基孔肯亚假病毒模型的建立及其在模拟检测中的应用

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。

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

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.