表达西尼罗病毒囊膜蛋白重组狂犬病病毒的构建与应用研究

发布时间：2018-09-01 08:53

【摘要】：西尼罗病毒病是由西尼罗病毒(West Nile virus,WNV)感染引起的急性人兽共患传染病。近20年,WNV在全球扩散,不断引起人、畜疾病的暴发流行,成为世界性公共卫生问题。2011年,梁国栋等从我国新疆维吾尔自治区采集的蚊虫标本中分离到WNV,大量血清学结果证明当地不仅存在WNV感染所致疾病,还发生过WNV感染引发的病毒性脑炎流行。到目前为止,尚无获批的针对WNV的特效药物和有效疫苗。本研究利用狂犬病病毒(Rabies virus,RABV)弱毒疫苗SRV9株反向遗传操作系统,通过PCR的方法,在SRV9株基因组的P与M基因间插入西尼罗病毒E基因(WNV E),构建表达WNV E蛋白的全长质粒pD SRV9 WNV E。将构建的全长质粒pD SRV9 WNV E与表达狂犬病病毒核蛋白(N蛋白)、磷蛋白(P蛋白)、转录大蛋白(L蛋白)、糖蛋白(G蛋白)的辅助质粒通过脂质体共同传染BSR细胞,进行重组病毒的拯救。直接免疫荧光结果表明:成功拯救获得了具有感染活性的重组狂犬病病毒rSRV9 WNVE。间接免疫荧光、Western blot、PCR等结果表明:重组病毒rSRV9 WNVE可成功表达WNV E蛋白。此外,重组病毒的生长曲线表明:外源基因的表达不影响重组病毒的体外生长特性。分别将母本病毒SRV9和重组狂犬病病毒rSRV9 WNVE经脑内注射途径接种小鼠,每天观察并对小鼠的精神状态、存活等情况进行记录,连续观察21天。结果表明:与母本病毒SRV9相比,重组狂犬病病毒rSRV9 WNVE表达WNV E蛋白后对小鼠的致病性降低,呈现高度的安全性。分别将母本病毒SRV9和重组狂犬病病毒rSRV9 WNVE以5×105TCID50/只和5×106TCID50/只的剂量通过肌肉注射途径免疫小鼠,初次免疫后两周,以相同的剂量和途径加强免疫一次,分别于一免和二免后2周采集小鼠血清,并测定血清中狂犬病病毒和WNV的抗体效价。结果表明:重组病毒免疫小鼠后,既可刺激机体产生狂犬病病毒中和抗体,也可刺激机体产生WNV特异性抗体,表明重组病毒具有很好的免疫原性。综上所述,表达WNV E蛋白的重组狂犬病病毒r SRV9 WNVE对小鼠具有的良好的安全性和免疫原性,具有作为疫苗的潜力,为进一步研制西尼罗病毒病新型疫苗奠定了基础。
[Abstract]:West Nile virus disease is an acute zoonotic disease caused by West Nile virus (West Nile virus,WNV) infection. In the past 20 years, WNV has spread in the world, causing outbreaks of human and animal diseases, and has become a worldwide public health problem. A large number of serological results of WNV, isolated from mosquito specimens collected from Xinjiang Uygur Autonomous region of China show that there are not only diseases caused by WNV infection but also viral encephalitis caused by WNV infection. So far, there is no approved WNV specific drug and effective vaccine. In this study, using reverse genetic operation system of SRV9 strain, a attenuated rabies virus (Rabies virus,RABV) vaccine, a full-length pD SRV9 / WNV E. was constructed by inserting WNV E), (WNV E),) between P and M genes of SRV9 strain by PCR. The constructed full-length plasmid pD SRV9 WNV E was co-transmitted to BSR cells through liposome with the co-expression plasmids of nucleoprotein (N protein), phosphoprotein (P protein), large transcription protein (L protein) and glycoprotein (G protein) of rabies virus. Carrying out the rescue of the recombinant virus. The results of direct immunofluorescence showed that the recombinant rabies virus rSRV9 WNVE. with infectious activity was successfully saved. The results of indirect immunofluorescence assay showed that the recombinant virus rSRV9 WNVE could express WNV E protein successfully. In addition, the growth curve of recombinant virus showed that the expression of exogenous gene did not affect the growth characteristics of recombinant virus in vitro. The female virus SRV9 and the recombinant rabies virus rSRV9 WNVE were inoculated into mice by intracerebral injection respectively. The mental state and survival of the mice were observed and recorded for 21 days. The results showed that compared with female SRV9, the pathogenicity of recombinant rabies virus rSRV9 WNVE to mice was decreased after expression of WNV E protein, which showed a high level of safety. The female SRV9 and recombinant rabies virus rSRV9 WNVE were immunized intramuscularly with 5 脳 105TCID50/ and 5 脳 106TCID50/, respectively. Two weeks after the first immunization, the mice were immunized with the same dose and route. Mouse serum was collected two weeks after the first and second immunizations, and the antibody titers of rabies virus and WNV in the serum were determined. The results showed that the recombinant virus could not only stimulate the production of rabies virus neutralizing antibody, but also stimulate the production of WNV specific antibody, which indicated that the recombinant virus had good immunogenicity. In conclusion, the recombinant rabies virus r SRV9 WNVE expressing WNV E protein has good safety and immunogenicity to mice, and has the potential as a vaccine, which lays a foundation for the further development of a new vaccine for West Nile virus disease.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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