猪瘟病毒C株全长cDNA感染性克隆的构建及病毒拯救  

Construction of the Full Length Infectious cDNA Clones of CSFV C Strain and Virus Rescue

作　　者：邹兴启[1] 赵启祖[1] 范运峰[2] 朱元源[1] 王琴[1] 徐璐[1] 范学政[1] 宁宜宝[1] 

机构地区：[1]中国兽医药品监察所,北京100081 [2]中国动物疫病预防控制中心,北京100026

出　　处：《中国农业科学》2011年第2期409-416,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(30571377)

摘　　要：【目的】建立猪瘟病毒研究技术平台,为进一步研究猪瘟病毒C株在细胞中的增殖机制及猪瘟病毒标记疫苗奠定基础。【方法】本试验以猪瘟病毒C株为研究材料,经RT-PCR扩增获得涵盖全长的6个片段,用合适的酶切位点连接,成功构建了C株全长感染性克隆pAC-CS。体外转录得到RNA,然后将其分别转染BHK-21和SK6细胞。拯救的病毒通过上清传代(常规病毒传代)和带毒细胞传毒繁殖。【结果】通过RT-PCR、免疫过氧化酶单层细胞试验和兔体发热试验检测,表明病毒拯救成功。经比较以电转的方式转染SK6的效果较好。通过带毒细胞传代,至12代时病毒滴度稳定达104TCID50.mL-1,而上清传代至第3代时用免疫过氧化酶单层细胞试验(IPMA)不能检测出病毒。【结论】成功构建了猪瘟病毒C株感染性克隆;拯救C株时以SK6细胞电转较好;带毒细胞传代培养C株有利于获得较高滴度的病毒。【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.

关 键 词：猪瘟病毒 C株 感染性克隆构建 拯救方法 传代 

分 类 号：S852.65[农业科学—基础兽医学]