人巨细胞病毒UL135蛋白的原核表达及其多克隆抗体的制备和鉴定

摘要
图/表
参考文献
相关文章 (6)

全文: PDF (1616 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要目的：制备人巨细胞病毒（HCMV）UL135原核表达蛋白及其多克隆抗体。方法：生物信息学分析HCMV UL135蛋白的跨膜区结构，经原核密码子优化后对胞外序列进行全基因合成，将序列克隆至原核表达载体构建pET21a（+）/HCMV UL135重组质粒，IPTG诱导蛋白表达后，Ni-NTA亲和层析法纯化UL135重组蛋白，免疫新西兰兔制备多克隆抗体，ELISA、Western blot法检测抗体灵敏度和特异性。结果：HCMV的UL135基因在原核表达系统中成功表达，经Ni-NTA亲和纯化后获得高纯度的目的蛋白；免疫新西兰兔诱导产生特异性的多克隆IgG抗体，且效价为1:12 800；经ELISA以及Western blot证实制备的兔抗多克隆抗体可特异性识别UL135蛋白。结论：成功实现了HCMV UL135的重组表达并制备了UL135兔多克隆抗体，为进一步研究UL135蛋白的生物学功能奠定基础。

	服务
	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	胡盈盈
	郭刚强
	叶思思
	孙祥威
	毛晨晨
	薛向阳
	吕杰强

关键词 ：人巨细胞病毒, UL135蛋白, 多克隆抗体

Abstract： Objective: To construct a prokaryotic expression vector expressing UL135 protein of human cytomegalovirus (HCMV) and prepare a UL135 specific polyclonal antibody. Methods: Bioinformatics methods were used to analyze the transmembrane domain of UL135 protein, and then the cytoplasmic domain synthesized by fully-synthetic gene was cloned into vector pET21a (+) after the prokaryotic codon optimization. The pET21a (+)/UL135 recombinant plasmid was transformed into E.coli BL21 (DE3) and then induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). To prepare specific polyclonal antibody, New Zealand rabbits were immunized by the UL135 recombinant protein which was purified by Ni-NTA affinity chromatography primarily. The sensitivity and the specificity of the antibody were determined by ELISA and Western blot assays. Results: HCMV UL135 protein was successfully expressed in prokaryotic system and highly purified target protein was obtained through Ni-NTA affinity chromatography. New Zealand rabbits could generate specific polyclonal IgG antibody after immunized. ELASE and Western blot analysis determined that the preparative polyclonal IgG antibody could specifically identify the UL135 protein. Conclusion: HCMV UL135 protein is successfully expressed in prokaryotic expression system, and anti-UL135 rabbit polyclonal antibody is successfully prepared for the further research.

Key words： human cytomegalovirus UL135 protein polyclonal antibody

收稿日期: 2016-10-25

基金资助:国家自然科学基金资助项目（81472308）。

通讯作者: 吕杰强，教授，博士生导师，Email：jieqianglu@ 126.com。

作者简介: 胡盈盈（1992-），女，浙江永嘉人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I5/336

[1] BOECKH M, GEBALLE A P. Cytomegalovirus: pathogen,paradigm, and puzzle[J]. J Clin Invest, 2011, 121(5): 1673-1680.
[2] STERN-GINOSSAR N, WEISBURD B, MICHALSKI A, et al. Decoding human cytomegalovirus[J]. Science, 2012, 338(6110): 1088-1093.
[3] YAMIN R, LECKER L S, WEISBLUM Y, et al. Hcmvvcxcl1 binds several chemokine receptors and preferentially attracts neutrophils over nk cells by interacting with cxcr2[J]. Cell Rep, 2016, 15(7): 1542-1553.
[4] GRIFFITHS P D. Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation[J]. Lancet Infect Dis, 2012, 12(10): 790-798.
[5] CHEN H P, JIANG J K, CHEN C Y, et al. Human cytomegalovirus preferentially infects the neoplastic epithelium of colorectal cancer: A quantitative and histological analysis[J]. J Clin Virol, 2012, 54(3): 240-244.
[6] CHEN W, LIN K, ZHANG L, et al. The cytomegalovirus protein UL138 induces apoptosis of gastric cancer cells by binding to heat shock protein 70[J]. Oncotarget, 2016, 7(5):5630-5645.
[7] COBBS C S, HARKINS L, SAMANTA M, et al. Human cytomegalovirus infection and expression in human malignant glioma[J]. Cancer Res, 2002, 62(12): 3347-3350.
[8] SAMANTA M, HARKINS L, KLEMM K, et al. High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma[J]. J Urol, 2003, 170(3): 998-1002.
[9] STANTON R J, PROD’HOMME V, PURBHOO M A, et al. HCMV pUL135 remodels the actin cytoskeleton to impair immune recognition of infected cells[J]. Cell Host Microbe,  2014, 16(2): 201-214.
[10] PAWELEC G, DERHOVANESSIAN E. Role of CMV in immune senescence[J]. Virus Res, 2011, 157(2): 175-179.
[11] HALWACHS-BAUMANN G. Recent developments in human cytomegalovirus diagnosis[J]. Expert Rev Anti Infect Ther, 2007, 5(3): 427-439.
[12] 王练英, 李正, 阮强. 婴儿先天性巨结肠与巨细胞病毒感染[J]. 中华小儿外科杂志, 1996, 17(1): 3-5.
[13] 卢光荣. Torch宫内感染孕妇临床筛查分析[J]. 中国实用医药, 2009, 24(4): 86-87.
[14] PARADOWSKA E, STUDZINSKA M, SUSKI P, et al. Human cytomegalovirus UL55, UL144, and US28 genotype distribution in infants infected congenitally or postnatally[J].  J Med Virol, 2015, 87(10): 1737-1748.
[15] DARGAN D J, DOUGLAS E, CUNNINGHAM C, et al. Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture[J]. J Gen Virol, 2010, 91(Pt 6): 1535-1546.
[16] MONTAG C, WAGNER J A, GRUSKA I, et al. The latency-associated UL138 gene product of human cytomegalovirus sensitizes cells to tumor necrosis factor alpha (Tnf-alpha) signaling by upregulating Tnf-alpha receptor 1 cell surface expression[J]. J Virol, 2011, 85(21): 11409-11421.
[17] Wills M R, Ashiru O, Reeves M B, et al. Human cytomegalovirus encodes an Mhc class I-like molecule  (UL142) that functions to inhibit Nk cell lysis[J]. J Immunol, 2005, 175(11): 7457-7465.
[18] LE V T, TRILLING M, HENGEL H. The cytomegaloviral  protein pUL138 acts as potentiator of tumor necrosis factor (Tnf) receptor 1 surface density to enhance ULb’-encoded modulation of Tnf-signaling[J]. J Virol, 2011, 85(24): 13260-13270.
[19] 陈文静, 黄崇安, 陈静, 等. 潜伏相关抗原ul138蛋白的制备及其用于人巨细胞病毒感染血清学检测的评价[J]. 温州医科大学学报, 2015, 45(10): 703-708.