nucleotide sequence of the fusion gene introduced with the enzyme cutting site is shown as SEQ ID NO: 5(W6G') and SEQ ID NO: 6(GW 6').
In the present invention, the rabies virus full-length cDNA plasmid is preferably pBNSP. The pBNSP is constructed in the laboratory and is publicly reported in the prior art (rescue of recombinant rabies virus carrying GFP gene and biological characteristic research thereof, Chinese veterinary medicine, 2018,45(7): 1965-charge 1971.). The insertion of the fusion gene between N and P is advantageous for the expression of the foreign gene, and the expression amount of the inserted foreign gene is increased as compared with other insertion sites (between G and L genes).
In the present invention, the recombinant plasmid is rescued by a reverse genetic manipulation system, preferably by referring to the Matthias Schnell method.
In the present invention, an in vitro mineralization treatment is also performed after the rescue. The method for the in vitro mineralization treatment comprises the steps of treating the recombinant rabies viruses by using a mineralization A liquid, and then adding a mineralization B liquid for treatment; the treatment time of the mineralized liquid A is 50-70 min, and the temperature is 3-5 ℃; the mineralized A liquid is Na-containing 2 HPO4 0.36g/L、NaH 2 PO 4 0.30g/L of aqueous solution with the pH value of 8; the treatment time of the mineralized B liquid is 50-70 min, and the temperature is 3-5 ℃; the mineralized B solution is 3.33g/L CaCL with the pH value of 8 2 An aqueous solution. The in vitro mineralization treatment is beneficial to combining elements such as calcium, phosphorus and the like on the surface of the recombinant rabies virus, and the heat-resistant stability of the recombinant rabies virus is further improved.
In the invention, the prepared recombinant rabies virus is subjected to passage and identification, Western blotting identifies the expression condition of the G protein, shows that the recombinant rabies virus can express a target protein, and detects the genetic stability of the virus of passage 1, 5, 10 and 15 respectively, and the result shows that the fusion gene of each generation of virus is not mutated, thus showing that the genetic stability is good.
The pathogenicity research of the recombinant rabies virus in a mouse body shows that the recombinant rabies virus has no pathogenicity to the mouse, proves that the recombinant rabies virus taking SAD-B19 as a framework is a low virulent virus, and can be used for preparing vaccines to immunize animals against rabies. The immune challenge experiment shows that the challenge protection rate of the inactivated virus to the mice is 100 percent, and the negative control mice are all killed.
In view of ideal heat-resistant stability, genetic stability, higher immunogenicity and growth performance of the recombinant rabies virus, the invention provides the application of the recombinant rabies virus or the recombinant rabies virus obtained by the construction method in preparing a vaccine for preventing and controlling rabies.
In the invention, the transport temperature of the rabies prevention and control vaccine is not higher than 38 ℃.
In the invention, the virus attack protection rate of the inactivated vaccine is 100%, which indicates that the inactivated vaccine can resist the attack of rabies viruses and meets the standard of more than 80% of the international required immune virus attack protection rate in a virus attack test.
The fusion gene for improving the heat stability and immune effect of rabies virus, the recombinant rabies virus and the application thereof provided by the invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the invention.
Example 1
Construction method of recombinant vector containing mineralized gene and G gene
1. The strategy for constructing recombinant plasmids pBNSP-RV-GW6 and pBNSP-RV-W6G is shown in figure 1.
(1) The synthesized genes (sequences shown in SEQ ID NO:5 and SEQ ID NO:6) and pBNSP plasmid (recombinant plasmid carrying the SAD-B19 strain full-length cDNA) were treated with BsiWI and NheI restriction endonucleases, and the cleavage system is shown in Table 1.
TABLE 1 enzyme digestion System
| Component name | Volume of |
| |
5μL |
| Bsiw1 | 2μL |
| Nhe1 | 2μL |
| BSA | 0.5μL |
| pBNSP plasmid | 42.5μL |
After the systems are mixed uniformly, the incubation is carried out for 2h at 37 ℃, then the incubation is carried out for 2h at 55 ℃, and the gel recovery kit is used for recovery. The recovered products were ligated, and the ligation system is shown in Table 2.
TABLE 2 connection System
| Component name | Volume of |
| T4ligase buffer(10×) | 2μL |
| T4 DNA ligase | 1μL |
| DNA fragment | 5μL |
| pBNSP DNA | 12μL |
The above systems were mixed well and ligated overnight at 16 ℃. Transferring the ligation product into DH5 alpha competent cells, culturing for 16h in a 37 ℃ incubator, selecting a single clone, inoculating the single clone into a carbenicillin resistant LB culture medium for shaking culture, performing shaking culture at 37 ℃ for 20h, performing enzyme digestion identification on the small-sized plasmid by BsiWI/NheI, wherein an enzyme digestion system is shown in Table 1, performing electrophoresis detection on an enzyme digestion result, performing double enzyme digestion on pBNSP-RV-GW6 and pBNSP-RV-W6G plasmids to respectively show 15254bp and 1658bp target fragments, and sending the plasmid with correct enzyme digestion to Beijing Jinzhinzhi Biotech Limited for sequencing. The results are shown in FIG. 2.
The clone with correct enzyme cutting identification and sequencing is extracted with reference to the instruction of a plasmid mass extraction kit of Qiagen company, the final plasmid concentration is adjusted to 1 mug/muL, and the plasmid is stored at 4 ℃ for standby.
Example 2
Recombinant plasmid pBNSP-RV-GW6 and pBNSP-RV-W6G are used for rescuing and screening recombinant viruses
1. Transfection and Virus screening methods were performed according to the Matthias Schnell method
Transfection: (1) preparing Vero-E6 cells: the cell density of the 6-hole cell culture plate is 5 multiplied by 10 5 Each well, the culture solution contains 10 percent of fetal calf serum; after culturing at 37 ℃ for 24h, transfection is carried out when the cells are about 75%.
(2) The transfection reagent X-tremeGene 924. mu.l was added to a 1.5mL centrifuge tube containing 600. mu.l Optimem medium, mixed well and left at room temperature for 5 minutes.
(3) 5. mu.l of pBNSP-RV-GW6 and pBNSP-RV-W6G, 2.5. mu.l of pTIT-N, 1.25. mu.l of pTIT-P, 1.25. mu.l of pTIT-L, 1. mu.l of pTIT-G plasmid, 1.5. mu.l of pCAGGS-T7 were mixed in another 1.5mL centrifuge tube.
(4) The plasmid was added to the tube of transfection reagent, mixed well and left to stand at room temperature for 15 min.
(5) The plasmid and transfection reagent mixture was added to cells in 6-well plates at 100. mu.l/well and then placed at 34 ℃ with 5% CO 2 Culturing for 96h in an incubator.
And (3) virus screening:
on day 5 post-transfection, cells in 6-well plates were individually transferred to 60mm dishes for expansion. And (4) on 8 days after transfection, harvesting virus liquid, and performing immunofluorescence assay to identify whether the recombinant virus is successfully rescued. The method comprises the following steps: the harvested virus solution was added to a monolayer of cells grown in 12-well plates, and then placed at 34 ℃ in 5% CO 2 Culturing in incubator for 24h, collecting supernatant, filling with 80% precooled acetone, and keeping at 4 deg.C for 30 min. Discarding acetone, slightly air-drying, adding 500 mu L of rabies virus resistant N protein fluorescent antibody and CDV fluorescent antibody into each hole, incubating at 37 ℃ for 1-2 h, discarding dissolving solution, washing cells for 3 times by using deionized water, observing under a fluorescence microscope, and showing a large amount of green fluorescent spots, wherein the obtained virus is named as figure 3: rBNSP-RV-GW6 and rBNSP-RV-W6G.
Example 3
Passage and identification of recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G
1. The titer of rescued recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G was determined, and confluent monolayers of VeroE6 of cells were inoculated at an MOI (multiplicity of infection) of 0.1 and placed at 34 ℃ in 5% CO 2 Culturing for 72h in incubator, harvesting virus, supplementing culture solution, and continuously standing at 34 deg.C and 5% CO 2 The virus was cultured in an incubator for about 72 hours and then harvested again, thus harvesting the virus 3 times in succession. Subpackaging at-80 deg.C for use. Sequentially passaging to 15 times, and detecting the virus titer, wherein the titer of the two recombinant viruses can reach 1X 10 7 FFU/ml。
Western blotting is used for identifying the expression condition of the G protein, and the result is shown in figure 4, which shows that the recombinant virus can express the target protein.
The genetic stability of GW6 and W6G genes in the recombinant virus was identified by RT-PCR. The primers are as follows: an upstream primer: 5'-GGCTGAACTGACAAAGACTGAC-3' (SEQ ID NO: 5);
a downstream primer: 5'-CAGCCATCTCAAGATCGGCCAG-3' (SEQ ID NO: 6).
50 μ L reaction: 25 μ L of Taq premixed enzyme, 1 μ L of each of the upstream and downstream primers (50 pmoL/. mu.L), 2 μ L of cDNA, and 21 μ L of sterile water. Reaction procedure: 95 ℃ for 5 min; {94 ℃, 1 min; 57 ℃ for 1 min; 72 ℃ for 2 min; 35 Cycles; 72 ℃ for 10 min.
The detection results are shown in FIG. 5, which indicates that the recombinant virus can be stably passaged.
Example 4
In vitro growth Curve study of recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G on Vero cells
1. Vero cells were plated in 6-well plates at 5X 10 cell number 5 Culturing in a well for 24h, then infecting cells with recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G at MOI10, placing at 34 deg.C and 5% CO 2 Culturing, sampling 100 μ L supernatant culture solution at 2h, 24h, 48h, 72h and 96h after virus infection, and finally performing immunofluorescence assay, determining and drawing a virus growth curve.
Results FIG. 6 shows that the titers of the recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G are slightly lower than those of the recombinant viruses carrying the GFP gene constructed in this laboratory, and the highest level of virus titer was observed at 72h after infection.
Example 5
Pathogenicity research of recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G in Balb/c mice
1. The toxic price reaches 1 x 10 7 FFU/mL recombinant virus was serially diluted 10-fold in 2% FBS PBS with a gradient of 10 dilution -1 ～10 -5 6 BALB/c mice of 6-8 weeks old are inoculated in the brain of each diluted virus, each mouse is inoculated with 0.03mL, observation is carried out day by day, mice dying within 3 days are not caused by virus infection and are non-specific death, the final result is not counted, and observation is carried out for 14 days.
The results show that all mice grow healthily in the observation period, no macroscopic clinical symptoms appear, the recombinant virus is non-pathogenic to the mice, and the recombinant rabies virus with SAD-B19 as a framework is proved to be a low virulent virus and can be used for immunizing wild animals with rabies by oral vaccines.
Example 6
Inactivated vaccine of recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G, immunogenicity and challenge protection research
1. Inactivation of recombinant rabies virus and preparation method of vaccine
(1) Adding beta-propiolactone into recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G according to the proportion of 0.025 percent, oscillating for 24 hours at 4 ℃, and then hydrolyzing for 2 hours at 37 ℃ to complete the inactivation of the viruses.
(2) Safety test of inactivated virus: respectively culturing inactivated virus solution and inactivated rabies virus solution and BSR cells on 96-well cell culture plate at 34 deg.C and 5% CO 2 After the culture in the incubator is carried out for 4d, an immunofluorescence test is carried out to detect whether rabies viruses grow, and if the inactivated virus samples can detect green fluorescence, the inactivation is not thorough. As a result, inactivated viruses have no fluorescent spots, and all inactivated viruses have green fluorescent spots.
(3) Adopting Montanide GEL adjuvant of SEPPIC company of France, mixing with inactivated antigen according to proportion of 5% -20%, emulsifying at 200 r/min, stirring at low speed for 10min, subpackaging the vaccine into sterilized vaccine bottles, covering and sealing, and storing at 4 deg.C for use.
2. Vaccine immunization test:
BALB/C mice were immunized with rBNSP-RV-GW6, rBNSP-RV-W6G inactivated antigen and its vaccine prepared with Montanide GEL adjuvant, respectively, 200. mu.L of each mouse was inoculated, 20 mice were inoculated per group, and a negative control mouse (injected with 200. mu.L of PBS) was also set. Mice in each group were immunized 2 times on days 0 and 14. 3 mice were bled 7d, 21d and 28d after the first immunization, and the level of anti-rabies glycoprotein antibody was determined by an indirect ELISA method established in this laboratory.
The results show that the inactivated antigen and the vaccine prepared by Montanide GEL can generate higher antibody level against rabies virus glycoprotein after being used for immunizing mice (see figure 7).
To verify that mice acquired high levels of immunity had immunoprotection against rabies virus challenge, mice were inoculated with 50LD intracranially at 28d post-immunization 50 CVS-1130. mu.L of (5), 14 days of continuous observation, and the morbidity and mortality of the mice were recorded.
The results are shown in fig. 8, the mice immunized with the vaccine prepared by the inactivated antigen and the Montanide GEL adjuvant all survived the rabies virus challenge, the protection rate of the mice immunized with the inactivated antigen alone was 100%, and the negative control mice all died.
The results show that the inactivated vaccine prepared from the rBNSP-RV-GW6 and the rBNSP-RV-W6G can resist the attack of rabies viruses after being used for immunizing mice, and the standard of more than 80 percent of immune attack protection rate meeting the international requirement in an attack test is met.
Example 7
To examine the heat-resistant stability of the recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G at different temperatures after carrying the mineralized group, the virus titer was examined after placing the recombinant viruses rBNSP-RV-GW6 and rBNSP-RV-W6G and the recombinant viruses rBNSP-RV-hG and rBNSP-RV-G which do not carry the mineralized group at 4 ℃, 25 ℃ and 37 ℃ respectively for 7 days.
The results are shown in table 3 below, which shows that the recombinant virus has certain heat-resistant stability characteristics after the chimeric mineralization gene.
TABLE 3 thermal stability test results
Example 8
In vitro mineralization methods and heat stability tests
1) Preparing a mineralized solution: respectively preparing solution A and solution B, wherein the compositions of the solutions are as follows:
mineralization of the solution A: separately weighing Na 2 HPO 4 0.36g，NaH 2 PO 4 0.30g, dissolved in 1L deionized water, mixed, adjusted pH to 8.0, and filtered with 0.45 μm filter for use.
Mineralization of the liquid B: weighing CaCL 2 3.33g of the extract was dissolved in 1L of deionized water, mixed, adjusted to pH 8.0, and sterilized by filtration through a 0.45 μm filter.
2) The in vitro mineralization method comprises the following steps: firstly, 8ml of mineralized A liquid is added into a 15ml centrifuge tube, then 2ml of recombinant rabies virus is added, the mixture is stirred for 1 hour at 4 ℃ by a magnetic stirrer, then 1ml of mineralized B liquid is added, the mixture is stirred for 2 hours by the magnetic stirrer at 4 ℃, and the virus in-vitro mineralization treatment is completed.
3) Heat resistance stability test:
recombinant rabies viruses rBNSP-RV-GW6 and rBNSP-RV-W6G are mineralized and treated respectively according to the in vitro mineralization method, and recombinant rabies viruses rBNSP-G (recombinant viruses obtained by rescuing only G gene inserted according to the construction method of rBNSP-RV-GW 6) and rBNSP-hG (signal peptide of G gene inserted on the basis of rBNSP-G is replaced by signal peptide of human IgG) without inserted mineralized gene are mineralized and treated as comparison to obtain the heat resistance of the mineralized gene W6. The mineralized virus rBNSP-RV-GW6 is recorded as rBNSP-GW6, and the mineralized virus rBNSP-RV-W6G is recorded as rBNSP-W6G. The above-mentioned mineralized samples were placed at 4 ℃ and 37 ℃ respectively, and the virus titer was measured for 5 days.
The results are shown in FIG. 9. Experiments show that after the mineralization gene is inserted, the recombinant virus is mineralized in vitro, the virus titer is basically not reduced after the recombinant virus is placed for 5 days at 37 ℃, and the heat-resistant stability is obvious.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
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cgtacgatga ttcctcaagc tctgttgttt gtacctcttc tggtttttcc attgtgtttc 60
gggaaattcc ccatttacac gataccagac aaactcggcc cctggagccc gatcgatata 120
cataacctca gctgtccgaa caatctggtt gtggaggacg aaggatgtac caatctgtca 180
ggattctcat acatggagct taaagtagga tatatttcgg ccataaaggt gaacgggttc 240
acttgtacgg gtgtggtaac ggaagcagaa acctacacta actttgtcgg ttatgtcacc 300
accacgttta agagaaagca cttccgacca acaccggatg catgcagatc agcatacaat 360
tggaagatgg caggtgaccc cagatatgaa gagtctctgc acaatcccta tcctgattat 420
cattggctcc ggactgtaaa aaccaccaaa gagtctgttg ttatcatatc tccaagtgtg 480
gcagacttag acccgttcga taaatcactt cattcgagag tttttcctag aggaaaatgc 540
tcaggaataa cggtgtcttc tgcctactgc tctaccaacc atgattatac catctggatg 600
cctgaaaatc ctagactggg gacctcttgt gatattttca ccaacagcag agggaagaga 660
gcatccaaag ggagcaagac ctgtggattt gtggatgaga gaggcttgta caaatctcta 720
aaaggagcat gcaaactgaa gctgtgtgga gttcttggac ttaggcttat ggacggaaca 780
tgggtcgcga ttcagacatc aaacgagacc aagtggtgcc ctcctgatca actcgtgaat 840
ctacatgact ttcattcaga tgagattgaa catcttgttg tggaggagtt ggttgagaag 900
agggaggagt gtctagatgc actggagtcc atcatgacca ccaatcccgt gagtttcaga 960
cgtctcagtc ccttgaggaa gcttgtgcct ggatttggaa aagcatacac catattcaac 1020
aagaccttaa tggaggctga tgctcactac aaatcggtcc aaacttggga tgagatcatc 1080
ccctcgaaag ggtgtttaag agtcggggcg agatgtcatc ctcatgtgaa cggagtattt 1140
ttcaatggta tcatcctagg ccctgacggc catgtcttaa tcccggaaat gcagtcatcc 1200
ctcctccagc gctggcggct tgaaggcaca gacgataagg aggagccaga aagtcaaaga 1260
agaataggaa gattcggaca gcatatggag ttgttggaat cctcggtcat ccccttaatg 1320
catcccttgg cagatccatc aacggttttt aaagatggtg acgaggtgga ggattttgtt 1380
gaggttcacc ttccagatgt gcataagcag gtctcagggg ttgatctcgg tctcccaaac 1440
tgggggaagg atgtgttgat gggcgcaggc gttttgacgg cactgatgtt gatgattttc 1500
ctaatgacgt gttgccgaag gactaataga gcagaatcaa tacaacacag tcttggagag 1560
acagggagga aagtgtcggt gacctcccaa agcgggaggg tcatatcttc atgggagtca 1620
tataaaagcg gaggtgagac caagctgtaa gctagc 1656
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggctgaactg acaaagactg ac 22
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cagccatctc aagatcggcc ag 22
Claims (10)
1. A fusion gene for improving heat-resistant stability and immune effect of rabies virus is characterized by comprising a mineralization gene W6 and a G gene;
the nucleotide sequence of the mineralization gene W6 is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the G gene is shown as SEQ ID NO: 2, respectively.
2. The fused gene of claim 1, wherein the fused gene comprises W6G and GW 6;
the nucleotide sequence of the W6G is shown as SEQ ID NO: 3 is shown in the figure;
the nucleotide sequence of GW6 is shown as SEQ ID NO: 4, respectively.
3. A recombinant rabies virus carrying a mineralized gene, wherein the recombinant rabies virus comprises the fusion gene of claim 1 or 2;
the fusion gene is inserted between the N and P genes of the backbone rabies virus genome.
4. The recombinant rabies virus of claim 3, wherein said fusion gene is inserted between N and P of the backbone rabies virus genome via BsiW I and Nhe I cleavage sites.
5. The recombinant rabies virus of claim 3, wherein the backbone strain of the recombinant rabies virus is rabies virus SAD-B19 strain.
6. The recombinant rabies virus of claim 5, wherein said recombinant rabies virus comprises rBNSP-RV-GW6 and rBNSP-RV-W6G.
7. The method for constructing recombinant rabies virus of any one of claims 3 to 6, comprising the following steps:
1) inserting the fusion gene of claim 1 or 2 between the N gene and the P gene of the full-length cDNA plasmid of the rabies virus to obtain a recombinant plasmid;
2) and rescuing the recombinant plasmid by a reverse genetic operation system to obtain the recombinant rabies virus.
8. The construction method according to claim 7, characterized in that an in vitro mineralization treatment is further performed after the rescue;
the method for the in vitro mineralization treatment comprises the steps of treating the recombinant rabies viruses by using a mineralization A liquid, and then adding a mineralization B liquid for treatment;
the treatment time of the mineralized liquid A is 50-70 min, and the temperature is 3-5 ℃; the mineralized A liquid is Na-containing 2 HPO40.36g/L、NaH 2 PO 4 0.30g/L of aqueous solution with the pH value of 8;
the treatment time of the mineralized B liquid is 50-70 min, and the temperature is 3-5 ℃; the mineralized B solution is 3.33g/L CaCL with the pH value of 8 2 An aqueous solution.
9. Use of the recombinant rabies virus according to any one of claims 3 to 6 or the recombinant rabies virus obtained by the construction method according to claim 7 or 8 in the preparation of a vaccine for preventing and controlling rabies.
10. The use of claim 9, wherein the delivery temperature of the rabies vaccine does not exceed 38 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958919A (en) * | 2022-05-09 | 2022-08-30 | 军事科学院军事医学研究院军事兽医研究所 | Rabies virus recombinant filovirus or arenavirus pseudovirus and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104096235A (en) * | 2013-04-10 | 2014-10-15 | 浙江大学 | Genetic engineering heat stable vaccine and preparation method thereof |
| CN104630270A (en) * | 2015-01-28 | 2015-05-20 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for biologically mineralizing surface of influenza virus |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104096235A (en) * | 2013-04-10 | 2014-10-15 | 浙江大学 | Genetic engineering heat stable vaccine and preparation method thereof |
| CN104630270A (en) * | 2015-01-28 | 2015-05-20 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for biologically mineralizing surface of influenza virus |
Non-Patent Citations (1)
| Title |
|---|
| 刘翠: "重组狂犬病病毒的拯救及其矿化疫苗的生物学特性研究", 中国农业科学院学位论文, 15 December 2018 (2018-12-15) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114958919A (en) * | 2022-05-09 | 2022-08-30 | 军事科学院军事医学研究院军事兽医研究所 | Rabies virus recombinant filovirus or arenavirus pseudovirus and preparation method and application thereof |
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