外源HCV核心基因表达载体在HepG2中蛋白表达的亚细胞定位

Construction of HCV core genes eukaryotic expression vectors and subcellular localization of the proteins in HepG2 cells

目的研究HCV全长(天然)核心蛋白(C蛋白)和成熟C蛋白在HepG2细胞内的亚细胞定位,为进一步研究C蛋白与宿主细胞蛋白因子相互作用提供实验依据。方法设计HCV全长和成熟核心基因引物,以pBRTM/HCV1-3011为真核表达载体为模版行PCR;将PCR扩增产物酶切纯化后定向插入pEGFP-C1的BeglⅡ和EcoRⅠ位点,得到能表达全长HCV核心蛋白(C191aa.)和成熟核心蛋白(C173aa.)的真核表达载体pEGFP-C1-C191、pEGFP-C1-C173;将扩增pEGFP-C1-C191、pEGFP-C1-C173质粒转染到HepG2细胞中,28h、5d在荧光显微镜下观察两种C蛋白的亚细胞定位。5d后再用免疫细胞化学法观察两种C蛋白在HepG2细胞中着色现象。结果HCV核心基因PCR产物和构建的pEGFP-C1-C191、pEGFP-C1-C173经扩增测序证实。pEGDP-C1-C191转染HepG2细胞28h后融合荧光全长C蛋白C191主要出现在核膜和胞质中,少部分出现在胞核及其膜中;而pEGDP-C1-C173转染HepG2细胞28h后主要定位在核中和核膜上。但pEGDP-C1-C191转染HepG2细胞5d后,其表达的融合荧光蛋白主要出现在核内和核膜上,少量在胞质中;而成熟C蛋白C173则仍然在胞核内及核膜上。pEGDP-C1-C转染HepG2细胞5d后进行免疫细胞化学显示,C191蛋白和C173蛋白主要在核中、核膜着色,胞质有少量着色。结论HCV全长C蛋白的亚细胞定位是一动态过程,先出现在胞质中和核膜上,最后定位在核中和核膜上;成熟C蛋白主要定位在核中和核膜上。这种现象为解释瞬时转染HCV不同长度C基因对p53/p21通路作用出现相反结果奠定基础。

Objective To study subcellular localization of hepatitis C Virus（HCV） innate form and mature core protein in HepG2 cell line and provide experimental data for investigating the interaction of core protein with host cell protein. Methods The primers of HCV core genes were designed and the core genes were amplified from the pBRTM/ HCV1-3011 vectors by PCR. The amplified products（corel91 and core173） were digested by enzymes and were purified. Then the products were directly inserted into the Begl Ⅱ and EcoR Ⅰ sites of pEGFP-C1 ,so the eukaryotic expression vectors pEGFP-C1-C191 （ corel91 gene）, pEGFP-C1-C173 （ core173 gene） were constructed . After that the plasmids were trasnfected into HepG2 cell line and the localizations of the fluorescence-core fusion protein were detected by invert fluorescent microscopy at 28h,5d after transfection. Also the stain for these two kinds of core proteins were performed by the method of immunocytochemistry at 5d after transfection. Results A 591bp and a 537bp（including enzyme sequence） core cDNA were amplified by PCR; the recombinant plamids pEGFP-C1-C191 ,pEGFP-C1-C173 were identified by DNA sequencing. The innate form of core protein was localized in the cytoplasm and nuclear membrane at 28h, whereas at 5d the innate form of core protein primarily localized in nucleus and nuclear membrane after pEGFP-C1- C191 transfected into HepG2 observing under fluorescent microscopy. However, the mature form core protein was mainly localized in the nucleus and nuclear membrane of the cells whenever at 28h or 5d after pEGFP-C1-C173 transfection . The primary positive nucleus and nuclear membrane staining for innate form, mature form of core protein was detected by immunocytochemistry at 5d after core 191 and core 173 transfection into HepG2, and simultaneously small amounts and lightcytoplasm staining was also detected. Conclusion The localization of HCV full length core protein is a dynamic process, in cytoplasm and nuclear membrane at early stage, then mainly in nucleus and nuclear membrane of HepG2; whereas the mature form core protein mainly localized in nucleus and nuclear membrane of HepG2.