人葡萄糖调节蛋白78基因磷酸化位点的真核突变载体的构建及表达  

Construction and expression of eukaryotic vectors for human glucose-regulated protein 78 gene with mutation at phosphorylation sites

作　　者：宋慧娟[1] 赵颂[1] 苏荣健[1] 

机构地区：[1]辽宁医学院辽宁省高校分子细胞生物学与新药开发重点实验室,辽宁锦州121001

出　　处：《中国生物制品学杂志》2014年第7期881-884,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(81172048)

摘　　要：目的构建人葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)基因编码区磷酸化位点的真核突变载体,并在肝癌SMMC-7721细胞中进行表达。方法利用定点诱变技术将GRP78基因的两个磷酸化位点进行突变,即203位苏氨酸(T)用丙氨酸(A)替换,466位酪氨酸(Y)用苯丙氨酸(F)替换,以质粒pEGFP-N1-GRP78为模板,进行PCR扩增后,构建突变质粒pEGFP-N1-GRP78(T203A)、pEGFP-N1-GRP78(Y466F)和pEGFP-N1-GRP78(T203A/Y466F),并进行DNA测序分析。将测序正确的突变质粒转染肝癌SMMC-7721细胞,荧光显微镜下观察绿色荧光蛋白表达,Western blot法检测融合蛋白在细胞中的表达。结果突变质粒pEGFP-N1-GRP78(T203A)、pEGFP-N1-GRP78(Y466F)和pEGFP-N1-GRP78(T203A/Y466F)经双酶切及测序鉴定,证明构建正确;突变质粒组荧光蛋白表达分布于细胞质内,细胞核内无表达产物分布;3个突变质粒转染组在相对分子质量约105 000处均可见目的蛋白条带,大小与预期相符。结论成功构建了人GRP78单突变T203A、Y466F和双突变T203A/Y466F真核表达载体,并可在肝癌SMMC-7721细胞中表达,为进一步研究GRP78基因磷酸化位点T203、Y466在肝肿瘤侵袭和转移中的作用奠定了基础。Objective To construct a eukaryotic vector for human glucose-regulated protein 78（GRP78）gene with mutation in the coding region of phosphorylation sites and express in liver cancer SMMC-7721 cells. Methods Mutations from T to A and Y to F were induced at phosphorylation sites 203 and 466 of GRP78 gene by site-directed mutagenesis,respectively. The mutant genes were amplified by PCR using plasmid pEGFP-N1-GRP78 as a template,based on which mutant plasmids pEGFP-N1-GRP78（T203A）,pEGFP-N1-GRP78（Y466F）and pEGFP-N1-GRP78（T203A / Y466F）were constructed and identified by DNA sequencing,then transfected into SMMC-7721 cells. The expression of green fluorescent protein（GFP）was observed by fluorescent microscopy,while that of fusion protein by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmids pEGFP-N1-GRP78（T203A）,pEGFP-N1-GRP78（Y466F）and pEGFP-N1-GRP78（T203A / Y466F）were constructed correctly. The expressed GFPs in SMMC-7721 cells transfected with the mutant plasmids were located in cytoplasm but not in nuclei. The target protein with a relative molecular mass of about105 000 was observed in the cells transfected with any of the three recombinant plasmids,which was consistent with that expected. Conclusion Eukaryotic expression vectors for human GRP78 with single mutant（T203A,Y466F）and double mutants（T203A / Y466F）were constructed successfully and expressed in SMMC-7721 cells,which laid a foundation of further study on the role of phosphorylation sites of GRP78 gene in the invasion and metastasis of liver tumor.

关 键 词：葡萄糖调节蛋白78 磷酸化位点 定点诱变 真核细胞 基因表达 

分 类 号：Q782[生物学—分子生物学]