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 adenovirus pAd-GRP78 of glucose regulated protein 78 (GRP78) and to infect HepG2 cells to establish a cell model capable of high expression of GRP78, and to study the effect of recombinant GRP78 adenovirus on proliferation of HepG2 cells The impact of ability. Methods: The shuttle plasmid pAdTrack-TO4-GRP78 carrying GRP78 gene was constructed by adenoviral shuttle plasmid pAdTT-TO4. The vector sequence was identified by gene sequencing. The vector was linearized with Pme I digestion and gene homologous recombination with Ad Easy-BJ5183 competent cells. The recombined adenoviral vector was linearized by Pac Ⅰ digestion and then transfected into HEK293 cells for packaging. The packaging of recombinant adenovirus was determined according to the expression of enhanced green fluorescent protein (EGFP). GRP78 recombinant adenovirus was extracted by repeated centrifugation and cryogenic lysis of cells. Human hepatoma HepG2 cells were targeted for virus infection. GRP78 gene expression was detected by qRT-PCR and GRP78 protein expression by Western blot. HepG2 cells were finally infected and the effect of recombinant adenovirus GRP78 on cell proliferation was detected by MTS colorimetric assay. Results: The sequence of pAd Track-TO4-GRP78 was verified by sequencing. The recombinant adenovirus vector pAd-GRP78 was successfully constructed. The pAd-GRP78 was packaged in HEK293 cells and the GPF fluorescence assay indicated that the virus was successfully packaged. The human hepatocellular carcinoma HepG2 cells were infected with pAd-GRP78. The overexpression of GRP78 gene was detected by qRT-PCR and the expression of GRP78 protein was detected by Western blot. MTS test results showed that recombinant adenovirus GRP78 could promote proliferation of HepG2 hepatoma cells. CONCLUSION: The recombinant adenovirus GRP78 was successfully constructed and verified that the constructed adenovirus vector can collect and infect HepG2 cells expressing GRP78 protein. It is proved that recombinant adenovirus GRP78 can promote the proliferation of hepatoma cells. Which laid the foundation for further study on the function of GRP78.