微小隐孢子虫重组BCG-CP15/60-23疫苗的构建及鉴定  

Construction and identification of recombinant BCG vaccine with Cryptosporidium parvum CP15/60-23 antigen gene

作　　者：李瑾[1] 黄炳成 魏庆宽[1] 肖婷[1] 崔勇[1] 傅婷霞[1] 江洪涛[1] 胡颖新[1] 文桂香[1] 张佃波[1] 韩广东[1] 刘克义[1] 

机构地区：[1]山东省寄生虫病防治研究所,山东济宁272033

出　　处：《中国热带医学》2008年第6期917-919,921,共4页China Tropical Medicine

基　　金：山东省自然科学基金项目(NO.Y2004C22)

摘　　要：目的构建和鉴定微小隐孢子虫CP15/60-23基因重组卡介苗。方法以pET-30 a-CP15/60-23质粒为模板,PCR扩增CP15/60-23基因片段,然后克隆至TA载体;通过酶切、测序鉴定后,将CP15/60-23基因片段用限制性内切酶切下定向克隆至大肠埃希菌-分枝杆菌穿梭表达载体pMV262结核杆菌热休克蛋白(HSP)启动子下游,构建重组质粒pMV262-CP15/60-23,用电穿孔将该质粒导入野生卡介苗(BCG-WT)构建微小隐孢子虫CP15/60-23基因重组卡介苗。结果扩增出792bp的微小隐孢子虫CP15/60-23基因片段,成功构建pMV262-CP15/60-23质粒,并通过PCR扩增和提取质粒、酶切等表明该质粒被正确导入BCG-WT中。结论成功构建微小隐孢子虫CP15/60-23基因重组卡介苗,为研究隐孢子虫病新的防治方法打下了基础。Objective To construct and identify recombinant BCG vaccine with Cryptosporidium parvum. Methods CP15/60 - 23 gene fragment was amplified from pET - 30a - CP15/60 - 23 by PCR. The purified PCR fragment was ligated into the pMD18 -T Simple Vector and then subcloned into the downstream location of Mycobacterium tuberculosis HSP60 promoter ofEscherichia coli - Mycobacterium shuttle plasmid to construct recombinant pMV262 - CP15/60 -23. The plasmid was transformed into BCG - WT by electroporation to construct recombinant BCG - CP15/60 - 23. Results The 792bp inserted into shuttle plasmid pMV262 and confirmed by PCR, double enzymes digestion and DNA sequencing. Conclusion Recombinant BCG vaccine with Cryptosporidium parvum CP15/60 -23 antigen gene is successfully constructed and this may provide a new way to control cryptosporidiosis.

关 键 词：微小隐孢子虫 CP15/60—23基因 重组卡介苗 构建 鉴定 

分 类 号：R382.9[医药卫生—医学寄生虫学]