重组人高密度脂蛋白B族Ⅰ型清道夫受体基因腺相关病毒血清型2/1杂合载体在HepG2肝细胞的表达

Expression of Adeno-associated Virus Vector Serotype 2/1 Containing Human Scavenger Receptor Class B Type Ⅰ Gene and Its Expressionin in HepG2 Hepatoma Cells

【目的】构建含绿色荧光蛋白(EGFP)重组人高密度脂蛋白B族I型清道夫受体(SR-BI)基因腺相关病毒(rAAV)2/1杂合载体,观察其在HepG2肝细胞的表达。【方法】以pCMV.SPORT6-SR-BI质粒为模板PCR扩增目的基因cDNA,将其克隆至pSNAV2.0-IRES-EGFP-LacZa载体中,经酶切及测序鉴定后,脂质体介导转染BHK21细胞,经G418筛选得到含SR-BI基因的BHK/SR-BI-IRES-EGFP载体细胞株,用携带rep2-cap1基因的辅助病毒(rHsv/r2cl)感染该细胞株。通过细胞孵育、裂解和病毒纯化,得到杂合载体rAAV2/1-SR-BI-IRES-EGFP病毒。免疫荧光显微镜检测绿色荧光表达,计算转染效率,Western Blot检测转染后SR-BI蛋白的表达。【结果】酶切鉴定表明pSNAV2.0-SR-BI-IRES-EGFP重组成功,基因测序显示装入pSNAV2.0-IRES-EGFP-LacZa质粒中的SR-BI基因正确;成功构建了rAAV2/1-SR-BI-IRES-EGFP病毒载体。转染HepG2肝细胞后免疫荧光显微镜检测最佳MOI值为1×105时,以此值转染HepG2肝细胞荧光高效表达;Western Blot检测未转染组及rAAV2/1-IRES-EGFP组HepG2肝细胞均有SR-BI蛋白表达,rAAV2/1-SR-BI-IRES-EGFP组SR-BI蛋白表达水平较rAAV2/1-IRES-EGFP组及未转染组显著增加。【结论】成功构建了rAAV2/1-SR-BI-IRES-EGFP杂合载体系统,转染后SR-BI蛋白在HepG2肝细胞高效表达,为SR-BI生理学作用机制及临床应用的深入研究奠定基础。

[Objective] The recombinant adeno-associated virus serotype 2/1 （rAAV2/1） hybrid vector containing human scavenger receptor class B type I gene （SR-BI） was constructed and its expression was examined in human HepG2 hepatoma cells. [Methods] The SR-BI eDNA which was obtained by PCR from pCMV.SPORT6-SR-BI plasmid was inserted into the AAV vector plasmid pSNAV2.0 -IRES-EGFP-LacZa. The recombinant plasmid pSNAV2.0-SR-BI-IRES-EGFP was transfected into BHK21 cells by LipofaetamineTM2000 after being identified by restriction endonuelease digestion and DNA sequencing. The rAAV vector producing cells were selected with G418. The G418 resistant cells were infected by helper recombinant rHSV/rep2capl viruses containing rep gene from AAV2 and cap gene from AAV1. The Cells were cultured and purified to obtain rAAV2/1-SR-BI-IRES- EGFP. The expression of recombinant vectors rAAV2/1-SR-BI-IRES-EGFP in human HepG2 hepatoma cells was examined by immunofluoreseent microscopy and Western blot analysis. [Results] The recombinant plasmid pSNAV2.0-SR-BI-IRES-EGFP was confirmed by digestion with restriction enzyme and the sequence of subcloned SR-BI eDNA was identical with that published on GenBank. The hybrid vectors rAAV2/1-SR-BI-IRES-EGFP was successfully constructed. The best MOI value in HepG2 hepatoma cells was 1 × 10^5. The fluorescence was highly expressed after transfected in HepG2 hepatoma cells. Higher levels of SR-BI protein expression were detected in the rAAV2/1-SR-BI-IRES-EGFP transfected HepG2 cells than those in the negative control and rAAV2/1-IRES-EGFP transfected HepG2 cells （P 〈 0.01 ）. [ Conclusions ] The rAAV2/I-SR-BI-IRES-EGFP hybrid vectors were successfully constructed and high efficiency expressed in human HepG2 hepatoma cells. Thus, the rAAV2/1-SR-BI-IRES-EGFP vector may be an interesting tool for specific transgene expression approach to atherosclerosis diseases.