Ripalti A, Ruan Q, Boccuni M C, Campanini F, Bergamini G, Landini M P
Department of Microbiology, School of Medicine, University of Bologna, Italy.
J Clin Microbiol. 1994 Feb;32(2):358-63. doi: 10.1128/jcm.32.2.358-363.1994.
We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides, can be valuable diagnostic material in the serology of HCMV infection (M. P. Landini, M. X. Guan, G. Jahn, W. Lindenmaier, M. Mach, A. Ripalti, A. Necker, T. Lazzarotto, and B. Plachter, J. Clin. Microbiol. 28:1375-1379, 1990; M. P. Landini, T. Lazzarotto, A. Ripalti, M. X. Guan, and M. La Placa, J. Clin. Microbiol. 27:2324-2327, 1989; A. Ripalti, M. P. Landini, E. S. Mocarski, and M. La Placa, J. Gen. Virol. 70:1247-1251, 1989). In this work we addressed the question of whether the expression of more than one linear epitope on a single fusion protein could increase the reactivity of genetically engineered antigenic material with human antibody. To answer this question we fused sequences expressing two different epitopes contained in the basic phosphoprotein of 150 kDa encoded by UL32 (M. S. Chee, A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, T. Cerny, T. Hornsel, C. A. Hutchinson, T. Kouzarides, J. A. Martignetti, and B. G. Barrell, Curr. Top. Microbiol. Immunol. 154:125-169, 1990; G. Jahn, T. Kouzarides, M. Mach, B.-C. Scholl, B. Plachter, B. Traupe, E. Preddie, S. C. Satchwell, B. Fleckenstein, and B. G. Barrell, J. Virol. 61:1358-1367, 1987), ppUL32, which was repeatedly shown to be the strongest immunogen present in the viral particle. We also made fusions with sequences expressing a single epitope repeated once, twice, or three times. The different fusion proteins were tested with HCMV-positive human sera. We found that fusion proteins expressing different epitopes together were recognized by a larger number of serum specimens and with more intense reactions in Western blot (immunoblot) experiments. We also found evidence that expression on the same polypeptide of the two distinct epitopes produced a stronger antigen than the mere addition of two fusion proteins which each carried one copy of one of these epitopes. Furthermore, we found that while the same epitope expressed two or three times on the same fusion protein was not better recognized by immunoglobulin G than the single epitope, immunoglobulin M reactivities to the double and triple epitopes were enhanced.
我们之前已经表明，主要人类巨细胞病毒（HCMV）抗原的单个线性表位，以融合蛋白形式表达或合成为寡肽时，在HCMV感染血清学中可能是有价值的诊断材料（M.P.兰迪尼、M.X.关、G.扬、W.林德迈尔、M.马赫、A.里帕尔蒂、A.内克、T.拉扎罗托和B.普拉赫特，《临床微生物学杂志》28:1375 - 1379，1990；M.P.兰迪尼、T.拉扎罗托、A.里帕尔蒂、M.X.关和M.拉普拉卡，《临床微生物学杂志》27:2324 - 2327，1989；A.里帕尔蒂、M.P.兰迪尼、E.S.莫卡尔斯基和M.拉普拉卡，《普通病毒学杂志》70:1247 - 1251，1989）。在这项工作中，我们探讨了在单个融合蛋白上表达多个线性表位是否会增加基因工程抗原材料与人类抗体的反应性这一问题。为了回答这个问题，我们将表达UL32编码的150 kDa碱性磷蛋白（M.S.奇、A.T.班基尔、S.贝克、R.博尼、C.M.布朗、T.切尔尼、T.霍恩塞尔、C.A.哈钦森、T.库扎里德斯、J.A.马丁内蒂和B.G.巴雷尔，《微生物学与免疫学当前专题》154:125 - 169，1990；G.扬、T.库扎里德斯、M.马赫、B - C.朔尔、B.普拉赫特、B.特劳佩、E.普雷迪、S.C.萨奇韦尔、B.弗莱肯施泰因和B.G.巴雷尔，《病毒学杂志》61:1358 - 1367，1987）中包含的两个不同表位的序列进行融合，ppUL32，该蛋白已多次被证明是病毒颗粒中最强的免疫原。我们还将表达单个表位重复一次、两次或三次的序列进行融合。用HCMV阳性人类血清对不同的融合蛋白进行检测。我们发现，一起表达不同表位的融合蛋白在蛋白质免疫印迹（免疫印迹）实验中被更多血清标本识别，且反应更强烈。我们还发现有证据表明，在同一多肽上表达两个不同表位所产生的抗原比仅仅添加分别携带其中一个表位一个拷贝的两种融合蛋白更强。此外，我们发现，虽然在同一融合蛋白上表达两次或三次的相同表位在免疫球蛋白G的识别上并不比单个表位更好，但免疫球蛋白M对双表位和三表位的反应性增强。
