乙脑病毒E蛋白的原核表达、纯化及初步鉴定.doc

乙脑病毒E蛋白的原核表达、纯化及初步鉴定
作者:高淑娴,张伟,任君萍,雷迎峰,丁天兵,宋建华,马文煜,徐志凯
【Abstract】 AIM: To construct gene encoding Japanese encephalitis virus (JEV) envelope glycoprotein and to express E protein in BL21(DE3). METHODS: Using RTPCR, the gene encoding E protein was amplified. The gene was cloned into pET28a(+) and the binant plasmid pET286HisE was transformed into BL21(DE3). The 6HisE protein expressed in BL21(ED3) was determined by SDSPAGE and Western Blotting. The expression product in inclusion body was purified by NiNTA chromatography. RESULTS: Sequencing of E gene revealed that the mutation rate was ﹪(3/1500) and the base mutation didnt change the amino acid nonsense. The binant expression vector pET286HisE was constructed and the 6HisE protein was essfully expressed in an insoluble form. After expression and purification by metal chelate affinity chromatography, purified 6HisE fusion protein was obtained. CONCLUSION: The E protein can be expressed in prokaryotic cell and would be useful for further research on the receptor of JEV and its infection mechanisms.
【Keywords】 encephalitis virus, Japanese; viral envelope proteins; binant fusion proteins
【摘要】目的: 构建乙脑病毒(JEV)E蛋白重组载体并在原核细胞BL21(DE3)中表达. 方法: 采用RTPCR扩增片段,定向克隆入pET28a(+)中;重组载体pET28a6HisE转化BL21(DE3),通过酶切、SDSPAGE和Western Blotting检测其载体构建和蛋白表达;表达产物包涵体经NiNTA亲和层析纯化. 结果: 构建得到原核融合重组载体pET28a6HisE;诱导后表达得到6HisE融合蛋白,纯化得