蓝舌病毒HbC₃株S7基因5′非编码区序列分析

蓝舌病毒HbC 3 株S7基因5′非编码区序列分析 Sequence Analysis of S7 5′-NCR Gene of Bluetongue Virus-HbC 3 Strain 桂亦瑞 GUI Yi-rui 1 2 first-author 董长垣 DONG Chang-yuan 1 2 陈晓 CHEN Xiao 1 2 张蔚英 ZHANG Wei-ying 1 2 刘军 LIU Jun 1 2 武汉大学医学院 武汉大学医学院 School of Medicine, Wuhan University School of Medicine, Wuhan University 武汉大学医学院,湖北,武汉,430071 武汉大学医学院,湖北,武汉,430071 根据已发表的蓝舌病毒（bluetonguevirus,BTV）10型标准株S7基因设计合成一对与其5′ NCR（non codingregion）序列同源的引物,经反转录 聚合酶链反应（RT PCR）扩增出HbC3株和10型标准株长度分别为277bp和290bp的S7基因5′非编码区cDNA片段,以建立起dsRNA体外扩增系统.将扩增的BTV HbC3毒株的S7基因5′非编码区片段通过粘端连接克隆到pUCm T载体中,用PCR技术和限制性内切酶分析鉴定,表明获得重组质粒pUCm T BTV HbC3 S7.通过核苷酸序列分析方法分别将2个毒株的S7基因5′非编码区与呼肠孤病毒 3（reovirus 3）型比对,发现BTV HbC3株与呼肠孤病毒 3L2基因5′非编码区基因具有完全的同源性.将BTV HbC3毒株接种在不同细胞系如猴肾传代细胞（Vero）、人肝癌细胞（Hep 3B）和小鼠成纤维细胞（L929）等细胞株上,比较BTV HbC3在不同种系细胞上的增殖特征,并且用双向免疫扩散试验证实BTV HbC3和BTV 10型之间的血清学关系.结合本实验室的研究结果提示BTV HbC3株可能是蓝舌病毒的一个新的基因型. Using primers complementary to the conserved sequence previously published of BTV genomic dsRNA segment,S7 5′-NCR of BTV-HbC 3 and BTV-10 were synthesized and amplified by RT-PCR method resulting in a 277 bp and 290 bp products. And the 277 bp product was then cloned into pUCm-T vector. The partial sequence of BTV-HbC 3 had a comparison with that of Reovirus-3 L2 5′-NCR revealing 100% nucleotide similarity,while it has no origin with BTV-10 in this part of sequence.Perhaps BTV-HbC 3 and BTV-10 originated from two gene pools or some sequences of Reovirus-3 were inserted into BTV-HbC 3 undergo genetic drift which contributed to diversification of individual gene segments of field strains of BTV. With the replicated and morphologic characteristics of BTV-HbC 3 infecting different cells such as Vero,Hep-3B and L 929 ,all the cells displayed a rounded, granular phenotype having the same CPE（characteristics of cytopathic effects） with that of BTV-10.Through agar gel immunodiffusion test,the cross-reactivities were detected between BTV-HbC 3 and BTV-10 indicating that the common antigenic determinants on the VP7 proteins of BTV-HbC 3 and BTV-10 shared some epitopes.All these findings showed that BTV-HbC 3 was the prototype virus of the Orbivirus genus, probably a new genome type of bluetongue virus. 蓝舌病毒 S7基因 克隆 基因型 bluetongue virus S7 clone genome type S852.65 国家自然科学基金资助项目（30271471） 2004-02-01 2021-04-01 2