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.
出处
《中国农业科学》
CAS
CSCD
北大核心
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)
关键词
猪瘟病毒
C株
感染性克隆构建
拯救方法
传代
classic swine fever virus
Chinese strain
infectious clone construction
rescue method
passage
