摘要
目的利用纯化的兔抗重组生殖支原体黏附素蛋白（rMgPa）的多克隆抗体（pAb）从噬菌体展示随机12肽库筛选MgPa的模拟表位。方法用纯化的兔抗rMgPa的pAb对噬菌体随机12肽库进行生物淘洗，随机挑取噬菌体克隆进行DNA测序与分析，并用MIMOX对噬菌体克隆所展示的肽序列进行生物信息学分析。用ELISA、竞争性结合试验和Westernblot检测噬菌体与pAb结合的特异性。结果4轮生物淘洗后特异性噬菌体克隆得到了明显的富集。依据氨基酸组成的不同，74个噬菌体克隆所展示的肽序列可大致分为3组。经对肽序列进行比较分析以及用MIMOX进行生物信息学分析，结果表明这3组的核心序列分别为：P—S-A-A／V—X—R—F／w—E／S．L—S—P、A—K—L／L-T／Q-X—T—L—X—L和K-S-L-S—R—X—D—X-I。ELISA、竞争性结合试验和Westernblot方法检测的结果表明噬菌体能与pAb发生特异性结合，说明其可能足MgPa的模拟表位。结论成功筛选到MgPa的3个可能的模拟表位，为研制基于Mg抗原表位的诊断试剂与多肽疫苗奠定了一定的实验基础。
Objective To screen a 12-mer phage display peptide library by the polyclonal antibod- y （pAb） against the recombinant adhesion protein of Mycoplasma genitalium （rMgPa） in order to obtain the antigenic mimic epitopes of MgPa. Methods The purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library. Seventy-four recombinant phage clones were randomly selected, and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX. The binding speci- ficities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA, competitive ELISA and Western blot analysis. Results After four rounds of biopanning, a significant enrichment of phages was achieved, the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences. Amongst 45 peptides, 36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa, lmmunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M. genitalium, P-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X- D-X-I. Results of bioinformatics analysis by MIMOX demonstrated that S, A, F for cluster 1, A, K, I, T and L for cluster 2, K, S, L, R, D and ] for cluster 3, may be the key consensus amino acid residues in the aligned mimotopes, respectively. Conclusion Antigenic mimics on MgPa were successfully identified and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent the immunodominant mimic epitopes of MgPa. And S, A, F K, I, T, L, R and D may be the key amino acid residues for the epitopes of MgPa.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2012年第1期84-90,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金项目（30801064）
湖南省研究生科研创新项目（CX20108381）
湖南省高校科技创新团队（湘教通2010-212号）
关键词
生殖支原体
黏附蛋白
噬菌体展示肽库
模拟表位
Mycoplasrna genitalium
Adhesin protein
Phage display random peptide library
Mimic epitope
