【摘要】 本文旨在分析含红色荧光蛋白mCherry基因的重组柯萨奇病毒B组3型(CVB3)基因组的稳定性。用重组质粒pCVB3-mCherry转染HeLa细胞,观察细胞病变和mCherry的表达。收获病毒后,用噬斑形成实验纯化病毒并测定病毒毒力。将重组病毒CVB3-mCherry在HeLa细胞中连续传代,提取第2～6代重组病毒总RNA,经反转录-聚合酶链反应(RT-PCR)扩增出报告基因mCherry及CVB3部分序列,进行测序分析。结果表明,pCVB3-mCherry转染的HeLa细胞出现细胞病变并表达红色荧光蛋白mCherry;从第2代开始,CVB3-mCherry出现报告基因mCherry及部分CVBVP4基因序列丢失,基因序列丢失导致病毒开放读码框架移位。本研究表明,mCherry基因序列的插入导致CVB3基因组不稳定。随着病毒的传代逐渐丢失插入的报告基因mCherry及CVB3基因组的部分序列,病毒读码框架移位,产生致死性突变株。因此,应用CVB3-mCherry时,病毒的传代次数不超过2代,否则应重新从重组质粒中收获病毒,并对每代重组病毒进行纯化和毒力测定。更多还原

【Abstract】 The genome stability of a recombinant coxsackie virus B3(CVB3) with a red fluorescent reporter gene(mCherry) was evaluated.Recombinant virus CVB3-mCherry was produced by transfecting HeLa cells with pCVB3-mCherry.The virulence of the recombinant virus was determined by plaque forming unit(PFU) assay,and the expression of mCherry in the infected HeLa cells was detected with fluorescence microscopy.The existence of the inserted mCherry sequence in the CVB3-mCherry collected from a sequencial passages in HeLa cells was determined by reverse transcriptase-polymerase chain reaction(RT-PCR) and sequencing.The results showed that both cytopathic effect and mCherry expression were observed in HeLa cells transfected with the pCVB3-mCherry.Instability of CVB3-mCherry genome was observed as early as in the second passage.Deletions of the mCherry gene as well as the CVB3 genome sequence were detected.The deletions may cause lethal mutations.In conclusion,the recombinant CVB3-mCherry could be used to study the infection of CVB3,and the recombinant virus showed an increased instability upon passages due to the insertion of mCherry open reading frame(ORF).Therefore,for the application of CVB3-mCherry,the first two passages are desirable and recovering the virus from the recombinant plasmid is recommended if longer period of application is considered.更多还原