肺炎支原体重组P1蛋白抗体研究

Preparation of specific antibodies against the recombinant P1 protein of Mycoplasma pneumoniae

目的研制针对肺炎支原体(Mycoplasma pneumoniae,Mp)主要粘附蛋白-P1蛋白的特异性抗体。方法用纯化后重组P1蛋白免疫BALB/c小鼠,获得多克隆抗体(Polyclonal Antibody,PcAb),通过传统的杂交瘤技术制备抗P1重组蛋白单克隆抗体(Monoclonal Antibody,McAb),采用ELISA和IFA法对单抗和多抗进行反应性和特异性检测。结果ELISA法检测P1重组蛋白抗原性显示,P1蛋白与Mp抗血清有较好的反应性,蛋白敏感性大于1ng/ml;在重组P1蛋白免疫小鼠获得的PcAb与Mp的反应中,检测到的抗体滴度可达到1∶1600;获得针对Mp重组P1蛋白的特异性McAbs2株,在ELISA检测中均与P1抗原有较好的反应特异性,抗体滴度可达到1∶800～1600;IFA结果显示,上述PcAb和McAbs均与Mp有较好反应性,与生殖支原体等无交叉反应。结论本研究选取的重组P1蛋白具有较好的免疫原性;其特异性PcAb和McAbs与Mp有较好的特异性,为临床Mp感染的抗原快速、早期诊断方法的建立打下了基础。

To prepare specific antibodies against P1 protein of Mycoplasmas pneumoniae （Mp）for clinical diagnosis and correlative research. Based on the purification of protein, the antigens were emulsified with FCA or FIA and injected to BALB/ c mice to obtain polyclonal antiserum against P1 protein. The McAbs obtained from the traditional hybridization technique were selected with recombinant P1 ELISA, and the specificity and sensitivity of the PcAb and McAbs were identified by ELISA and IFA. Result indicated the sensitivity of recombinant protein detected by ELISA was more than lng/ml. The titer of P1 antibody （in mouse serum） identified by Mp-ELISA reached 1 ： 1 600. And we obtained two groups of specific and stable McAbs against P1 protein of M. pneumoniae. ELISA analysis showed two McAbs had high affinity and specificity with recombinant P1 protein（5μg/ml） ,the titer of IgG reached 1 ： 800-1 ： 1 600. The PcAb and McAbs showed better affinity and specificity with Mp in IFA. In conclusion, the recombinant express protein show high immunogenicity and immunoreactivity. The anti-Mp P1 PcAb and McAbs has high specificity with Mp. The recombinant Plprotein and the McAbs obtained from this study might be bases for the early clinical detection of Mp infection.