大鼠骨形成蛋白4基因重组腺病毒的构建

目的 利用Cre-loxP特异性位点基因重组技术构建大鼠骨形成蛋白BMP4的重组腺病毒.方法 高保真PCR得到大鼠BMP4 cDNA,克隆到质粒pCR-Blunt Ⅱ-TOPO中,然后将目的基因片段连接到中间载体质粒pDNR-CMV,测序后在Cre重组酶作用下,将BMP4 cDNA转移到腺病毒载体pLP-Ade-no-X-CMV.在HEK 293细胞中扩增重组腺病毒,并检测BMP4基因重组腺病毒在细胞中的表达和功能.结果 PCR法和限制性内切酶Xhol I消化法均证实BMP4重组腺病毒的成功构建,RT-PCR和Western Blot检测证实该重组腺病毒显著性增强了CFSC-2G细胞中BMP4的mRNA和蛋白表达水平.结论 Cre-loxP特异性位点基因重组技术是一种快速、高效构建基因重组腺病毒的方法,大鼠BMP4基因重组腺病毒的成功构建为进一步研究BMP4的细胞调控功能和BMP4基因治疗提供了良好的工具.

Construction of recombinant adenovirus carrying rat bone morphogenetic protein-4 by Cre- loxP site-specific recombination

Objective] To construct a recombinant adenovirus carrying the rat bone morphogenetic protein BMP4 by Cre- loxP site-specific recombination. Methods] The rat BMP4 amplified by high-fidelity PCR was cloned into the plasmid of pCR-Blunt II-TOPO, then the target gene was transferred into the shuttle vector of pDNR-CMV. After DNA sequencing, the rat BMP4 was inserted into the adenoviral vector of pLP-Adeno-X-CMV under the Cre- loxP recombination. The recombinant adenovirus was amplified in HEK 293 cells, its mRNA and protein expression functions were tested in the cell of CFSC-2G. Results] The sequence and the construction of the recombinant BMP4 adenovirus were confirmed by PCR and the restriction enzyme Xhol I digestion, and its proper mRNA and protein expression functions were identified in the cells of CFSC-2G. Conclusion] The Cre- loxP site-specific recombination is a quick technology with high efficiency to construct a recombinant adenovirus. The successful construction of rat BMP4 recombinant adenovirus provided us a powerful tool to furtherly investigate the regulation functions and the gene therapy roles of BMP4.