葡萄糖调节蛋白GRP78与HBV的PreS1相互作用位点的初步研究
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摘要
目的:初步研究78 k D的葡萄糖调节蛋白(the 78 k D glucose-regulated protein,GRP78)与乙型肝炎病毒(hepatitis B virus,HBV)的前S1蛋白(Pre S1)的相互作用位点。方法:利用PCR技术扩增GRP78的基因,将扩增的目的基因克隆至p W28载体质粒,在大肠杆菌(Escherichia coli,E.coli)B834中表达,经过镍离子亲和层析柱纯化GRP78蛋白;将Pre S1 3个截短片段的重组质粒(p GST-Pre S1-X1/X2/X3)在B834中表达后,经过GST亲和层析柱纯化相应蛋白;利用蛋白质体外结合实验(pull down)、微量热泳动(microscale thermophoresis,MST)检测GRP78与Pre S1 3个截短片段的相互作用。结果:成功构建重组质粒p W28-GRP78;获得GRP78蛋白及Pre S1 3个截短片段的融合蛋白;pull down及MST实验验证了GRP78可以与Pre S1的3个片段结合,且GRP78与GST-Pre S1-X1结合效果最好。结论:利用分子克隆技术及蛋白质表达纯化技术,获得GRP78蛋白及Pre S1截短片段的融合蛋白,并初步筛选了Pre S1与GRP78的相互作用位点,为后续研究打基础。
Objective:To study interaction sites of the glucose-regulated protein 78 k D(GRP78)with pre-S1 protein(Pre S1)of hepatitis B virus(HBV). Methods:Being amplified by PCR,the open reading frame of GRP78 was subcloned into p W28 and expressed in Escherichia coli(E.coli)B834. The GRP78 protein was purified by nickel affinity chromatography column. Three truncated plasmids of HBV Pre S1,including p GST-Pre S1-X1,p GST-Pre S1-X2,p GST-Pre S1-X3,were expressed in B834 and then the bacterial protein was purified by GST affinity chromatography column. Interactions between GRP78 and Pre S1 truncations were detected by pull down and microscale thermophoresis(MST). Results:The recombinant plasmid of p W28-GRP78 was successfully constructed. The GRP78 protein and the fusion protein(GST-Pre S1-X1,GST-Pre S1-X2,GST-Pre S1-X3)were obtained. The directly interaction between GRP78 and fusion protein were detected by pull down and MST,suggesting that the 1-65 amino acid residues of Pre S1 play a key role for GRP78 binding to Pre S1. Conclusion:Thanks to the molecular cloning and purified technology of protein purification,the fusion protein of GRP78 protein and the Pre S1 truncated segments is formed. And interaction sites of GRP78 protein and the Pre S1 is primarily compared,providing preliminary data for future study.
Objective:To study interaction sites of the glucose-regulated protein 78 k D(GRP78)with pre-S1 protein(Pre S1)of hepatitis B virus(HBV). Methods:Being amplified by PCR,the open reading frame of GRP78 was subcloned into p W28 and expressed in Escherichia coli(E.coli)B834. The GRP78 protein was purified by nickel affinity chromatography column. Three truncated plasmids of HBV Pre S1,including p GST-Pre S1-X1,p GST-Pre S1-X2,p GST-Pre S1-X3,were expressed in B834 and then the bacterial protein was purified by GST affinity chromatography column. Interactions between GRP78 and Pre S1 truncations were detected by pull down and microscale thermophoresis(MST). Results:The recombinant plasmid of p W28-GRP78 was successfully constructed. The GRP78 protein and the fusion protein(GST-Pre S1-X1,GST-Pre S1-X2,GST-Pre S1-X3)were obtained. The directly interaction between GRP78 and fusion protein were detected by pull down and MST,suggesting that the 1-65 amino acid residues of Pre S1 play a key role for GRP78 binding to Pre S1. Conclusion:Thanks to the molecular cloning and purified technology of protein purification,the fusion protein of GRP78 protein and the Pre S1 truncated segments is formed. And interaction sites of GRP78 protein and the Pre S1 is primarily compared,providing preliminary data for future study.
引文
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[2]El-Serag HB,Rudolph KL.Hepatocellular carcinoma:epidemiology and molecular carcinogenesis[J].Gastroenterology,2007,132(7):2557-2576.
[3]Venook AP,Papandreou C,Furuse J,et al.The incidence and epidemiology of hepatocellular carcinoma:a global and regional perspective[J].Oncologist,2010,15(S4):5-13.
[4]Gish R,Jia JD,Locarnini S,et al.Selection of chronic hepatitis Btherapy with high barrier to resistance[J].Lancet Infect Dis,2012,12(4):341-353.
[5]Ni M,Lee AS.ER chaperones in mammalian development and human diseases[J].FEBS Lett,2007,581(19):3641-3651.
[6]Munro S,Pelham HR.An Hsp70like protein in the ER:identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein[J].Cell,1986,46(2):291-300.
[7]Yang J.Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone Bi P[J].Structure,2015,23(12):2191-2203.
[8]Lee AS.GRP78 induction in cancer:therapeutic and prognostic implications[J].Cancer Res,2007,67(8):3496-3499.
[9]Miao YR,Eckhardt BL,Cao Y,et al.Inhibition of established micrometastases by targeted drug delivery via cell surface-associated GRP78[J].Clin Cancer Res,2013,19(8):2107-2116.
[10]Wang M,Wey S,Zhang Y,et al.Role of the unfolded protein response regulator GRP78/Bi P in development,cancer,and neurological disorders[J].Antioxid Redox Signal,2009,11(9):2307-2316.
[11]Zhang NQ,Zheng ZH,Xu HX,et al.Glucose-regulated protein 78demonstrates antiviral effects but is more suitable for hepatocellular carcinoma prevention in hepatitis B[J].Virol J,2017,14(1):77.
[12]Huang KL,Lai YK,Lin CC,et al.Involvement of GRP78 in inhibition of HBV secretion by Boehmeria nivea extract in human Hep G22.2.15 cells[J].J Viral Hepat,2009,16(5):367-375.
[13]Ma Y,Yu J,Chan HL,et al.Glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis B virus[J].Mol Cell Proteomics,2009,8(11):2582-2594.
[14]Pontisso P,Alberti A.The role of pre S1 in the interaction of hepatitis B virus with human hepatocytes[J].Hepatology,1991,14(2):405-406.
[15]杨凤,丁岗强,唐霓,等.GRP78在Hep G2细胞的亚细胞定位及真核表达[J].解放军医学杂志,2009,34(11):1328-1332.
[16]丁岗强,张君,杨凤,等.HBV前S1蛋白与Hep G2细胞膜蛋白结合位点鉴定[J].生物技术通报,2009,25(8):120-124.
[17]丁岗强,张君,杨凤,等.Hep G2细胞膜上乙型肝炎病毒前S1蛋白(pre S1)结合蛋白的研究[J].中国生物工程杂志,2009,29(9):71-74.
[18]Neurath AR,Kent SB,Strick N,et al.Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus[J].Cell,1986,46(3):429-436.
[19]Glebe D,Urban S.Viral and cellular determinants involved in hepadnaviral entry[J].World J Gastroenterol,2007,13(1):22-38.
[20]Rehman SU,Sarwar T,Ishqi HM,et al.Deciphering the interactions between chlorambucil and calf thymus DNA:a multi-spectroscopic and molecular docking study[J].Arch Biochem Biophys,2015,566:7-14.
[21]Yan H,Zhong G,Xu G,et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[J].Elife,2012,1:e00049.
[22]Stetz G,Verkhivker GM.Dancing through life:molecular dynamics simulations and network-centric modeling of allosteric mechanisms in Hsp70 and Hsp110 chaperone proteins[J].PLo S One,2015,10(11):e143752.
[23]Zhang A,Guo Y,Zhang S,et al.Cytokine effects and cellular signaling pathways of grass carp HSP70 in head kidney leukocytes[J].Fish Shellfish Immunol,2015,46(2):550-556.
[24]王良宏,杨丽,潘卫,等.与HBV pre S1黏附相关的细胞受体的研究[J].重庆医学,2014,43(10):1221-1223.
[25]Pan XW,Zhao XH.In vitro proliferation and anti-apoptosis of the papain-generated casein and soy protein hydrolysates towards osteoblastic cells(h FOB1.19)[J].Int J Mol Sci,2015,16(6):13908-13920.