pAd-SIG腺病毒质粒构建及其在MCF-7细胞中的表达

Construction of Recombinant Adenovirus Plasmid-Bearing hSSTr2 and Its Expression in MCF-7 Cells

目的含人生长抑素受体2亚型(human somatostatin receptor subtype2,hSSTr2)和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的腺病毒重组质粒的构建及表达。方法采用PCR方法将hSSTr2以及IRES-EGFP基因亚克隆至穿梭质粒pShuttle-CMV中,获得pShuttle-CMV/hSSTr2-EGFP,经PmeⅠ酶切,去磷酸化后转化含pAdeasy-1的超感受态BJ5183菌,细菌内同源重组构建腺病毒质粒pAdeasy-1/hSSTr2-EGFP(pAd-SIG),将其转染HEK293细胞进行病毒包装与滴度测定。重组腺病毒感染MCF-7细胞,流式细胞术检测其表达。结果酶切、PCR及基因测序证实重组腺病毒质粒pAd-SIG构建成功,病毒滴度为8.2×109IU/ml,流式细胞术结果显示hSSTr2在MCF-7细胞中表达阳性率为86.59%。结论成功构建了重组腺病毒质粒pAd-SIG,并能在真核细胞表达,为体内外研究hSSTr2效应提供了基础。

Objective To prepare a recombinant adenovirus plasmid bearing human somatostatin receptor subtype 2 （hSSTr2） and （enhanced green fluorescent protein, EGFP） and to evaluate the expression of hSSTr2 in eukaryocyte in vitro. Methods hSSTr2 and IRES-EGFP genes were amplified by PCR and subcoloned into plasmid of pShuttle-CMV to yield the recombinant plasmid of pShuttle-CMV/hSSTr2-EGFP, pShuttle-CMV/hSSTr2-EGFP was linearized with Pme I and trans- formed into competent BJ5183 containing pAdeasy-1 vector. Positive clone of homologous recombination （pAd-SIG） was transfected into packaging 293 cells to generate virus. After tittering the virus, the recombinant adenovirus was transfected into MCF-7 cells and the expression of hSSTr2 was detected by flow cytometry. Results The recombinant adenoviral plasmid of pAd-SIG was constructed correctly as confirmed by restricted endonuclease enzymes analysis, PCR and DNA sequencing. The virus titer was 8.2 109 U/mL. After the transfection, the positive expression of hSSTr2 was 86.59% as detected by flow cytometry, Condusion The recombinant adenoviral plasmid of pAd- SIG was successfully constructed and hSSTr2 was expressed in eukaryocyte, which provides a basis for further study of hSSTr2 in vitro and in vivo.