关键词: 猪瘟病毒, C株, 感染性克隆构建, 拯救方法, 传代

Abstract:

【Objective】 A reverse genetic technical platform of the classic swine fever virus (CSFV) Chinese strain was established and used to study the propagated mechanism of C strain of CSFV in cell culture and to develop DIVA marker vaccine. 【Method】A full length infectious clone pAC-CS was constructed successfully based on the CSFV C strain which cultured in primary bovine testicular cells. Linearized plasmid pAC-CS, transcripted with T7 RNA polymerase in vitro, and then transfected into BHK-21 and SK6 cells respectively by electropration. The rescued viruses were harvested from routine culture on SK6 cells and the supernatant of virus harboring SK6 cells, respectively. 【Result】 The viruses were detected through RT-PCR, immunoperoxidase monolayer assay (IPMA) and rabbit fever reaction. The results indicated that the virus was successfully rescued,and the efficiency of transfection in SK6 was higher than in BHK-21. After 12 “passages” of the virus harboring SK6 cell, the virus titer in the supernatant was 104TCID50.【Conclusion】A full length infectious clone of CSFV C strain was successfully constructed. The efficiency of transfection into SK6 by electropration was better. High titer viruses could be obtained from the supernatant of the virus harboring SK6 cell.

Key words: classic swine fever virus, Chinese strain, infectious clone construction, rescue method, passage