人胃蛋白酶原A基因原核表达载体构建及鉴定

目的构建人胃蛋白酶原A(Pepsinogen A)原核表达载体,用于下一步人胃蛋白酶的表达。方法以Pepsinogen A CDNA为模板,利用PCR技术扩增Pepsinogen A基因,与pMD18-T载体相连构建载体pMD18-T/Pepsinogen A,提取质粒进行EcoRⅠ和XhoⅠ双酶切并测序鉴定,插入原核表达载体pET30a的相应位点,构建原核表达载体pET30a-Pepsinogen A,经菌落PCR、EcoRⅠ和XhoⅠ双酶切分析鉴定重组质粒。结果 PCR扩增出约1 000bp的Pepsinogen A基因片段,经酶切和测序验证Pepsinogen A基因正确;克隆至表达载体后,菌落PCR及双酶切证明pET30a-Pepsinogen A原核表达载体构建成功。结论成功构建了pET30a-Pepsinogen A原核表达载体,为进一步分析研究人胃蛋白酶提供了实验基础。

Construction and identification of prokaryotic expression vector of native human pepsinogen A gene

Objective To construct the prokaryotic expression vector of native human pepsinogen A for the product of protein in the future.Methods PCR was carried out by the cDNA of Pepsinogen A gene as a template.Then the interim vector of pMD18-T/Pepsinogen A was finished through link between the target PCR fragment and pMD18-T vector.After that suspected positive cloning was cultivated,extracted plasmid,identified by digestion with EcoRⅠand XhoⅠ,and performed with sequence analysis finally.Next stage the pET30a-pepsinogen A,an prokaryotic expression vector,was identified by the clony PCR technology and similar digestion with EcoRⅠand Xhol.Results We got a PCR fragment about 1000bp of the pepsinogen A gene,which was apparently identified by digestion and sequence analysis.Finally the expression vector pET30a-Pepsinogen A,which was identified by clony PCR technology and the similar digestion,was successfully constructed.Conclusions The prokaryotic expression vector pET30a-Pepsinogen A has been successfully constructed,which will lay the foundation for the further study of native human pepsinogen A in the future.