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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 the recombinant adenovirus of rat Smad1 by Cre-loxP site-specific gene recombination technology. Methods The entire open reading frame of rat Smad1 was obtained by nested PCR and cloned into plasmid pCR-BluntⅡ-TOPO. The target gene fragment was ligated to the intermediate vector pDNR-CMV. After sequencing, Smad1 cDNA Transferred to the adenovirus vector pLP-Adeno-X-CMV. The recombinant adenovirus was packaged and amplified in HEK293 cells and the expression and function of Smad1 gene recombinant adenovirus in rat hepatic stellate cell line HSC-T6 was detected. Results PCR and restriction endonuclease XholI digestion confirmed the successful construction of Smad1 recombinant adenovirus. RT-PCR and Western Blot showed that the recombinant adenovirus significantly enhanced Smad1 mRNA and protein expression in HSC-T6 cells Its phosphorylation level. Conclusion Cre-loxP site-specific gene recombination technology is a rapid and efficient method for constructing recombinant adenovirus. The successful construction of recombinant adenovirus Smad1 gene in rat provides good signal transduction and gene therapy related to Smad1 tool.