Cloning and prokaryotic expression of the multi-copies of the human proinsulin-like gene

根据大肠杆菌密码子偏好性,优化人胰岛素原基因序列,同时用2个赖氨酸取代C链。重叠PCr扩增法克隆优化的类人胰岛素原基因,PCr扩增引物中引入胰蛋白酶酶切位点和核酸限制性内切酶识别位点。经酶切、拼接获得类人胰岛素原多拷贝基因,并构建人胰岛素原基因多拷贝原核表达载体,转化感受态大肠杆菌诱导表达融合蛋白。表达产物经SdS-PAgE电泳,考马斯亮兰染色和融合蛋白染色条带光密度分析表明,含有4拷贝的类人胰岛素原重复基因序列原核表达载体的诱导表达类人胰岛素原效率最高,是单拷贝类人胰岛素基因原核表达载体表达效率的3倍左右。表达产物经质谱鉴定,确定为优化设计的类人胰岛素原,表明构建的多拷贝类人胰岛素原基因表达载体可应用于后续人胰岛素原的高效表达生产研究。The human pre-insulin gene was redesigned for optimal expression in Escherichia coli through the alteration of its codon bias to reflect were redesigned according to the Codon Preference Parameter of Escherichia coli codon preference,and the C peptide of human preinsulin was replaced with Lys-Lys.Then the redesigned human pre-insulin gene(hINS)was cloned using Polymerase Chain Reaction(PCR),and further performed as a template on which based the multi-copy genes of hINS(hINS-M)was cloned by PCR with several couples of special primers.Besides,sequences of both the Restriction Enzyme and Trypsin cutting sites were included in the primers.Accordingly,restriction Endonucleases were employed to cut the PCR products,and T4DNA Ligase was then employed to tape the cut PCR produces to the differently designed multi-copies of the hINS-M.The prokaryotic expression vectors of these multi-copies genes were constructed and transformed into host Escherichia coli strains for expression.Furthermore,dodecyl sufate,sodium salt-polyacrylamide gel electrophoresis(SDS-PAGE),coomassie brilliant blue staining,photodensitometry,and mass spectrometry were employed to analyze the the expression products of the hINS-M.Our results indicated that the expression efficiency of the 4-copies gene of hINS-M was significantly higher than that of the others.The ratio of the expression product of the 4-copies gene hINS-Mthe human preinsulin to the total expression proteins of the host strains was over 28.0%.The ratio was about 3times of that of the expression product of the 1-copy gene of hINS-M.Moreover,the expression products from the different mutil-copes genes of hINS-M were all indentified to be the re-designed human preinsulin.Therefore,the prokaryotic expression vectors of the multi-copies genes of hINS-M could be used to improve the expression efficiency of the recombinant human preinsulin.国家自然科学基金资助项目(31201969); 厦门市科技计划项目(3502Z20093041

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