【作者】 袁子国；

【导师】 扈荣良；

【作者基本信息】 吉林大学 ， 预防兽医学， 2008， 博士

【摘要】 为了研制一种新型的抗狂犬病疫苗,本研究通过同源重组的方法成功的构建了表达狂犬病病毒糖蛋白膜外区的两株重组伪狂犬病病毒rPrV/eGFP/rgp、rPrV/rgp),并对获得的重组病毒进行了犬科动物的免疫试验。本研究首先对PrV疫苗株Bartha-K61在犬科动物体内的安全性和有效性进行了评价。结果显示:该疫苗株在犬科动物体内安全,且能有效的刺激机体产生免疫应答。通过在Bartha-K61的PK基因处分别插入eGFP/rgp和rgp两个表达盒,构建了表达RV糖蛋白的两株重组病毒。犬的免疫试验结果表明:肌肉注射和口服免疫5周时,均能刺激机体产生抗体滴度大于0.5 IU/mL具有保护水平的抗RV的中和抗体,针对PrV的中和抗体滴度在1:64～1:128之间,且均可维持在26周以上。表明,两个重组病毒均可有效的刺激机体产生抗RV的保护性免疫应答。更多还原

【Abstract】 Rabies is an zoonosis, caused by rabies virus. The fatality is almost 100% when rabies was happened in infected people. So far, it is an acute infection disease of supreme case-fatality rate in human history. The happening trend of rabies has been rising up year after year in recently years. The death of people is approximately 55,000 in the world every year. Ninety nine percent cases are happened in Asia and Africa. The dead cases are about 35,000～40,000 every year in Asia, at the same time, more than 45,000,000 people are in the station of post-exposure of rabies. There are some reasons, except for poor immuno-measure, one of the most reason is the single lower immune effect. Therefore, it should be an urgent problem to develop a kind of safe, effective and economical vaccine.Rabies virus is made up of glycoprotein, neucleoprotein, matrix protein, phosphorylated protein and large transcriptase. The glycoprotein is the mainly virulence factor and, correlated with viral tropism of nerves and pathopoiesis mechanism. It is the only viral structural protein, including signal peptide of hydrophobicity, intramembrane domain, transmembrane domain, ectodomain, antigen epitopes are mainly concentrated in ectodomain, and possesses higher conservation.The research of rabies vaccine experiences belowed processed, including killed vaccine, attenuated vaccine, subunit vaccine, genetic engineering vaccine, antiidiotypic antibody vaccine, live virus vaccine and DNA vaccine. In developed country, the purified and concentrated killed vaccine is usually used. Lots of disadvantages are displayed, including complex preparation technology and higher costs. Due to the limitation of economical condition, it hasn’t been used all the time. So far, the attenuated viccine occupied the mainly position in China, it is easy to prepare, possesses long immuno period, but exists the risk of reversion to virulence. Therefore, the attenuated vaccine had been forbidden in developed country. Simultaneously, it is poined that we should research novel and safe vaccine to prevent rabies. Besides killed vaccine and attenuated vaccine, the glycoprotein and neucleoprotein is the mainly interested target genes. The glycoprotein is usually used in the study of subunit vaccine and genetic engineering vaccine, possess the easy preparation, but have the disadvantages of poor immunogenicity, and hav’nt been used in practical application. The technology of antiidiotypic antibody vaccine doesn’t mature in the large scale of production. The recombinant human adenovirus type 5 expressing the rabies virus glycoprotein and neucleoptotein possessed the better immunity protection, but existed the problem of the second immunological rejection. It plays an important role in the preservation trial of rabies. There are lots of paper about DNA vaccine, and few vaccines have began the clinical test, but the low expression of foreign antigen and sigle preventing schedule limits its development. So, we should discuss the agenda about the research a kind of vaccine that is suitable with our history.Recently, to apply the non-pathogenicity or attenuated virus as the live vector, and clone the other antigen genes into this vector. Because, the sites of recombinant are the non-essential regions and not effect the propagation of virus. Therefore, it has been the focus to construct the recombinant live virus vaccine. Pseudorabies virus belongs to a member of herpesviridae, possesses large genome, and many non-essential regions of replication, potency of expression foreign genes. So it is regarded as an ideal live vector. The gene of protein kinase is the non-essential gene and also essential composition of neurovirulence. By mutation and deletion to PK gene, the virus virulence degrades and the immunogenicity doesn’t change. Therefore, the region of PK is the ideal inserting region.In our research, we used PrV Bartha-K61 vaccine strain as the vector, and constructed the two recombinant pseudorabies virus (PrV/eGFP/rgp、PrV/rgp) expressing the glycoprotein of rabies virus first times and carried out the immunity test in canine animals.In order to estimate safety and efficacy for vaccine strain Bartha-K61 of pseudorabies virus in canine animals. We immunized dogs by two routes (intramuscular injection, intranasal and oral vaccination) with 2 mL (107.0/0.1mL TCID50). At the ended of trial, we detected a serial indexes, including pathological section, immunohistochemistry, clinical behavior, birth performance, viral persistence in excreta, we found that no antigen existed in sensitive organs and, body temperature was normal and, mental state, birth were also well and, no virion was found in excreta. Meanwhile, we detected the level of cell mediated immunity, humoral-mediated immunity and neutralization test that could induce effective immune response administred by three immune routes and neutralization antibody arrived at higher level than control group. From above experiments, we domonstrated the Bartha-K61 was safety and utility in vaccinated dogs and, accordingly, to provide a scientific basis for recombinant live vaccine that expressed the antigens of dogs using the PrV as the vector.We selected PK gene of pseudorabies virus as the homologous recombinant region. During the experiments, we used the vector of p8-AA that was previously constructed by our lab. We respectively inserted three whole expression cassettes of LacZ, eGFP/rgp and eGFP-/rgp, including human cytomegalovirus promoter and polyadenylic acid (polyA), and gained three intermedial transfer vectors of p8AA-LacZ, p8AA-eGFP/rgp and p8AA-rgp. To obtain the recombinant virus, we used the cotransfection method of plasmid with the whole genome of pseudorabies virus by liposome mediation. To gain the recombinant virus of PrV/LacZ by the test of plaque clone, the method was briefly described, we transfected the PK-15 cells with the plasmid of p8AA-LacZ and the genome of Bartha-K61, the rate was approximately 3:1, and overlapped the nutrient agar inclucing X-gal, to picked the blue plaque cells, froze thawing it and re-inoculated the new PK-15 cells until all the cytopathic cell displayed the blue under X-gal, puried it and stored it at -80℃. We used the rPrV/LacZ as the parental virus strain and extracted its genome, to use the same method previously mentioned to gain the recombinant virus of PrV/eGFP/rgp, screening it until all the cells infected by rRPrV/eGFP/rgp radiated the green fluorescence under the fluorescent microscope, puried it and stored it at -80℃. The recombinant virus of PrV/rgp was also gained by the same method previously explained. Briefly, to extract the genome of rPrV/eGFP/rgp and plasmid of p8AA-rgp and, transfected the PK-15 cells with p8AA-rgp and the genome of rPrV/eGFP/rgp by liposome, identified it and purified it until the pathological cells could all radiate the green fluorescence under the fluorescent microscope through anti-screening rPrV/rgp and purifing it. To test the transcription, expression of foreign genes and genetic stability of recombinant virus. The result showed that the foreign genes could be correctly expressed and react with the RV positive serum. The two recombinant virus were genetic stability when they were passaged to 30th generations on PK-15 cells.To inoculate dogs administrated with two routes (intramuscular injection and oral vaccination) with two recombinant virus (rPrV/eGFP/rgp, rPrV/rgp). The results indicated that the body could be induced the production of antibody against the RV glycoprotein by Western blot and Mouse intracerebral. From the neutralization test of virus, we could find the neutralizing antibody titers against rabies virus reached >0.5 IU/mL, protective level neutralizing antibody were detected, and 1:64～1:128 serum dilution against pseudorabies virus by 5 weeks post-vaccination in all dogs by administration with intramuscular inoculation and oral vaccination. The result also indicated that the pseudorabies virus vector could infect dogs and replicate in vivo, and that the rabies virus glycoprotein had been expressed and an effective immune response elicited in the body. Antibody titers were maintained for over 6 months. The research provide the scientific basis for the research of injection vaccine and oral vaccine of rabies, and bring the hope for precaution of wild animals rabies.To above mentioned, we gained two recombinant virus expressing rabies virus glycoprotein, and carried out the identification of bionomics and immunity test for them. The results revealed that the two recombinant virus are safe and effective in dogs. Meanwhile, the protective neutralizing antibody to RV could be induced in dogs administrated with intramuscular injectiong and oral vaccination. The two recombinant virus expressing the rabies virus glycoprotein will play an active role in the precaution and control of rabies in China.更多还原