MAO Shanshan,WANG Lude,ZHOU Meng, et al. Expression of EBV LMP1 C-terminal protein and preparation of its polyclonal antibody[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2018, 48(7): 469-473.
Abstract：Objective: To prepare EBV LMP1 C-terminal (187-386 aa) protein by prokaryotic expression system and polyclonal antibody against it and verify its biological character. Methods: A prokaryotic vector pET21a(+)/EBV LMP1 C-terminal (187-386 aa) was constructed and transformed into E.coli BL21 (DE3), and then the recombinant protein was induced by IPTG and purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot analysis demonstrated the purified LMP1 C-terminal protein was expressed correctly. To obtain the polyclonal antibody, the rabbit was immunized with the purified LMP1 C-terminal recombinant protein. An analysis of polyclonal antibody was made by ELISA, Western blot and immunofluorescence technique. Results: The recombinant protein of LMP1 C-terminal was successfully expressed and purified by the prokaryotic expression system. The polyclonal antibody was obtained by immunizing the rabbits with the recombinant protein. Conclusion: The recombinant protein of LMP1 C-terminal has strong immunogenicity and the polyclonal antibody of LMP1 C-terminal has high titer and specificity.
[1] RAAB-TRAUB N. Epstein-Barr virus in the pathogenesis of NPC[J]. Semin Cancer Biol, 2002, 12(6): 431-441.
[2] MIDDELDORP J M, PEGTEL D M. Multiple roles of LMP1 in Epstein Barr virus induced immune escape[J]. Semin Cancer Biol, 2008, 18(6): 388-396.
[3] HENNESSY K, FENNEWAID S, HUMME I M, et al. A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection[J] . Proc Natl Acad Sci U S A, 1984, 81(22): 7207-7211.
[4] LI H P, CHANG Y S. Epstein-Barr virus latent membrane protein 1: structure and functions[J]. J Biomed Sci, 2003, 10 (5): 490-504.
[5] MORRIS M A, DAWSON C W, YOUNG L S. Role of the Epstein-Barr virus-encoded latent membrane protein-1, LMP1, in the pathogenesis of nasopharyngeal carcinoma[J]. Future Oncol, 2009, 5(6): 811-825
[6] TSAO S W, TRAMOUTANIS G, DAWSON C W, et aI. The significance of LMP1 expression in nasopharyngeal carcino-ma[J]. Semin Cancer Biol, 2002, 12(6): 473-487.
[7] LEE D Y, SUGDEN B. The latent membrane protein 1 oncogene modifies B-cell physiology by regulating autophagy[J]. Oncogene, 2008, 27(20): 2833-2842.
[8] WANG D, LIEBOWITZ E, KIEFF E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells[J]. Cell, 1985, 43(3 Pt 2): 831-840.
[9] SHAIR K H, SCHNEGG C I, RAAB-TRAUB N. Epstein-Barr virus latent membrane protein-1 effects on junctional plakoglobin and induction of a cadherin switch[J]. Cancer Res, 2009, 69(14): 5734-5742.
[10] HUEN D S, HEDNERSON S A, CROOM-CARTER D, et al.
Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-kappa B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain[J]. Oncogene, 1995, 10(3): 549-560.
[11] MOSIAIOS G, BIRKENBACH M, YAIAMANCHIII R,
et al. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor famiIy[J]. Cell, 1995, 80(3): 389-399.
[12] RUSSELL S M, TAYEBI N, NAKAJIMA H, et al. Mutation of Jak3 in patient wilh SClD: essential role of Jak3 in lymphoid development[J]. Science, 1999, 270(5237): 797-800.
[13] 余琳, 张雪雁, 李孜. EB病毒LMP1-CTAR2结构域的原核表达及蛋白质纯化[J]. 中国卫生检验杂志, 2010, 20(7): 1583-1585.
[14] 张雪雁, 杨英, 马桂璋. EB病毒LMP1蛋白CTAR23结构域的原核表达及纯化[J]. 热带医学杂志, 2004, 4(2): 117-121.
[15] 林勇平, 吴淑华, 杨英, 等. EBV-LMP1 cDNA的克隆测序及原核表达质粒构建[J]. 热带医学杂志, 2002, 2(4): 337-340.
