摘要
目的通过反向遗传学技术构建感染性质粒并拯救一株基孔肯雅病毒(Chikungunya virus,CHIKV)。方法通过PCR、琼脂糖凝胶电泳、同源重组等分子克隆实验,构建CHIKV(Caribbean strain,isolate M100,Genbank序列号为LN898093.1)全长感染性克隆;通过体外转录、脂质体转染等方法将病毒mRNA转染至BHK-21细胞以拯救病毒;应用TCID50法和Reed-Muench法计算病毒滴度。通过Western blot和RT-qPCR检测CHIKV在感染BHK-21及Vero E6细胞后不同时间点的病毒蛋白及mRNA水平的变化。结果成功构建了CHIKV感染性克隆全长cDNA编码质粒,并成功拯救出子代病毒。通过RT-qPCR和Western blot验证了病毒在感染过程中NS1蛋白的mRNA水平及BHK-21细胞中NS3、E1蛋白表达水平均显著上升。结论成功构建CHIKV感染性克隆质粒并成功拯救病毒,为后续CHIKV基因结构、蛋白质功能和致病机制等研究提供了良好的工具。
Objective To rescue a strain of Chikungunya virus(CHIKV)using reverse genetics system.Methods Molecular cloning experiments,including PCR,agarose gel electrophoresis and homologous recombination were employed to generate an infectious cDNA clone(pFK-CHIKV-LN)derived from the isolated Caribbean Chikungunya strain(Caribbean strain,isolate M100,Genbank LN898083.1).The mRNA obtained from vitro transcription were transfected into BHK-21 to rescue the CHIKV.The viral titer was determined by 50%tissue culture infection dose(TCID50),with calculations performed using the Reed-Muench method.Western blot(WB)and real time quantitative polymerase chain reaction(RT-qPCR)were employed to assess the levels of viral protein and mRNA at various time points during infection.Results The full-length cDNA clone of CHIKV was successfully constructed,enabling the rescue of CHIKV progeny.RT-qPCR and WB confirmed the significant increasing expression levels of NS1 mRNA and proteins(NS3,E1)of BHK-21 during the infection process.Conclusion The full-length infectious clone of CHIKV has been constructed successfully,which provides a good tool for subsequent studies on the gene structure,protein function and pathogenic mechanism of CHIKV.
作者
曹恒
刘娜
王永康
王漫兮
龙钢
CAO Heng;LIU Na;WANG Yong-kang;WANG Man-xi;LONG Gang(Shanghai Institute of Infectious Disease and Biosecurity,Fudan University,Shanghai 200032,China;Department of Medical Microbiology and Parasitology,Key Laboratory of Medical Molecular Virology(MOE/NHC/CAMS),School of Basic Medical Sciences,Fudan University,Shanghai 200032,China)
出处
《复旦学报(医学版)》
北大核心
2025年第6期811-818,共8页
Fudan University Journal of Medical Sciences
基金
上海市市级科技重大专项(ZXS002R2-3)。
