论文部分内容阅读
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 efficient, rapid and convenient His-tag prokaryotic expression system and clone the 24414 functional domain of the Mucuna C91-3 apoptosis-related gene. Methods Based on the gene sequence of apoptosis related gene 24414 in Mushroom C91-3, specific upstream and downstream primers were designed respectively. The 24414 gene sequence was amplified by PCR using 24414 as a template. The functional domain gene was ligated into the pMD19-T Simple Vector cloning vector, blue-white screening was performed, positive colonies were selected, and the plasmid was digested with restriction enzyme and verified by PCR. The target gene on the cloning vector was ligated into the prokaryotic expression vector pET-32a to construct the recombinant plasmid. Transformed into E. coli JM109 host bacteria, double digestion verified, further sequencing identification verification recombinant plasmid. Results The gene fragment of 747 bp in length was amplified by PCR. The result of sequencing showed that the homology of functional fragment of the 24414 gene was 100%, and the prokaryotic expression system was successfully constructed. Conclusion The successful construction of recombinant prokaryotic expression vector pET-32a-24414 lays the foundation for the further study on the expression, purification and biological activity of the 24414 functional domain of the apoptosis-related gene of C91-3 in shiitake mushrooms.