融合表达对人工重组多表位蛋白M．RCAg-1构象及免疫效能影响的研究  

Study of fusion expression effect on conformation and immune activity of polyepitope protein, M. RCAg-1

作　　者：王健[1] 蔺亚辉[2] 陈祥鹏[1] 李莉莉[1] 李军[1] 王恒[2] 张振龙[1] 

机构地区：[1]北京生物制品研究所第四研究室,100024 [2]北京协和医学院基础学院病原生物学系

出　　处：《中华微生物学和免疫学杂志》2012年第1期58-64,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家863计划重大专项（2006AA02A224）;传染病防治重大专项（2008ZX10004-015）

摘　　要：目的研究载体融合肽段对人工重组多表位蛋白M．RCAg-1构象、免疫原性及体外保护性的影响。方法将人工多表位抗原M．RCAg-1基因通过不同的酶切位点克隆到原核表达载体pDS—ex上，从而获得带有不同载体融合标签或没有标签的蛋白，表达产物经纯化得到P312-1、P312—2、P312-3这3种多表位蛋白，利用生物信息学和色谱技术对制备的3种蛋白的二级及三级结构进行了分析，并将这3种纯度大于95％的重组蛋白与弗氏佐剂乳化后分别免疫接种小鼠和新西兰白兔，免疫后血清按常规进行ELISA间接法检测抗体滴度，采用酶联免疫斑点试验（ELISPOT）检测分泌细胞因子的特异性淋巴细胞克隆数，同时进行了间接免疫荧光分析（IFA）以及对体外培养的恶性疟原虫的生长抑制试验（GIA）。结果P312—1、P312-2、P312-3这3种多表位蛋自在动物模型体内诱导的抗体滴度水平并无差异（P〉0．05），但蛋白免疫小鼠后却诱导了不同类型的T细胞反应，而且3种蛋白免疫血清IgG体外生长抑制率出现明显差异（分别为93．9％、14．7％、54．3％）。利用生物信息学和色谱技术分析，发现从不同表达载体制备的重组蛋白在二级和三级结构上出现极大差异，预测蛋白质分子的空间构象发生了改变，而且一些表位在分子中的位置也有明显的改变。结论不同的载体肽融合表达对多表位蛋白M．RCAg-1结构及免疫活性的影响会产生不同的作用，直接影响多表位蛋白的构象，进而会增强或抑制多表位蛋白的免疫效应。Objective To explore the effects of vector fusion peptides on the conformation and im- mune reactivity of recombinant polyepitope antigens, M. RCAg-1. Methods We subcloned polyepitope arti- ficial antigen gene, M. RCAg-1, into prokaryotic expression vectors, pDS-ex, that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site. Three recombinant proteins expressed by these vectors, named P312-1, P312-2, and P312-3, were purified and purity is greater than 95%. Then BALB/c mice were vaccinated with the three proteins formulated with Freund＇s adjuvant through intranasal. Serum were collected to detect specific antibodies of M. RCAg-1 and individual epitope by ELISA; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lym- phecyte activation was detected by enzyme-linked immunosorbent spot test （ELISPOT） ; and the growth of Plasmodiumfalciparum in vitro to evaluate by growth inhibition assay（ GIA）, and analyze secondary and ter- tiary structures of recombinant proteins from different expression vectors by bioinformatics and circular di- chroism technique. Results The P312-1, P312-2 have almost the same amino acid sequence, and the three proteins have the same immunogenicity in animal models （P〉0.05）, however, the different proteins elicited various T-cell responses, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro （respectively, 93.9%, 14.7% ,54.3% ）. The significant differ- ences of secondary and tertiary structures were shown in recombinant proteins from different expression vec- tors, analyzed by bioinformatics and circular dichroism technique, which demonstrated the change of protein molecule spaces conformation, and the obviously change of some epitope locations. Conclusion These re- suits suggest that the expressed polyepitope artificial antigens originating from the different vector fusion pep-tides indeed affect the protein

关 键 词：多表位蛋白 载体肽 融合表达 

分 类 号：R392[医药卫生—免疫学]