«上一篇/Previous Article|本期目录/Table of Contents|下一篇/Next Article»

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

23卷

期数:

2017年04期

页码:

290-294

栏目:

基础医学

出版日期:

2017-07-02

文章信息/Info

Title:

Construction of human GRP94 lentivirus vector and its application in screening human cervical cancer cell line HeLa for stable expression

文章编号:

1006-8147(2017)04-0290-05

作者:

杨娅楠; 王 媛; 左志宇; 王鑫廷; 杨 洁

( 天津医科大学免疫学系，天津 300070)

Author(s):

YANG Ya-nan;  WANG Yuan;  ZUO Zhi-yu;  WANG Xin-ting;  YANG Jie

(Department of Immunology，Tianjin Medical University, Tianjin 300070, China)

关键词:

葡萄糖调节蛋白94; 慢病毒载体; HeLa; SND1

Keywords:

GRP94; lentiviral vector;  HeLa;  SND1

分类号:

Q7

DOI:

-

文献标志码:

A

摘要:

目的:构建人葡萄糖调节蛋白 94(GRP94)基因慢病毒载体，筛选稳定表达GRP94蛋白的人宫颈癌细胞株，筛选GRP94结合蛋白，为探讨GRP94对宫颈癌的调控作用提供细胞模型。方法:采用RT-PCR法从HeLa细胞中扩增GRP94基因片段，连接到慢病毒载体pLVX-IRES-Hyg中，获得重组载体。瞬时转染293T细胞，采用Western blotting 法检测GRP94蛋白表达量。pLVX-FLAG-GRP94重组质粒通过与包装质粒共转染293T细胞，获得重组慢病毒。以慢病毒感染HeLa细胞，筛选并鉴定稳定表达GRP94蛋白的细胞株。用稳定表达FLAG-GRP94的HeLa细胞株，银染后利用质谱筛选结合蛋白，并经免疫共沉淀验证。结果:重组慢病毒载体经双酶切和基因测序比对鉴定正确。HeLa细胞经慢病毒感染，药物筛选后获得的稳定表达株中GRP94蛋白表达量高于野生型HeLa细胞(P<0.01)。成功钓取出一种与肿瘤发生发展密切相关的SND1蛋白。结论:成功构建了GRP94基因慢病毒载体pLVX-FLAG-GRP94，并筛选出稳定表达GRP94蛋白的HeLa细胞株，为进一步明确肿瘤的发生、发展机制奠定了基础。

Abstract:

Objective: To construct human GRP94 gene lentiviral vector and screen the GRP94 cell line stably expressing GRP94 protein, to provide a cell line model for the study of GRP94 gene regulation in cervical cancer. Methods: The GRP94 gene fragment was amplified from HeLa cells by RT-PCR and ligated into lentiviral vector expression plasmid pLVX-IRES-Hyg to obtain recombinant lentiviralvector pLVX-FLAG-SND1. After transfection of 293T cells with pLVX-FLAG-GRP94 for 48h, the expression of GRP94 protein was detected by Western blotting. PLVX-FLAG-GRP94 recombinant plasmid was co-transfected with 293T cells to obtain recombinant lentivirus carrying FLAG-GRP94. The expression of GRP94 protein was detected by Western Blotting method. The GRP94-binding protein lysate was obtained by FLAG-small peptide elution using HeLa cell line which could stably express FLAG-GRP94. SDS-PAGE electrophoresis and silver staining were performed to select the binding protein.Results:Recombinant lentiviral vector was identified by restriction enzyme digestion and gene sequencing. The expression of GRP94 protein in transfected 293T cells was higher than that in wild type HeLa cells (P <0.01). The expression of GRP94 protein in HeLa cells was significantly higher than that in wild-type HeLa cells (P<0.01). The SND1 protein, which was closely related to tumor development and progression, was successfully detected by mass spectrometry and verified by immnoprecipitation. Conclusion: The GRP94 gene lentiviral vector pLVX-FLAG-GRP94 could be successfully constructed, and the HeLa cell line stably expressing GRP94 protein is screened, which lays a foundation for further study of the mechanism of tumorigenesis and development.

参考文献/References:

[1]Fu Y,Lee A S.Glucose regulated proteins in cancer progression, drug resistance and immunotherapy[J].Cancer Biol Ther,2006,5(7):741

[2]Lee A S. The glucose-regulated proteins: stress induction and clinical applications[J].Trends Biochem Sci,2001,26(8):504

[3]Dollins D,Warren J,Immormino R, et al.Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones[J].Mol Cell,2007,28(1):41

[4]Takano S,Wadhwa R,Mitsui Y, et al.Identification and characterization of molecular interactions between glucose-regulated proteins (GRPs) mortalin/GRP75/peptide-binding protein 74 (PBP74) and GRP94[J].Biochem J,2001,357(Pt 2):393

[5]Ramakrishnan M,Sch?nthal A,Lee A.Endoplasmic reticulum stress-inducible protein GRP94 is associated with an Mg2+-dependent serine kinase activity modulated by Ca²⁺ and GRP78/BiP[J].J Cell Physiol,1997,170(2):115

[6]Lee S,Song R,Lee M, et al.A molecular chaperone glucose-regulated protein 94 blocks apoptosis induced by virus infection[J].Hepatology,2008,47(3):854

[7]Oda T,Kosuge Y,Arakawa M, et al.Distinct mechanism of cell death is responsible for tunicamycin-induced ER stress in SK-N-SH and SH-SY5Y cells[J].Neurosci Res,2008,60(1):29

[8]Lev A,Dimberu P,Das S, et al.Efficient cross-priming of antiviral CD8+ T cells by antigen donor cells is GRP94 Independent[J].J Immunol,2009,183(7):4205

[9]Mimnaugh E,Xu W,Vos M, et al.Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity[J].Mol Cancer Ther,2004,3(5):551.

[10]Dressel R,Grzeszik C,Kreiss M, et al.Differential effect of acute and permanent heat shock protein 70 overexpression in tumor cells on lysability by cytotoxic T lymphocytes[J].Cancer Res, 2003,63(23):8212

[11]Tomida A,Suzuki H,Kim H, et al.Glucose-regulated stresses cause decreased expression of cyclin D1 and hypophosphorylation of retinoblastoma protein in human cancer cells[J].Oncogene,1996, 13(12):2699

[12]Cai J,Henderson B,Shen J, et al.Induction of glucose regulated proteins during growth of a murine tumor[J].J Cell Physiol, 1993, 154(2):229

[13]Wu YD,Song JD. The expression of glucose regulated protein-94 in colorectal carcinoma cells treated by Sodium butyrate[J].Cell Res,2000,10(2):115

相似文献/References:

[1]杨 扬,白 虹,辛灵彪.卵巢癌细胞系OVCAR3过表达SND1稳定株的构建[J].天津医科大学学报,2018,24(01):7.
　YANG Yang,BAI Hong,XIN Ling-biao.Construction of ovarian cancer OVCAR3 cell line for the stable expression of SND1[J].Journal of Tianjin Medical University,2018,24(04):7.

备注/Memo

备注/Memo:

基金项目 国家杰出青年基金资助项目(31300709)

作者简介 杨娅楠（1987-），女，研究实习员，硕士在读，研究方向：免疫学；

通信作者：王鑫廷，E-mail：wangxinting@tmu.edu.cn。

常用功能

更新日期/Last Update: 2017-06-30