柯萨奇病毒B组3型中国分离株VP1基因真核表达系统的克隆

Clone of eukaryotic expression system for VP1 gene of Coxsackievirus group B type 3 Chinese strain

目的探讨构建柯萨奇病毒Ｂ组３型（ＣＶＢ３）基因疫苗的可行性。方法应用逆转录ＰＣＲ技术扩增ＣＶＢ３中国分离株ＶＰ１基因，通过ＴＡ克隆法构建ｐＣＲ２．１－ＣＶＢ３ＶＰ１，将ＣＶＢ３ＶＰ１亚克隆至真核表达载体ｐＣＥＰ４，构建真核表达系统ｐＣＥＰ４－ＣＶＢ３ＶＰ１，并进行酶切和测序鉴定。结果发现ＣＶＢ３的中国分离株与Ｎａｎｃｙ株的ＶＰ１基因基本一致，均编码２９３个氨基酸，其中有５９处核苷酸存在变异，但仅改变了１０处氨基酸编码，证实克隆的片段为ＣＶＢ３ＶＰ１基因，表达载体为ｐＣＥＰ４。结论实验成功地构建了ＣＶＢ３中国分离株的真核表达系统ｐＣＥＰ４－ＣＶＢ３ＶＰ１。

Objective To construct the gene vaccine of Coxsackievirus group B type 3(CVB3).Methods The VP1 gene of CVB3 Chinese strain was amplified by means of reverse transcript polymerase chain reaction (RT-PCR) and pCR2.1-CVB3VP1 was constructed by TA cloning strategy.The newly eukaryotic expression system pCEP4-CVB3VP1 was constructed by subcloning,and confirmed by restrict analysis and sequencing.Results The VP1 gene of CVB3 Chinese strain has 59 bases differences from that of CVB 3 Nancy strain,some of them cause only 10 amino acid changes and others are nonsense mutations.Conclusions Our results may be useful for research of CVB3 immunological characteristics.