甘丙肽重构基因GAL(intronⅡ)的克隆及其过表达转基因载体的构建

摘要

目的 克隆甘丙肽重构基因GAL(intronⅡ),构建小鼠神经系统特异性表达甘丙肽(galanin,GAL)的转基因载体pUC18/PDGF-β-promoter/GAL(intronⅡ),为制备GAL转基因小鼠做准备.方法 通过常规PCR、RT-PCR及重叠延伸PCR(overlap extension PCR,OE-PCR)扩增出插入GAL第二内含子的GAL全长cDNA的重构基因GAL(intronⅡ),将GAL(intronⅡ)克隆入T载体进行酶切、测序鉴定;同时从psisCAT6α中切下血小板源性生长因子β链(platelet-derived growth factor beta polypeptide,PDGF-β)启动子片段连接到空载体pUC18上构建出重组载体pUC18/PDGF-β-promoter;然后将GAL(intronⅡ)定向克隆于pUC18/PDGF-β-promoter下游,构建转基因载体pUC18/PDGF-β-promoter/GAL(intronⅡ).结果 PCR和限制性内切酶分析鉴定,表明获得转基因载体pUC18/PDGF-β-promoter/GAL(intronⅡ).通过对重构基因GAL(intronⅡ)的GALcDNA和第二内含子序列测序证实其与GenBank中的标准序列一致,GAL(intronⅡ)中第二内含子插入的位置准确,且无移码.结论 使用重叠延伸PCR扩增重构基因GAL(intronⅡ)并成功构建转基因载体pUC18/PDGF-β-promoter/GAL(intronⅡ),为转基因小鼠制备奠定一定的实验基础.%Objective To clone the recombinant molecule GAL (intronⅡ) and to construct the vector of transgenic mice model.Methods Firstly, the recombinant molecule GAL (intronⅡ) was amplified by RT-PCR, PCR and overlap extension PCR ( OE-PCR), then the GAL (intronⅡ) which the second intron of GAL was inserted into the GAL full-length cDNA was cloned into pM D19-T simple vector.Secondly, the platelet-derived growth factor beta polypeptide(PDGF-β)promoter from the psisCAT6α and the recombinant molecule GAL(intronⅡ) was inserted into pUC18 vector in the correct order to form a recombinant vector pUC18/PDGF-β-promoter/GAL (intronⅡ).Results The recombinant vector PUC18/PDGF-β-promoter/GAL(intronⅡ) was obtained, positive clones were screened and identified by PCR and digestion with restriction enzyme.The sequence of GALcDNA and intronⅡ was in accordance with the expected one, the insertion position of the second intron in the GAL (intronⅡ) was accurate, and there was no base malposition.Conclusion The recombinant molecule GAL (intronⅡ) is obtained and the transgenic expression vector of GAL recombinant is successfully constructed, which may provide a basis for further researches.