摘要
目的 为风疹病毒（RV）感染提供特异性及敏感性高的检测手段。方法采用PCR扩增RV包膜糖蛋白E1基因202—353aa片段，并将其插入表达载体pET30a（＋），构建pET30a（＋）-E1，转化BL21（plysS）菌株，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western Blot初步分析蛋白表达及抗原性；并探讨温度、异丙基-β-D-硫代半乳糖苷（IPTG）浓度和诱导时间对目的蛋白表达产量的影响。结果获得相对分子质量约为16．6kD的融合蛋白，Western Blot证明其具有良好的抗原性，并确定该蛋白在30℃、0．6mmol／L IPTG的条件下诱导表达4h可获得最佳表达量。结论本研究为RV包膜糖蛋白E1抗原纯化及应用奠定了基础，亦为研制生产高特异性和敏感性的RV检测试剂盒提供了实验依据。
Objective To provide inspection means with high specificity and sensitivity for the rubella virus（RV） infection. Methods The target gene encoding RV E1 202-353aa was amplified by PCRand cloned into expression vector pE330a （ ＋ ） . The pET30a （ ＋ ）-E1 was established and the BL21 strains was transformated ,the expression and antigcnicity of recombinant protein was analyzed by SDS-PAGE and Western Blot assays. The effect of temperature, IPTC concentration and induction time to the production of target protein were analyzed. Results The fusion protein with the relative molecular mass about 16.6 kD was obtained, which showed good antigenicity by the Western Blot assays. Its best expression condition were 30 ℃ ,0.6 mmmol/L IPTG and 4 h. Conclusion This study can lay foundation for the purification and utilization of RV E1 antigen, and provide experiment foundation for the manufacture of RV detection kit with high specificity and sensitivity.
出处
《山东医药》
CAS
北大核心
2010年第3期18-19,共2页
Shandong Medical Journal
基金
山东省卫生厅青年基金资助项目(2007QZ024)
