摘要
目的构建高效快速且方便的His-标签原核表达体系,对香菇C91-3凋亡相关基因24414功能域进行克隆。方法根据香菇C91-3凋亡相关基因24414的基因序列,分别设计特异性上下游引物,采用PCR方法,以24414基因序列为模板,扩增出24414基因的功能域序列。并将功能域基因连入pMD19-T Simple Vector克隆载体中,进行蓝白斑筛选,挑选出阳性菌落,提取质粒经双酶切及PCR验证后,进行基因测序。将克隆载体上目的基因连入pET-32a原核表达载体,构建重组质粒。转化入E.coli JM109宿主菌,经双酶切验证后,进一步测序鉴定验证重组质粒。结果 PCR扩增出大小为747 bp的基因片段,测序结果显示与香菇C91-3凋亡相关基因24414的功能域片段同源性为100%,并成功构建了原核表达体系。结论重组原核表达载体pET-32a-24414的成功构建为进一步研究香菇C91-3凋亡相关基因24414功能域的表达纯化及生物学活性奠定了基础。
Objective To construct a prokaryotic expressing vector of the function domain in apoptosis protein 24414 from Lentinula edodes C91.3. Methods According to the gene sequence of apoptosis protein 24414 from Lentinula edodes C91.3 , a special pair of primers to ampplified the target genes apoptosis protein 24414 was designed. Then it was cloned into the cloning vector-pMD19-T Simple Vector and selected by the blue and white spot experiment. Next, the gene apoptosis protein 24414 was selected by restriction enzyme digestion and cloned into the expressing vector-pET-32a. Results The function domain gene in apoptosis protein 24414 was checked by PCR, double enzymatic digestion and gene sequence. Compared with the original genes, there was no change on the level of amino acid. Conclusion This study successfully constructed the expression vector of pET-32a-24414. All these lay a foundation for further researches on function domain gene in apoptosis protein 24414 biological functions and its mechanism on inducing autophagy and apoptosis to tumor cells.
出处
《中国微生态学杂志》
CAS
CSCD
2013年第11期1270-1273,共4页
Chinese Journal of Microecology
