蓝氏贾第鞭毛虫PPDK多肽抗体制备与定位研究  

Preparation of a polypeptide antibody against PPDK and cellular localization of PPDK in Giardia

作　　者：冯宪敏[1] 张宏梅[1] 李瑶[1] 鞠晓红[1] 卢思奇[2] 

机构地区：[1]吉林医药学院病原学教研室,吉林吉林132013 [2]首都医科大学病寄生虫学教研室,北京100069

出　　处：《中国病原生物学杂志》2015年第2期144-147,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81301450);吉林省科技厅国际合作项目(No.20130413035GH);吉林省教育厅项目(No.2014367)

摘　　要：目的制备蓝氏贾第鞭毛虫(简称贾第虫)磷酸烯醇式丙酮酸双激酶(PPDK)特异性多肽抗体并进行PPDK定位。方法采用DNAstar软件和BIOSUN生物医学软件,结合抗原表位分析的基本原理,对贾第虫PPDK的氨基酸序列进行抗原分析,最终确定贾第虫PPDK的优势抗原表位肽。将上述抗原表位肽与KLH进行偶联,获得3个偶联蛋白,经脱盐柱吸附、洗脱纯化后,对抗原表位肽-KLH偶联蛋白进行定量测定。将得到的3条多肽-KLH偶联物混合,用PBS稀释为1mg/ml,分别与第1、15、29和43d免疫4只新西兰白兔[于颈背部皮下多点(至少8点)注射多肽抗原2mg],获得抗PPDK抗血清,采用酶联免疫吸附试验(ELISA)和Western blot试验检测抗体的滴度和特异性。以R3为一抗,以DyLight 649抗兔IgG为二抗,采用免疫荧光法进行PPDK的细胞定位。结果根据PPDK抗原分析结果,最终确定PPDK的优势抗原表位3个,即p1:TPENQPANSELC(氨基酸位点12-23),p2:KTRYGRKTDPELC(氨基酸位点167-178)和p3:DLQKKLAEDMNKKHC(氨基酸位点641-654)。与KLH偶联获得3个多肽偶联蛋白,即P1、P2和P3。3个偶联蛋白联合免疫新西兰白兔获得4份抗PPDK抗血清,纯化后的抗体分别命名为R1、R2、R3和R4。Western blot检测显示,R3和R4可与PPDK特异性结合,无交叉反应。以R3为一抗进行免疫荧光试验,结果显示PPDK主要定位于贾第虫细胞的胞浆内。结论本研究获得2个可与贾第虫PPDK特异性结合的抗体,并初步判断PPDK主要分布于贾第虫细胞胞浆,为PPDK功能研究奠定了基础。Objectives To prepare a polypeptide antibody against PPDK fromGiardia lamblia and to study the location of PPDK in G.lamblia. Methods An antigenic analysis of PPDK fromG.lamblia was performed with DNAStar software and BIOSUN biomedical software.In conjunction with the basic principles of epitope analysis,the dominant epitopes of PPDK fromG.lamblia were ultimately determined.Based on that analysis,the dominant epitopes were synthesized and conjugated to keyhole limpet hemocyanin（KLH）to obtain 3coupled proteins.Four New Zealand white rabbits were subcutaneously immunized in the back of the neck with a 2-mg mixture of the 3peptides coupled to KLH on days 1,15,29,and 43 to obtain specific antisera.These candidate antisera were allowed to react with recombinant PPDK protein;the antibody titer was determined using ELISA and the specificity of the antiserum was determined using Western blotting.The location of PPDK in G.lamblia was detected by immunofluorescence using the R3 as the primary antibody and goat anti-rabbit IgG conjugated with DyLight 649 as the secondary antibody. Results The immunodominant epitopes of PPDK fromG.lamblia were predicted using several bioinformatic approaches,resulting in the identification of three candidate antigens, p1（TPENQPANSELC, 12-23 aa）, p2（KTRYGRKTDPELC, 167-178 aa） and p3（DLQKKLAEDMNKKHC,641-654aa）.Four antibodies,designated R1,R2,R3,and R4,were allowed to react with recombinant PPDK protein in Western blotting.All four antisera recognized a 97.8kd band.R1 and R 2cross-reacted with 76-95 kd proteins and R3 and R4were specific to the 97.8kd proteins.PPDK was mainly localized in the cytoplasm of G.lamblia. Conclusion This study obtained peptide antibodies against PPDK fromG.lambliaand this study revealed that PPDK was mainly located in the cytoplasm of G.lamblia.This study has laid the foundation for further study of PPDK.

关 键 词：蓝氏贾第鞭毛虫 磷酸烯醇式丙酮酸双激酶 多肽抗体 

分 类 号：R382.2[医药卫生—医学寄生虫学]