ZHANG Xue-han, HE Kong-wang, GUO Rong-li, NI Yan-fang, WANG Fang and YU Zheng-yu. Cloning and Expression of E Gene of Japanese Encephalitis Virus and Preliminary application[J]. Virologica Sinica, 2005, 20(5): 494-497.
Citation:
ZHANG Xue-han, HE Kong-wang, GUO Rong-li, NI Yan-fang, WANG Fang, YU Zheng-yu.
Cloning and Expression of E Gene of Japanese Encephalitis Virus and Preliminary application .VIROLOGICA SINICA, 2005, 20(5)
: 494-497.
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摘要
参照GenBank中的日本乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14株序列设计了一对特异性引物,用PCR方法从SA14-14扩增E基因全长,然后克隆到pMD18-T载体中,转化宿主菌DH5a,提取阳性克隆质粒进行双酶切鉴定,将目的片段定向克隆到pET32a(+)中,转化入BL21(DE3),经IPTG诱导可表达分子量约73ka的蛋白,Western blotting试验呈阳性,表明E基因得到表达。以纯化的表达产物为核心抗原,猪抗JEV血清为一抗,HRP标记羊抗猪IgG抗体为二抗建立间接ELISA方法,并初步检测了一些血清样品,结果提示表达的蛋白具有很好的应用开发价值。
Cloning and Expression of E Gene of Japanese Encephalitis Virus and Preliminary application
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Abstract
The whole cDNA of E gene was amplified by RT-PCR from JEV strain SA14-14,and cloned into the pMD18-T vector.The fragment was identified by restriction enzymes digestion with EcoR I and Xho I,cloned into the pET32a(+) vector.The recombinant plasmid was transformed into BL21 and the recombinant bacteria was induced by optimal concentration of IPTG.SDS-PAGE and Western blotting were performed to detect the E fusion protein.Our result indicates the molecular weight of E protein is of E protein 73kDa and its antigenicity is good.Indirect ELISA procedure has been developed with the purified E protein as antigen for detection of JEV.
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Proportional views
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