论文部分内容阅读
目的构建狂犬病病毒弱毒疫苗株SRV9全长cDNA感染性克隆,并建立其反向遗传操作系统。方法通过DNAStar对狂犬病病毒SRV9株全长基因组序列进行分析,利用单一的酶切位点,将SRV9全长cDNA分为4段,根据每段重叠区域的酶切位点拼接全长,分别连入pCI和pCDNA3.1(+)载体,并通过PCR方法分别在全长序列的3′端和5′端引入核酶HamRZ和HdvRZ序列,构建全长真核表达质粒pCI-SRV9和pD-SRV9。同时构建能表达狂犬病病毒核蛋白(N)、磷蛋白(P)、糖蛋白(G)和聚合酶蛋白(L)的4个辅助质粒。分别将pCI-SRV9或pD-SRV9与辅助质粒通过脂质体共转染BSR细胞,拯救重组病毒。结果两种质粒表达系统均可拯救到有感染活性的病毒粒子,但pD-SRV9表达质粒的拯救效率(8/8)较pCI-SRV9表达质粒拯救效率3/30高;重组病毒与母本野生病毒的体外生长动力学曲线相一致,相同培养时间的病毒滴度差异无统计学意义(P>0.05)。结论成功构建了狂犬病病毒弱毒疫苗株的反向遗传操作系统,为进一步研究狂犬病病毒致病机理、筛选新型狂犬病疫苗或开发基于狂犬病病毒载体的其他疾病疫苗奠定了基础。
Objective To construct the full-length cDNA clone of SRV9 attenuated rabies virus attenuated vaccine and establish its reverse genetic operating system. Methods The full length genomic sequence of rabies virus SRV9 was analyzed by DNAStar. The full length cDNA of SRV9 was divided into four segments by using a single restriction enzyme site. The full length of the SRV9 was spliced according to the overlap region pCI and pCDNA3.1 (+) vectors. The full-length eukaryotic expression plasmids pCI-SRV9 and pD-SRV9 were constructed by introducing the ribozyme HamRZ and HdvRZ sequences into the 3 ’end and the 5’ end of the full-length sequence respectively. Four helper plasmids expressing rabies virus nucleoprotein (N), phosphoprotein (P), glycoprotein (G) and polymerase protein (L) were also constructed. Respectively, pCI-SRV9 or pD-SRV9 and helper plasmid were co-transfected into BSR cells by liposome to rescue the recombinant virus. Results Both plasmid expression systems could rescue virulent virions, but the rescue efficiency of pD-SRV9 expression plasmid (8/8) was 3/30 higher than that of pCI-SRV9 expression plasmid. The recombination virus and the female wild Virus in vitro growth kinetics curve consistent with the same incubation time, the virus titer difference was not statistically significant (P> 0.05). Conclusion The reverse genetic operation system of rabies virus attenuated vaccine was successfully constructed, which lays the foundation for further study on the pathogenesis of rabies virus, screening of new rabies vaccine or developing other vaccine based on rabies virus vector.