葡萄球菌肠毒素A基因的克隆、原核表达和鉴定  

Cloning,Prokaryotic Expression and Identification of Staphylococal Enterotoxin A Gene

作　　者：徐水凌[1] 毛亚飞[2] 颜丹红[1] 项洪琴[1] 

机构地区：[1]嘉兴学院医学院,浙江嘉兴314001 [2]浙江大学医学院

出　　处：《浙江预防医学》2006年第7期4-5,8,共3页Zhejiang Journal of Preventive Medicine

基　　金：浙江省科技计划项目(2003C34010);嘉兴学院重点课题(70105007)

摘　　要：目的克隆葡萄球菌肠毒索A(SEA)基因,构建其原核表达系统,鉴定重组蛋白(rSEA)免疫原性。方法采用PCR技术,从产SEA的葡萄球菌标准菌株ATCC13565基因组DNA中扩增全长SEA基因片段,T-A克隆后测序,构建pET32a-SEA表达质粒,转化入E.coli BL21DE3宿主菌,通过IPTG诱导表达,分离、纯化及Western印迹法鉴定。结果PCR获得SEA基因片段,DNA测序结果与已报道的SEA基因序列(GenBank No:L22566,AP004828)一致,构建了pET32a-SEA表达质粒,并成功地诱导、纯化了rSEA蛋白,rSEA表达量约为细菌总蛋白的40%。结论成功克隆了SEA全长基因,构建了rSEA原核表达系统,为进一步研制SEA的单克隆抗体及诊断试剂盒奠定了基础。Objective To clone the staphylococal enterotoxin A （SEA） gene, construct its prokaryotic expressive system, and identify and immunogenicity of recombinant proteins of SEA. Methods By the use of PCR, the entir SEA gene was amplified from standard Staphylococcus aureus strain ATCC 13565, which was sequenced by T-A cloning. Its prokaryotic expressive system, plasmid pET32a-SEA was constructed and transformed into the host strains of E. coli BL21De3 that was consequently induced to express by isopropyl-β-D-thiogalactoside （IPTG）. The immunogenicity of recombinant proteins Of SEA was identified by western blotting after separation and purification. Results The SEA gene segment was successfully amplified, of which sequence accorded with that reported （C, enBank No.： L22566）. The recombinant proteins of SEA were also successfully iduced and purified, whose yield equaled to overall protein yield of host strains. Conclusions The amplification of the entire SEA gene is successful, as well as the construction of its prokaryotic expressive system, both of which establish a basis for the development of monoclonal antibodies and detective kits of SEA.

关 键 词：葡萄球菌肠毒素A 基因克隆 原核表达 

分 类 号：R378.11[医药卫生—病原生物学]