狂犬病病毒弱毒疫苗株反向遗传操作系统的建立  

Construction of a reverse genetics system of rabies virus attenuated vaccine strain

作　　者：金宏丽[1,2] 冯娜[2] 王化磊[2] 齐瑛琳[1,2] 梁萌[1,2] 李露[1,2] 郑学星[2] 赵永坤[2] 王铁成[2] 高玉伟[2] 黄耕[2] 杨松涛[2] 夏咸柱[2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]中国人民解放军军事医学科学院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国病原生物学杂志》2012年第7期481-485,共5页Journal of Pathogen Biology

基　　金：国家科技重大专项(No.2009ZX10602-14);国家自然科学基金青年基金项目(No.31101791);农业部公益性行业(农业)科研专项(No.201103032)

摘　　要：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 a full-length cDNA infectious clone and reverse genetics system for rabies virus attenuated vaccine strain SRV9.Methods The full-length genomic sequence of rabies virus attenuated vaccine strain SRV9 was analyzed with DNAStar and divided into four fragments based on unique restriction enzyme cleavage sites.The four fragments were connected together in accordance with the enzyme cleavage sites in two overlapping regions.To generate a full-length cDNA eukaryotic expression plasmid,full-length cDNA induced with HamRZ（3′） and HdvRZ（5′） was inserted into a pCI or pCDNA3.1（＋） vector,i.e.pC-SRV9 or pD-SRV9,respectively.Four helper plasmids coding for viral proteins N,P,G,and L were constructed.Liposome 2000 was used to transfect BSR cells with pCI-SRV9 or pD-SRV9 together with helper plasmids to rescue recombinant rabies virus.Results Both expression plasmids allowed successful recovery of rabies virus.However,pD-SRV9 had a significantly higher recovery efficiency（8/8） than did pCI-SRV9（3/30）.There were no significant differences in viral growth kinetics of the recombinant virus and parent virus（P0.05）.Conclusion A reverse genetics system was successfully constructed for rabies virus attenuated vaccine strain SRV9.It provides a platform for further study of the pathogenesis of the rabies virus,selection of new types of rabies vaccines,and development of recombinant vaccines against other infectious diseases based on rabies virus vectors.

关 键 词：狂犬病病毒 弱毒疫苗株 SRV9 反向遗传操作系统 

分 类 号：R373.9[医药卫生—病原生物学]