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猪囊尾蚴抗原基因GP50的克隆、表达及鉴定

Molecular Cloning, Expression and Identification of GP50 Gene from Cysticercus Cellulosae in Vitro

【作者】 蔡娟

【导师】 沈继龙; 汪学龙;

【作者基本信息】 安徽医科大学 , 病原生物学, 2004, 硕士

【摘要】 目的:从猪囊尾蚴cDNA中扩增出GP50编码基因,亚克隆至真核表达载体,利用所得的重组蛋白,为建立一种灵敏检测猪囊尾蚴感染的实验方法作准备。方法:设计并合成引物,从猪囊尾蚴cDNA文库中PCR扩增GP50编码基因,通过与线性克隆载体pGEM-T连接,经酶切和PCR鉴定及DNA测序证实后,将目的基因亚克隆入双表达载体pBK-CMV,将获得的pBK-CMV-GP50阳性重组子转化宿主菌E.coli BL21,IPTG诱导表达,Western blot鉴定。结果:PCR法扩增出了一897bp左右的DNA片断,将重组质粒pGEM-T-GP50、pBK-CMV-GP50分别作BamH Ⅰ和Xho Ⅰ双酶切,均能得到一个大小与PCR扩增产物一致的插入片断,对插入片断的测序结果表明,GP50具有一个长度为897bp的完整开放阅读框(ORF),编码298个氨基酸,理论分子量33kDa,与GenBank收录(编号为AY212945)的猪囊尾蚴GP50基因具有高度的同源性(99%)。表明GP50基因的克隆和亚克隆均获成功,用IPTG诱导带有重组质粒pBK-CMV-GP50的大肠杆菌,产物行SDS-PAGE,可得到一约36kDa融合蛋白,Western-blot法鉴定该蛋白能被猪囊尾蚴感染的人血清所识别。

【Abstract】 Objective: To genernate a quick and accurate detection of Cysticercosis by use of recombinant protein, GP50 was amplified from Cysticercus cellulosae and subcoloned into the expression vector pBK-CMV. Methods: The specific primers were designed and synthesized. The DNA fragment encoding GP50 was amplified by PCR from cDNA of C.cellulosae. The PCR products were cloned into pGEM-T vector and recombinants were screened by PCR, risfriction digestion followed by sequencing. Then the GP50 was subcloned into pBK-CMV expression vector and the recombinant plasmid was transformed into E. coli BL21,IPTG was added to induce fusion expression of fused GP50. The expression was identified by Western blot. Results: The size of pGEM-T-GP50,pBK-CMV-GP50 digested with BamH I and Xho I was identical to the length of the PCR generated products. DNA sequencing indicated that the GP50 gene has an open reading frame of 897bp,which encodes 298 amino acids with an approximate molecular weight of 33kDa and has high homology with the GP50 gene submitted to GenBank (AY212945). As consequence GP50 gene was successfully cloned and subcloned into the vectors. With the positive clones induced by IPTG, a fusion protein was expressed in E.coli BL21. The fusion protein could be recognized by the sera of patients infected with Cysticercus cellulosae.

【关键词】 猪囊尾蚴抗原基因GP50克隆表达
【Key words】 Cysticercus cellulosaeGP50gene cloninggene expression
  • 【分类号】R346
  • 【下载频次】43
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