溶藻弧菌热休克蛋白70基因真核表达载体的构建及鉴定  

Construction and identification of a eukaryotic expression vector of HSP70 from Vibrio alginolyticus

作　　者：刘含亮[1] 孙敏敏[1] 王红卫[1] 李庆雷[1] 杨春贵[2] 高红莉[2] 万文菊[2] 王纪亭[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271000 [2]泰山医学院,山东泰安271000

出　　处：《中国病原生物学杂志》2011年第12期889-890,911,共3页Journal of Pathogen Biology

基　　金：山东省优秀中青年科学家奖励基金项目(No.2007BS02017);泰山医学院博士启动金项目

摘　　要：目的构建溶藻弧菌热休克蛋白70(HSP70)真核表达载体pcDNA3.1。方法提取溶藻弧菌基因组DNA,PCR扩增HSP70基因片段,克隆至TA载体,通过PCR、酶切及测序鉴定后,将HSP70基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.1,构建pcDNA3.1重组质粒,通过PCR、酶切及序列分析对HSP70基因pcDNA3.1重组质粒进行鉴定。结果扩增出1 914bp的溶藻弧菌HSP70基因片段,并成功构建溶藻弧菌HSP70真核表达载体pcDNA3.1。结论成功构建了溶藻弧菌HSP70真核表达载体pcDNA3.1,为HSP70基因疫苗研制奠定了基础。Objective To construct and identify a eukaryotic expression vector pcDNA3.1 of HSP70 from Vibrio alginolyticus.Methods Genomic DNA was extracted from V.alginolyticus.The HSP70 gene was amplified from V.alginolyticus genomic DNA by PCR.The purified PCR fragment was ligated into the pMD18-T simple vector;the positive clones were screened and identified by PCR and restriction enzyme digestion and the positive clones were sequenced after identification.The HSP70 gene fragments from TA clones were cloned into the eukaryotic expression vector pcDNA3.1 using restriction enzymes.The recombinant plasmid pcDNA3.1was identified through PCR,enzyme digestion and sequencing.Results The HSP70 gene fragment was amplified correctly,as the size of gene was a 1 914 bp,and HSP70 eukaryotic expression vector pcDNA3.1 was successfully constructed. Conclusion Construction of the eukaryotic expression vector pcDNA3.1 of HSP70 from V.alginolyticus lays the foundation for the development of HSP70 vaccine.

关 键 词：溶藻弧菌 热休克蛋白70 真核表达载体 基因疫苗 

分 类 号：S941.42[农业科学—水产养殖]