小鼠窖蛋白-1 基因重组腺病毒载体的构建

目的应用AdEasy重组腺病毒载体系统,构建携带小鼠窖蛋白-1(caveolin-1)编码区基因的复制缺陷型重组腺病毒载体Ad-caveolin-1。方法将质粒pTRE2-caveolin-1中的小鼠caveolin-1编码序列亚克隆到穿梭质粒pAdtrack-CMV中构建重组穿梭质粒pAdtrack-CMV-caveolin-1,酶切线性化后转入已含有病毒骨架质粒pAdEasy-1的大肠杆菌中进行同源重组。筛得的病毒质粒pAd-caveolin-1经酶切鉴定后,在293细胞中包装成重组腺病毒。荧光显微镜观察绿色荧光表达。扩增并纯化病毒,测定病毒滴度和重组病毒插入片段的序列。结果重组腺病毒质粒经酶切鉴定证实成功构建了携带caveolin-1基因的重组腺病毒载体,转染293细胞后可出现明显的细胞病变效应,并观察到绿色荧光的表达,caveolin-1基因测序鉴定与GenBank中公布序列一致。重组腺病毒的滴度为:2×109pfu/mL。结论应用AdEasy系统成功地构建了重组腺病毒载体Ad-caveolin-1,通过该系统携带的绿色荧光蛋白(GFP)标记基因可直观检测靶细胞转染和外源基因表达的情况,为进一步研究caveolin-1在牙齿发育中的作用奠定了基础。

Construction of Recombinant Adenovirus Vector Carrying the Mouse Caveolin-1 in Gene

OBJECTIVE: To construct recombinant adenovirus carrying the mouse dentin caveolin-1 gene using the recombinant adenoviral vector system AdEasy. METHODS: The cDNA fragment of caveolin-1 was derived from pTRE2-caveolin-1 by restriction enzyme digestion and subcloned into shuttle plasmid pAdtrack-CMV. The resulting plasmid pAdtrack-CMV-caveolin-1, after linearized by digesting with restriction endonuclease Pme I, was transformed into E. coli 1 BJ5183 which had been transformed by adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-caveolin-1 was screened by kanamycin LB plate and then identified by restriction enzyme digestion. The linearized adenovirus plasmid pAd-caveolin-1 was packaged in 293 cells, then the recombinant adenovirus Ad-caveolin-1 was harvested. The expression of green fluorescence protein was observed under fluorescent microscope. With further amplification and purification, the titer of recombinant adenovirus was determined. RESULTS: The recombinant adenovirus was identified by restriction enzyme digestion analysis and gene sequencing. Cytopathic effect and the expression of GFP were observed in the infected 293 cells. The sequence of caveolin-1 gene insert was the same as that published in the GenBank. The titer of the recombinant adenovirus was 2 x 10(9) pfu/mL. CONCLUSION: The mouse caveolin-1 recombinant adenovirus was constructed successfully by using AdEasy adenovirus system. Cell transfection and expression of exogenous gene can be detected directly by observing the expression of GFP. These results provide the basis for the further study on the role of caveolin-1 gene in other scopes.