基孔肯亚假病毒模型的建立及其在模拟检测中的应用  

Chikungunya Pseudo-Virus Model and its Application in Simulating Detection of Chikungunya Virus

作　　者：刘大斌[1] 张昕[1] 安小平[1] 张宝中[1] 王盛[1] 周育森[1] 童贻刚[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2010年第5期641-645,共5页Letters in Biotechnology

摘　　要：目的:构建含基孔肯亚病毒核酸序列的重组慢病毒载体,以之转染细胞以分泌含基孔肯亚病毒核酸序列的假病毒,利用该病毒建立基孔肯亚病毒的模拟检测方法。方法:人工合成基孔肯亚病毒部分保守性核酸序列,连入慢病毒载体,构建含基孔肯亚病毒核酸序列的重组慢病毒质粒pLenti-chik,该质粒连同辅助质粒pLP1、pLP2、pLP/VSVG共转染293FT细胞;72 h后收集上清,用特异引物进行荧光定量RT-PCR检测。结果:建立了一个仅能进行一次复制、高度安全的基孔肯亚假病毒模型;同时采用荧光定量RT-PCR建立了较灵敏的检测病毒分泌的方法。结论:假病毒可以模拟基孔肯亚病毒分泌,但较真病毒安全性更高;用荧光定量的方法可以很灵敏地检测病毒的分泌,为突发基孔肯亚病毒感染的检测做好准备。Objective： To construct a recombinant letivirus vector containing Chikungunya virus genomic sequence and use it to transfect cells to produce Chikungunya pseudo-virus as a tool to develop a simulating detection method with real-time RT-PCR.Methods： The conservative nucleic acid sequences of Chikungunya virus were synthesized and cloned into a lentilirus vector to construct plasmid pLenti-chik,which was co-transfected with helper plasmid pLP1,pLP2 and pLP / VSVG into 293FT cell.Cell culture supernatants were collected after 72 hours,real-time RT-PCR was carried out with Chikungunya virus specific primers.Results： A Chikungunya pseudo-virus secretion model which allow single-cycle virus replication was eastablished,and a sensitive real-time RTPCR method for simulating detection of Chikungunya virus was developed.Conclusion： The pseudo-virus can simulate the real virus secretion and is much safer than real virus.The virus secretion can be easily detected by realtime RT-PCR,which provides a preparation for future Chikungunya virus infection detection.

关 键 词：基孔肯亚病毒 假病毒 检测 实时荧光定量RT-PCR 

分 类 号：Q75[生物学—分子生物学]