论文部分内容阅读
目的:构建含有定点突变的人胰岛素原基因的腺病毒表达载体。方法:首先利用RT-PCR方法从正常流产胎儿胰腺组织获取野生型人胰岛素原cDNA;然后通过重叠延伸PCR方法在人胰岛素原cDNA上产生2个突变位点,使突变的cDNA编码的蛋白质含弗林蛋白酶的识别位点,该突变型胰岛素原cDNA片段命名为INS-M2;最后将INS-M2亚克隆至腺病毒载体系统的穿梭载体的多克隆位点上,并与骨架载体共转染至细菌内,通过细菌内同源重组得到重组载体。结果:Hind Ⅲ和Xho I酶切、Pac I酶切及测序均证实载体pAd-INS-M2构建成功。结论:构建的含定点突变人胰岛素原基因腺病毒载体pAd-INS-M2为进一步转染非胰岛β细胞,对糖尿病进行基因治疗奠定了基础。
Objective: To construct adenoviral expression vector containing the mutated human proinsulin gene. Methods: Firstly, wild-type human proinsulin cDNA was obtained from normal aborted fetal pancreas by RT-PCR method. Then, two mutated sites were generated on human proinsulin cDNA by overlap extension PCR. The mutated cDNA- INS-M2. Finally, INS-M2 was subcloned into the multiple cloning site of the shuttle vector of the adenoviral vector system and co-transfected with the backbone vector to In bacteria, the recombinant vector is obtained by homologous recombination in bacteria. Results: Hind Ⅲ and Xho I digestion, Pac I digestion and sequencing confirmed that the vector pAd-INS-M2 was successfully constructed. CONCLUSION: The recombinant adenovirus vector pAd-INS-M2 containing the site-directed mutated human proinsulin gene provides a basis for gene therapy of diabetes for further transfection of non-islet β cells.