风疹病毒JR23株E1包膜糖蛋白的基因克隆与序列分析

Cloning and sequence analysis of envelope glycoprotein E1 gene of rubella virus,JR23 strain

目的 建立风疹病毒包膜糖蛋白E1的克隆载体 ;研究E1基因变异情况 ,并对其序列进行系统发生树分析。方法 利用RT PCR方法扩增并回收风疹病毒JR2 3株的包膜糖蛋白E1的基因片段 ,将其与PMD 18T载体连接 ,经氨苄青霉素筛选 ,酶切鉴定 ,以获得风疹病毒E1蛋白基因的克隆。将此基因测序后 ,利用DNASTAR和WINSTAR软件包绘制系统发生树进行序列之间的比较分析。结果 筛选出含有风疹病毒E1蛋白基因的克隆。序列分析及发生树的绘制表明 :JR2 3株与日本TCRB株及英国THOMAS株差别最小 ,分别为 0 .9%和 1.2 %。与北京BRD2株及香港XG379株差别最大 ,分别为 7.6 %和 7.3% ,与其它各株的差别均小于 3% (除NC株为 3.7%外 ) ,系统发生也与THO MAS株、TCRB株最近 ,与BRD2株最远。结论 克隆载体的建立为进一步研究E1基因与糖蛋白功能的关系提供基础。系统发生树表明中国不同地区风疹流行株基因序列存在明显差异。这对风疹病毒遗传与变异、分子流行病学研究 。

Objectives To construct the cloning vector of glycoprotein E1 gene of rubella virus, to study the mutation of glycoprotein E1 gene, and to analyze the sequence of E1 gene by the phylogenetic tree. Methods The envelope glycoprotein E1 gene was amplified from rubella virus, Jinan strain JR23, by RT PCR, and recombined with the PMD 18T vector. After amp r selection and analysis of restriction enzyme, the clones which carry the E1 gene were identified. After sequencing, this gene was handled by the software DNASTAR and WINSTAR and the phylogenetic tree was drawn. Results The clone of glycoprotein E1 was constructed. The analysis of the sequence and the map of the phylogenetic tree showed that strain JR23 has the least difference in sequence from strain TCRB of Japan and strain THOMAS of British with a value 0.9% and 1.2%, respectively. There is the largest difference of JR23 from strain BRD2 of Beijing and strain XG379 of Hong Kong with a value 7.6% and 7.3% respectively. The sequence differences between strain JR23 and the other strains were less than 3% except strain NC, which is 3.7%. The phylogenetic tree also showed the same trend. Conclusions The construction of glycoprotein E1 gene can serve as the foundation to study the relationship between E1 gene structure and function, and the structure of E1 glycoprotein and its function. The phylogenetic tree shows that there are significant differences in the sequences of the rubella viruses isolated in China. This can contribute to the researches on molecular genetics and epidemiology of the virus, and effective subunit vaccine.