【摘要】 本研究为高效构建狂犬病SRV9ψ区缺失株感染性cDNA克隆,在细胞内进行重组狂犬病病毒的拯救提供帮助。按照Isothermal in vitro recombination system or"Gibson Assembly"连接方法设计引物,分别扩增得到去除狂犬病SRV9标准疫苗株的伪基因区(ψ区)的首尾均存在互补序列的5个结构蛋白基因的目的片段。利用T5核酸外切酶、DNA聚合酶及T4连接酶的协同作用,两步即可获得狂犬病SRV9ψ区缺失株的感染性cDNA。本研究利用Gibson Assembly连接法成功高效构建了狂犬病SRV9ψ区缺失株的感染性cDNA。此方法避免了传统的酶切连接方法中繁琐的实验步骤,实现了多个基因片段间的无缝连接,大大提高了载体构建的效率,为进一步研究狂犬病病毒的基因功能提供高效的方法。同时也为Gibson Assembly法应用于其它载体的构建提供借鉴。更多还原

【Abstract】 In this study, in order to efficiently construct infectious cDNA for SRV9 rabies virus and to provide help for recombining rabies virus in vivo, primers were designed by Isothermal in vitro recombination system and"Gibson Assembly"connector. 5 target fragments of structural genes which have complementary series at both ends were obtained after removing the pseudo gene region(ψ region) of rabies SRV9. We used T5 exonuclease,DNA polymerase and T4 ligase to get rabies SRV9 ψ region deleted mutant infectious cDNA. This study constructed cDNA infectious cDNA of SRV9 rabies ψ region deleted strains by Gibson Assembly connection method. This method avoided redundant steps in the traditional enzyme-digestion connection method, and realized the seamless connection between multiple genes, which improved the efficiency of the carrier construction and provided a more efficient method for the further researched of rabies. Also it would provide reference for the construction of other carriers with the application of Gibson Assembly method.更多还原