副溶血弧菌tlh基因克隆载体和表达载体的构建
Construction of clone and expression vector of gene of vibrio parahaemolyticus
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摘要:目的 构建含副溶血弧菌(vibrio parahaemolyticus, VP)tlh基因克隆载体和表达载体, 检测并鉴定表达载体在转化菌的表达, 为制备单抗、诊断试剂及进一步深入研究不耐热性溶血毒素(TLH)的功能奠定基础.方法 根据Genbank的tlh全基因序列, 设计一对附加EcorⅤ和HindⅢ两个限制性内切酶酶切位点的特异性引物, 以VP基因组DNA为模板, 用高保真Taq酶扩增出tlh基因.连接pGEM-T载体构建克隆载体, 测序鉴定后, 再用EcorⅤ和HindⅢ酶切回收目的基因并克隆至表达载体pET32a+, 采用PCR和双酶切鉴定筛选阳性质粒, 转化DE 3, IPTG诱导, 用SDS-PAGE检测tlh的表达.结果 扩增的目的基因与GenBank的tlh比较具有99%的同源性, 重组表达载体转化DE 3, 能表达融合蛋白.结论 本研究成功扩增出完整的tlh基因, 构建了克隆载体和表达载体, 获得融合表达的目的蛋白.Abstract:Objective To construct clone and expression vector of tlh gene of vibrio parahaemolyticus and examine its expression in DE 3 for preparaing monoclonal antibody and dignosing reagent and studying function of tlh.Methods Using VP14-90 as template, the tlh gene was amplified by modified PCR with a pair of specific primer which contained the restrictive sites of Eco RV and HindⅢ, and then subcloned into plasmid pGEM-T and expression plasmid pET32a.The recombinant plasmids were identified by restiction endonuclease enzyme analysis and PCR.The recombinant protein expression was induced by IPTG.Results The amplified frament was about 1.3kb by showed on the agarose gel electrophoresis and had 99% homeologous with the tlh gene in Genbank.The recombinant protein was about 63 KD.Conclusion The PCR method amplified the tlh gene.A clone vector and a expression vector containing tlh gene was successfully constructed, and the cell DE 3 transformed with the expression vector was capable of expression tlh gene.
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