弓形虫CDPK5基因多克隆抗血清的制备及功能鉴定

Preparation of the polyclonal antibodies of CDPK5 gene from toxoplasma gondii and the identification of its functions

目的筛选弓形虫(Tg)CDPK5基因序列的免疫多肽,将合成多肽免疫新西兰白兔制备多抗血清,并对其功能进行鉴定。方法利用生物信息学的方法分析确定Tg CDPK5序列免疫多肽,再用合成的多肽免疫新西兰白兔制备多抗。收集多抗血清,酶联免疫吸附测定(ELISA)测定多抗滴度,蛋白免疫印迹法(Western blot)鉴定免疫活性,免疫荧光实验分析Tg CDPK5的亚细胞定位。结果通过生物信息学分析,选择Tg CDPK5序列N端一段长17bp的多肽序列作为免疫多肽;用合成的多肽免疫兔子,成功获得多抗血清。ELISA测定多抗血清效价为1∶640 000;Western blot证明该多抗血清能特异性识别Tg CDPK5(75.4×103)条带;免疫荧光实验结果表明,该多抗能特异性识别弓形虫内源Tg CDPK5蛋白。结论研究根据Tg CDPK5序列信息的分析,获得Tg CDPK5序列免疫多肽,并制备兔源多克隆抗体。

Objective Screening the immune polypeptide sequence of toxoplasma （Tg） CDPK5 gene,which were synthesized and then immunized the New Zealand white rabbit to prepare antiserum,and identification its function. Methods Bioinformatics analysis was used to determine the immune peptide of Tg CDPK5 sequence, which were artificially synthesized to immune white rab- bit to prepare antiserum. The titers of antibodies were determined by ELISA and the polyclonal antibodies were verified with CD- KP5 antigen by Western blot. The sub-cellular localization of Tg CDPK5 were obtained by immunofluorescence assay. Results 17 bp peptide sequence from the Tg CDPK5 N-terminal were chosen as immune polypeptide by bioinformatics analysis. Synthetic peptide were used to immune rabbit to obtain polyclonal antiserum. The result showed that the titer of the obtained ployantibody were 1 = 640 000% Western blot demonstrated that the antiserum could specifically recognize Tg CDPKS（75.4 × 10a） ;Immunofluorescence assay revealed this antibody could specifically recognize the endogenous Tg CDPK5 of Toxoplasma gondii. Conclusion According to the analysis of Tg CDPK5 sequence information, this study successful obtained Tg CDPK5 polyclonal antibody.