论文部分内容阅读
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。
Objective To construct recombinant retrovirus pLXSN-hIL-4 carrying human interleukin-4 (hIL-4) expression sequence and detect the expression of the target gene. Methods From June to December 2004, primers with restriction sites were designed in the School of Basic Medical Sciences, China Medical University. The DNA of the target gene was amplified by polymerase chain reaction and the target gene fragment was purified. pLXSN was cloned into the target gene fragment hIL-4, and the positive clones were screened out and identified. The recombinant retroviral vector pLXSN-hIL-4 was amplified and transfected into human synovial fibroblast-like cells cultured in vitro. Western blotting method was used to determine the protein level of target gene. Results The recombinant retrovirus containing the therapeutic gene was successfully constructed: rRV-hIL-4. Western blotting detected the expression of hIL-4. Conclusion A recombinant retrovirus containing therapeutic gene rRV-hIL-4 was successfully constructed. The hIL-4 gene can be introduced into human synovial fibroblast-like cells cultured in vitro by retroviral vector. The transfected cells can express hIL-4 protein.