表達西尼羅病毒囊膜蛋白重組狂犬病病毒的構(gòu)建與應(yīng)用研究
發(fā)布時間：2018-09-01 08:53
【摘要】：西尼羅病毒病是由西尼羅病毒(West Nile virus,WNV)感染引起的急性人獸共患傳染病。近20年,WNV在全球擴散,不斷引起人、畜疾病的暴發(fā)流行,成為世界性公共衛(wèi)生問題。2011年,梁國棟等從我國新疆維吾爾自治區(qū)采集的蚊蟲標本中分離到WNV,大量血清學(xué)結(jié)果證明當?shù)夭粌H存在WNV感染所致疾病,還發(fā)生過WNV感染引發(fā)的病毒性腦炎流行。到目前為止,尚無獲批的針對WNV的特效藥物和有效疫苗。本研究利用狂犬病病毒(Rabies virus,RABV)弱毒疫苗SRV9株反向遺傳操作系統(tǒng),通過PCR的方法,在SRV9株基因組的P與M基因間插入西尼羅病毒E基因(WNV E),構(gòu)建表達WNV E蛋白的全長質(zhì)粒pD SRV9 WNV E。將構(gòu)建的全長質(zhì)粒pD SRV9 WNV E與表達狂犬病病毒核蛋白(N蛋白)、磷蛋白(P蛋白)、轉(zhuǎn)錄大蛋白(L蛋白)、糖蛋白(G蛋白)的輔助質(zhì)粒通過脂質(zhì)體共同傳染BSR細胞,進行重組病毒的拯救。直接免疫熒光結(jié)果表明:成功拯救獲得了具有感染活性的重組狂犬病病毒rSRV9 WNVE。間接免疫熒光、Western blot、PCR等結(jié)果表明:重組病毒rSRV9 WNVE可成功表達WNV E蛋白。此外,重組病毒的生長曲線表明:外源基因的表達不影響重組病毒的體外生長特性。分別將母本病毒SRV9和重組狂犬病病毒rSRV9 WNVE經(jīng)腦內(nèi)注射途徑接種小鼠,每天觀察并對小鼠的精神狀態(tài)、存活等情況進行記錄,連續(xù)觀察21天。結(jié)果表明:與母本病毒SRV9相比,重組狂犬病病毒rSRV9 WNVE表達WNV E蛋白后對小鼠的致病性降低,呈現(xiàn)高度的安全性。分別將母本病毒SRV9和重組狂犬病病毒rSRV9 WNVE以5×105TCID50/只和5×106TCID50/只的劑量通過肌肉注射途徑免疫小鼠,初次免疫后兩周,以相同的劑量和途徑加強免疫一次,分別于一免和二免后2周采集小鼠血清,并測定血清中狂犬病病毒和WNV的抗體效價。結(jié)果表明:重組病毒免疫小鼠后,既可刺激機體產(chǎn)生狂犬病病毒中和抗體,也可刺激機體產(chǎn)生WNV特異性抗體,表明重組病毒具有很好的免疫原性。綜上所述,表達WNV E蛋白的重組狂犬病病毒r SRV9 WNVE對小鼠具有的良好的安全性和免疫原性,具有作為疫苗的潛力,為進一步研制西尼羅病毒病新型疫苗奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.65
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.65
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