携带hIL-4逆转录病毒重组体的构建及在类风湿关节炎中体外表达的研究

目的构建携带人白介素4(hIL-4)表达序列的重组逆转录病毒pLXSN-hIL-4并检测目的基因的表达。方法2004年6月至12月在中国医科大学基础医学院设计带有酶切位点的引物,含有目的基因的DNA进行聚合酶链反应扩增并纯化目的基因片段。pLXSN与目的基因片段hIL-4进行定向克隆连接,筛出阳性克隆并进行鉴定。扩增重组逆转录病毒载体pLXSN-hIL-4,并转染体外培养的人滑膜成纤维样细胞。Westernblotting法测定目的基因蛋白表达水平。结果成功构建了携带治疗基因的逆转录病毒重组体:rRV-hIL-4。Westernblotting法检测到了hIL-4的表达。结论成功构建了携带治疗基因的逆转录病毒重组体:rRV-hIL-4。以逆转录病毒为载体可以将hIL-4基因导入体外培养的人滑膜成纤维样细胞,转染后的细胞可以表达hIL-4蛋白。

Construction of the recombinent retroviral vector rRV - hIL - 4 and expression of rheumatoid arthritis in vitro

Objective To construct recombinent retroviral vectors containsins the expression sequence of hIL-4,and detect the expression of target genes.Methods Design the primer with restrictive enzyme spot and amplify target gene by PCR from plasmid including hIL-4 gene.Clone the target gene into retroviral vector pLXSN,and identify the aquired plasmid by sequencing.Transfect human synoviocytes with acquired virus in vitro.Detect the protein expresssion by western blotting.Results We successfully constructed the recombinant retrovirus-rRV-hIL-4.The hIL-4 gene was transduced into human synoviocytes by recombinant retrovirus in vitro.The protein expression of genes was detected by Western blotting.Conclusion Recombinant retrovirus rRV-hIL-4 is constructed successfully.The hIL-4 gene is transduced successfully into the human synoviocytes in vitro and the transduced synoviocytes can express hIL-4 protein.