柯萨奇B组病毒的RACE扩增与序列分析

Amplification of 3′cDNA end of coxsackievirus B by RACE and its sequence analysis

目的探讨3'RACE(rapidamplificationofcDNAends)技术检测柯萨奇B组病毒(CVB)的可行性,并建立相应的实验检测方法。方法首先自行设计柯萨奇B组病毒6个型别病毒基因组的一条通用引物sp1;然后从柯萨奇病毒(CVB1￣CVB6)感染的Hela细胞,提取总RNA,用引物sp1结合OligodT进行3'RACE扩增,将产物克隆到pGEM-T载体上,挑取阳性菌落进行序列测定,与标准株进行同源性比对。结果获得了柯萨奇B组病毒6个型别的3'末端扩增产物及其核苷酸序列,同源性分析的结果显示扩增毒株与标准株之间的核苷酸同源性在95%￣99%之间,氨基酸同源性在98%￣100%之间。结论设计的引物“sp1”是柯萨奇B组病毒6个型别病毒基因组的一条通用引物;该引物结合OligodT进行的3'RACE扩增可用于柯萨奇B组病毒的检测。

Objective To evaluate the possibility to detect the coxsackievirus B (CVB) by 3′RACE (rapid amplification of cDNA ends), and to construct a corresponding experiment method to detect CVB. Methods Using 3′RACE method, the special fragments of the 3′end of CVB were amplified from the total RNA extracted from the Hela cell infected by CVB1 ~ CVB6 separately and the positive products were cloned into pGEM-T vector and then sequenced, followed by comparison with the standard strains. Results The special fragment and the sequence of the 3′end of CVB1 ~ CVB6 was obtained, the homogeneity of nucleotide and amino acid of the positive clones to that the standard strains were 95% ~ 99% and 98% ~ 100%, respectively. Conclusions The primer sp1 is proved to be a current primer of CVB; and the method of 3′RACE detecting the CVB has been constructed.