微小隐孢子虫重组BCG-CP15／60-23疫苗的构建及鉴定

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-30a-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。

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

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.