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目的制备兔抗重组生殖支原体粘附素蛋白(rMgPa)的多克隆抗体(pAb),以期筛选MgPa的模拟表位。方法用rMgPa免疫新西兰兔以制备兔抗rMgPa的pAb,以此pAb为靶分子对噬菌体展示随机12肽库进行生物淘洗,随机挑取74个噬菌体克隆进行DNA测序与分析,用ELISA检测噬菌体与pAb结合的特异性。结果制备了兔抗rMgPa的特异性pAb,以此抗体为靶分子对噬菌体展示肽库进行生物淘洗后,特异性噬菌体克隆得到了明显富集。经对74个噬菌体克隆所展示的外源性多肽序列进行比较分析,共可大致分为3组一致性的核心序列: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和K-S-L-S-R-X-D-X-I。ELISA结果说明36个噬菌体克隆能与pAb发生特异性结合,因此,这三组核心序列可能是MgPa的模拟表位。结论成功制备了兔抗rMg-Pa的pAb,并筛选到MgPa的3个可能的模拟表位,为进一步研究Mg的诊断与预防提供一定的实验依据。
Objective To prepare polyclonal antibody (pAb) against rabbit reproductive mycoplasma adhesin protein (rMgPa) in order to screen mimotopes of MgPa. Methods New Zealand rabbits were immunized with rMgPa to prepare pAb of rabbit anti-rMgPa. The pAb was used as the target molecule to bio-elute the random peptide library of phage display. 74 phage clones were randomly selected for DNA sequencing and analysis. The specificity of pAb binding. Results The specific pAb of rabbit anti-rMgPa was prepared and the specific phage clones were enriched after biopanning the phage displayed peptide library with this antibody as target molecule. After comparative analysis of the exogenous polypeptide sequences displayed by 74 phage clones, we can roughly classify into three groups of core sequences: 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. The results of ELISA showed that 36 phage clones could specifically bind to pAb. Therefore, the three core sequences may be MgPa mimotopes. Conclusion The pAb of rabbit anti-rMg-Pa was successfully prepared and three possible mimotopes of MgPa were screened, which provided some experimental evidences for further study on the diagnosis and prevention of Mg.