人SMOC1重组腺病毒载体的构建及其在主动脉瓣间质细胞中的表达

目的:利用AdEasyTM vector system构建携带人SMOC1基因的重组腺病毒表达载体,感染人主动脉瓣间质细胞(hAVICs)并检测外源性SMOC1在细胞中的表达和对细胞增殖的影响?方法:用PCR的方法扩增SMOC1 cDNA,将其克隆到pGEM-T Easy载体中并测序鉴定?经NotⅠ和XhoⅠ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-CMV-SMOC1?将经PmeⅠ线性化的pShuttle-CMV穿梭载体与pAdEasyTM DNA电穿孔共转化BJ5183重组细菌,获取重组腺病毒质粒pAdEasy-SMOC1,再将经PacⅠ线性化的重组病毒骨架质粒转染HEK293A包装细胞,包装并扩增重组腺病毒(Ad-SMOC1)?用Ad-SMOC1感染hAVICs,以Western blot法和免疫荧光染色法检测重组SMOC1在hAVICs中的表达与定位,用MTS法检测SMOC1对主动脉瓣间质细胞增殖的影响?结果:成功构建并包装表达SMOC1蛋白的重组腺病毒,该重组腺病毒在体外能有效感染hAVICs并高表达SMOC1蛋白,且其介导表达的SMOC1蛋白主要定位于hAVICs的胞浆和胞膜上,MTS分析表明重组SMOC1能够显著促进主动脉瓣间质细胞的增殖? 结论:成功地构建了携带人SMOC1基因的重组腺病毒,并证实SMOC1具有促进增殖的作用?本研究为进一步研究SMOC1在瓣膜间质细胞中的作用奠定了实验基础?

Construction of recombinant human SMOC1 gene adenovirus vector and its expression in human aortic valve interstitial cells

Objective:To construct a recombinant adenovirus vector containing SMOC1 gene and to identify its expression in cultured human aortic valve interstitial cells (hAVICs) and effects on cell proliferation. Methods:The SMOC1 gene was amplified by PCR and inserted into pGEM-T Easy vector and sequenced. Double digested with Not I and Xho I,SMOC1 gene was inserted into pShuttle-CMV vector. This recombinant plasmid was linearized by Pme I and electronically transfected into BJ5183 germs to get the recombinant adenovirus vector (pAdEasy-SMOC1). Then the recombinant adenovirus expressing SMOC1 (Ad-SMOC1) was obtained by packaging in HEK293 A cells. After infecting hAVICs by Ad-SMOC1,the expression of recombinant SMOC1 was detected by Western blot and Immunofluorescence. The proliferation of hAVICs infected with Ad-SMOC1 was measured by MTS assay. Results:The recombinant adenovirus Ad-SMOC1 was successfully generated,which could effectively infect hAVICs and increase SMOC1 protein levels in hAVICs. Besides this,exogenous expressed SMOC1 was mainly located at the cytoplasm and membrane of hAVICs. MTS assay showed that recombinant SMOC1 promoted the proliferation of hAVICs. Conclusion:The bioactive recombinant adenovirus Ad-SMOC1 has been successfully constructed and recombinant SMOC1 promotes cell proliferation. This study laid a foundation for further research of the function of SMOC1 in hAVICs.