摘要
目的 扩增SjCL2基因 ,构建巴氏毕赤酵母表达载体 pPICZαB -SjCL2 ,为进一步研究其蛋白酶的功能奠定基础。方法 运用RT -PCR技术 ,从日本血吸虫 (中国大陆株 )成虫总RNA中扩增获得SjCL2基因 ;将其定向克隆至巴氏毕赤酵母表达载体pPICZαB质粒 ;经KpnⅠ、XbaⅠ双酶切分析和PCR鉴定筛选出阳性克隆 ,测序分析SjCL2基因并确定其读码框的正确插入。结果 利用RT -PCR技术从日本血吸虫成虫总RNA中扩增到约 1Kb大小的SjCL2基因 ,通过双酶切分析、PCR鉴定以及DNA序列分析确定SjCL2基因被克隆到巴氏毕赤酵母表达载体 pPICZαB中。 结论 成功构建了含有SjCL2基因编码区序列的巴氏毕赤酵母表达载体 pPICZαB
To construct the expression vector of Pichia pastoris yeast for the gene coding for proteinase cathepsin L2 of Schistosoma japonicum (SjCL2) for further studies of the biological function of this gene, the SjCL2 gene was amplified from the total RNA of S.japonicum by RT PCR technique, and was cloned into the expression plasmid pPICZaB of Pichia pastoris The recombinant plasmid pPICZaB SjiCL2 was identified by PCR,double digestion with Kpn I and Xba I as well as the sequence analysis. It was found that the SjCL2 of about 1Kb in length was amplified from the total RNA of S.japonicum, and was cloned correctly into the yeast expression plasmid pPICZaB.It concludes that the recombinant plasmid pPICZaB SjCL2 has been constructed successfully and it can be used for the further studies on the biological functions of SjCL2 gene.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第5期390-393,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目 (No 3 0 170 83 77)
广东省高教厅2 11工程重点学科建设基金资助项目
关键词
日本血吸虫
组织蛋白酶L2
基因克隆
巴氏毕赤酵母表达载体
Schistosoma japonicum
proteinase cathepsin L2
gene cloning
Pichia pastoris yeast expression vector
