重组表达小鼠MIP-1α和B7-1基因的慢病毒载体的构建及鉴定

Constructing and Identification of Lentivirus-mediated Mouse MIP-1α and B7-1 Gene Vectors

目的构建重组表达小鼠MIP-1α和B7-1基因的慢病毒载体,为淋巴瘤基因治疗的实验研究奠定基础。方法设计引物扩增获得目的基因小鼠MIP-1α和B7-1基因的全长编码序列cDNA,将目的基因与经酶切线性化的慢病毒载体进行定向连接,其产物转化感受态细胞,对长出的阳性克隆进行PCR鉴定和直接测序序列分析。MIP-1α和B7-1目的基因质粒转染293T细胞,观察绿色荧光蛋白(GFP)表达,采用Western Blot法检测其蛋白表达,实时荧光定量PCR,检测慢病毒浓缩液的滴度。结果成功构建了重组表达小鼠MIP-1α和B7-1基因的慢病毒载体,实时荧光定量PCR证实MIP-1α、B7-1基因重组慢病毒载体的滴度均达2.00E+8 TU/mL。结论本研究成功构建并包装出高滴度的小鼠MIP-1α和B7-1基因重组慢病毒载体,为淋巴瘤基因治疗的实验研究奠定了基础。

Objective To construct lentivirus-mediated mouse MIP-1α and B7-1 gene vectors and lay a foundation for gene therapy with lymphoma.Methods Mouse MIP-1α and B7-1 genes were synthesizeed and amplification by PCR.Target genes were directly connected with Lentivirus vector,the production of which were transformed into Bacterium coli DH5α cells,and the positive colones were identified by PCR and direct sequencing analysis.Then the plasmids of MIP-1α and B7-1 genes infected 293T cells,respectively,green fluorescence protein（GFP） in 293T cells was observed by fluorescence microscope;Western Blot was used to test protein expression of MIP-1α and B7-1 genes and Real-time PCR was used to detect the titer of lentivirus.Results Lentivirus-mediated mouse MIP-1α and B7-1 gene vectors were successfully constructed and the titer of which was 2.00E＋8 TU/ml tested by real-time PCR.Conclusion Lentivirus-mediated mouse MIP-1α and B7-1 gene vectors were successfully constructed and lay a foundation for gene therapy with lymphoma in the future.