癌胚抗原T、B细胞表位短肽诱导特异性体液免疫反应研究  

Specific Humoral Immune Response Produced in Rabbits Immunized with T,B Cell Epitope Peptide of CEA

作　　者：陈江[1] 肖德双[1] 吴乐[1] 蒋佩佩[1] 邵欢义[1] 应江辉[1,2] 朱珊丽[2] 薛向阳[2] 张丽芳[2] 朱冠保[1] 

机构地区：[1]温州医学院附属第一医院胃肠外科,325000 [2]温州医学院分子病毒与免疫研究所/微生物学与免疫学教研室

出　　处：《医学研究杂志》2012年第4期65-68,共4页Journal of Medical Research

基　　金：浙江省自然科学基金资助项目(Y2100660)

摘　　要：目的筛选癌胚抗原含T、B细胞表位的短肽并分析其诱导机体产生体液免疫的特性。方法利用免疫信息学方法预测CEA CTL表位,结合我们早期对CEA B表位预测的结果,选择含多个CTL表位肽和B表位肽的CEA氨基酸序列580～598处短肽作为目的基因。目的基因密码子优化后采用pET32a(+)原核表达系统表达融合蛋白,经SDS-PAGE和Western blot-ting鉴定融合蛋白的抗原性;经镍螯合亲和层析柱(Ni-NTA Agarose)纯化CEA580～598全长融合蛋白,纯化蛋白用佐剂乳化后经日本大耳白家兔背部皮下多点免疫注射,采用Western blotting检测血清抗体的特异性,采用ELISA法检测血清抗体水平及变化趋势。结果含多个T、B细胞表位的短CEA580～598在大肠杆菌中获得了高效表达,表达量占总蛋白的25.6%;表达产物的相对分子质量(Mr)约21kDa,与预期Mr相符;Western blotting分析结果显示,在21kDa处出现特异性条带。日本大耳白兔经CEA580～598融合蛋白免疫后产生高水平的特异性抗体,与空载体和PBS对照组比较具有显著性差异(P<0.05),制备的免疫血清不但能特异识别CEA580～598短肽,而且能特异识别天然CEA抗原。结论 CEA580～598短肽融合蛋白具有较强的免疫原性,诱导的兔免疫血清能特异性结合CEA抗原,为基于CEA表位疫苗的研究奠定理论和实验基础。Objective To screen the peptide with multi T, B cell epitopes from Carcinoembryonic antigen （CEA） and analyze the specific humoral immune response produced in rabbits by the peptide. Methods Immunization information methods were used to predict CEA CTL epitopes, combined with our earlier forecast of CEA B epitopes, select the table containing multiple CTL epitopes and B epitopes of the CEA peptide at the amino acid sequence 580 - 598 as the target gene. Multi - epitopes gene was then cloned into the pro- karyotic expression vector pET - 32a（ ＋ ） and then induced with IPTG. The fusion protein containing CEA580 - 598 was expressed and analyzed with both SDS - PAGE and Western blot and purified with Ni - NTA Agarose. CEA580 - 598 protein in Freund＇s adjuvant was prepared by the usual method. Rabbits were immunized with the prepared antigen by subcutaneous injection at various sites on the back. The sera of these immunized rabbits were collected separately for preparation of rabbit serum IgG. Specific binding of the rabbit serum antibody and the CEA580 -598 protein was analyzed with Western blotting and the titer was assayed with ELISA. Results Multi T, B cell epitopes in the short CEA580 - 598 in E. eoli were highly expressed by 25.6% of total protein. The relative molecular mass （Mr） of expression product was about 21kDa, matched with the expected Mr. Western blotting analysis showed that in about 21kDa the specific band occured. Rabbits immunized by CEA580 - 598 fusion protein produced high level of specific antibodies. As comported with the vector and PBS control group,it had significant difference （P 〈 0.05）. Serum prepared can not only specifically recognize short CEA580 - 598 peptide, and can specifically recognize the natural CEA antigen. Conclusion CEA580 -598 peptide fusion protein has strong immunogenieity. Rabbit antiserum can bind to CFA specificly,which provides theoretical and experimental basis for the study of vaccine based on CEA antigen epitope.

关 键 词：癌胚抗原 预测 ELISA 融合蛋白 CTL 

分 类 号：R392-33[医药卫生—免疫学]