大鼠Smad1基因重组腺病毒的构建

Construction of adenovirus vector carrying rat Smad1 by Cre-loxP site-specific recombination

目的利用Cre-loxP特异性位点基因重组技术构建大鼠Smad1的重组腺病毒。方法巢式PCR得到大鼠Smad1全部开放阅读框架序列,克隆到质粒pCR-BluntⅡ-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Smad1 cDNA转移到腺病毒载体pLP-Adeno-X-CMV。在HEK293细胞中包装和扩增重组腺病毒,并检测Smad1基因重组腺病毒在大鼠肝脏星状细胞系HSC-T6中的表达和功能。结果PCR法和限制性内切酶XholI消化法均证实Smad1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HSC-T6细胞中Smad1的mRNA、蛋白表达及其磷酸化水平。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Smad1基因重组腺病毒的成功构建为与Smad1有关的细胞信号调控以及基因治疗提供了良好的工具。

[Objective] To construct a recombinant adenovirus carrying rat Smadl gene by Cre-loxP site-specific recombination. [Methods] The cDNA sequence covering the full opening reading frame of rat Smadl amplified by high-fidelity nested-PCR was cloned into the plasmid of peR-Blunt II-TOPO, then the target gene was transferred into the shuttle vector of pDNR-CMV. After DNA sequencing, rat Smadl fragment was inserted into the adenoviral vector of pLP-Adeno-X-CMV through Cre-loxP recombination. The recombinant adenovirus was packaged and amplified in HEK 293 ceils. Smadl mRNA expression, protein expression and phosphorylation were tested in rat hepatic steUate cell line of HSC-T6. [Results] The sequence and the construction of the recombinant Smadl adenovirus were confirmed by PCR and the restriction enzyme Xhol I digestion, and its proper mRNA expression, protein expression and phosphorylation were identified in the cells of HSC-T6. [Conclusion] Cre-loxP site-specific recombination is a quick technology with high efficiency to construct a recombinant adenovirus. The successful construction of rat Smadl adenovirus provided us a powerful tool to further investigate the regulation functions and the gene therapy roles of Smadl.