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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。
Objective To prepare PPDK specific antibody against Giardia lamblia (Giardia lamblia) and locate its PPDK. Methods DNAstar software and BIOSUN biomedical software were used to analyze the antigenicity of the amino acid sequence of PPDK in Giardia so as to determine the dominant epitope of PPDK in Giardia. The antigen epitope peptide was coupled with KLH to obtain three coupled proteins, which were adsorbed on desalting column and purified by elution. The epitope peptide-KLH coupled protein was then quantified. The three polypeptide-KLH conjugates obtained were mixed and diluted to 1 mg / ml with PBS. Four New Zealand white rabbits were immunized with 1, 15, 29 and 43 d respectively [multiple subcutaneous injections (at least 8 o’clock) Peptide antigen 2mg], obtained anti-PPDK antiserum, the use of enzyme-linked immunosorbent assay (ELISA) and Western blot test antibody titer and specificity. Using R3 as primary antibody and DyLight 649 anti-rabbit IgG as secondary antibody, immunofluorescence assay was used to locate the PPDK cells. Results According to the results of PPDK antigen analysis, 3 dominant epitopes of PPDK were finally identified, namely p1: TPENQPANSELC (amino acid positions 12-23), p2: KTRYGRKTDPELC (amino acid positions 167-178) and p3: DLQKKLAEDMNKKHC 641-654). Four anti-PPDK antisera were obtained by conjugating KLH with three peptide-conjugated proteins, namely P1, P2 and P3, and New Zealand white rabbits. The purified antibodies were named as R1, R2, R3 and R4. Western blot showed that R3 and R4 could specifically bind PPDK without cross-reaction. With R3 as the primary antibody immunofluorescence assay, the results showed that PPDK mainly located in the cytoplasm of Giardia cells. Conclusions Two antibodies specifically binding to Giardia PPDK were obtained in this study. The results showed that PPDK was mainly located in the cytoplasm of Giardia so as to lay the foundation for the study of PPDK function.