GRP78重组表达腺病毒的构建及在HepG2细胞的表达及其相关功能研究  

Construction and expression of recombinant GRP78-expressed adenovirus and GRP78 expression in HepG2

作　　者：吴爽[1] 张祥[1] 魏杰[1] 汪德强[2] 罗淼 

机构地区：[1]重庆医科大学检验医学院,重庆400016 [2]感染性疾病分子生物学教育部重点实验室,重庆400016 [3]重庆市渝北区人民医院医学检验科,重庆400016

出　　处：《重庆医科大学学报》2016年第4期384-388,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81301395)

摘　　要：目的:构建葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)重组表达腺病毒pAd-GRP78,并感染HepG2细胞建立能够高表达GRP78的细胞模型,同时在HepG2细胞中研究重组表达GRP78腺病毒对细胞增殖能力的影响。方法:利用腺病毒穿梭质粒pAd Track-TO4构建载有GRP78基因的穿梭质粒pAd Track-TO4-GRP78载体,并通过基因测序鉴定载体序列正确。将载体用PmeⅠ酶切线性化,与Ad Easy-BJ5183感受态细胞进行基因同源重组。将重组后的腺病毒载体经PacⅠ酶切线性化后,转染到HEK293细胞中进行包裹,根据增强绿色荧光蛋白(enhance green fluorescent protein,EGFP)的表达判断重组腺病毒的包装情况。通过反复离心和低温裂解细胞提取GRP78重组表达腺病毒。以人肝癌HepG2细胞为目标,进行病毒感染。通过qRT-PCR检测GRP78基因表达情况,使用Western blot检测GRP78蛋白的表达。最后感染HepG2细胞,通过MTS比色法检测重组表达GRP78腺病毒对细胞增殖的影响。结果:测序验证pAd Track-TO4-GRP78构建成功,酶切验证重组腺病毒载体pAd-GRP78构建成功。在HEK293细胞中将pAd-GRP78包裹,GPF荧光检测结果表示病毒包装成功。使用pAd-GRP78感染人肝癌HepG2细胞,qRT-PCR检测到细胞中GRP78基因的过表达,Western blot方法检测到GRP78蛋白的高表达。MTS检测结果显示重组表达GRP78腺病毒对HepG2肝癌细胞有促增殖作用。结论:实验成功构建了GRP78重组表达腺病毒,并验证了构建的腺病毒载体可以收集并感染HepG2细胞表达GRP78蛋白。证明了重组表达GRP78腺病毒能够促进肝癌细胞的增殖。为进一步研究GRP78的功能奠定了基础。Objective：To construct recombinant glucose regulated protein（GRP78）-expressed adenovirus, to establish a cell model by infecting HepG2 cells, and to study the effect of recombinant GRP78 adenovirus on cell proliferation in the HepG2 cells. Methods：Plasmid pAd Track-TO4-GRP78 containing GRP78 gene was constructed by using adenovirus shuttle plasmid pAd Track-TO4,and the sequence was identified by restriction enzyme digestion method. After linearized by Enzyme digestion of PmeⅠ,it was homologous recombination with Ad Easy-BJ5183 cells. The recombinant adenovirus vector was linearized by PacⅠ enzyme and transfected into HEK293 cell. The packaging of recombinant adenovirus was judged by the expression of the enhanced green fluorescent protein.GRP78 recombinant adenovirus was extracted by repeated centrifugation and low temperature cracking. HepG2 cells were infected with recombinant adenovirus. The expression of GRP78 gene was detected by qRT-PCR. The expression of GRP78 protein was detected by Western blot. Then the GRP78 adenovirus infected HepG2 cells. The effect of recombinant GRP78 adenovirus on cell proliferation was detected by MTS colorimetric assay.Results：It was proved by gene sequencing that pAd Track-TO4-GRP78 was successfully constructed. And by enzyme digestion validation,the recombinant adenovirus vector pAd-GRP78 was successfully constructed. After pAd-GRP78 being transfected into HEK293 cells,the GPF fluorescence results indicated that the virus was successfully packaged. HepG2 cells were infected with recombinant virus. And the expression of GRP78 gene was confirmed by qRTPCR,and the expression of GRP78 protein was confirmed by Western blot. MTS showed that the recombinant expression of GRP78 adenovirus could promote the proliferation of HCC cells. Conclusion：GRP78 recombinant adenovirus vector was successfully con structed,and the recombinant adenovirus vector could infect HepG2 cells and express GRP78 protein. GRP78 adenovirus could promote the proliferation of HCC cells. This stud

关 键 词：腺病毒 葡萄糖调节蛋白78 HEPG2细胞 MTS 

分 类 号：Q71[生物学—分子生物学]