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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 To screen the immune polypeptide of CDPK5 gene sequence of Toxoplasma gondii (Tg) and immunize New Zealand white rabbits with synthetic peptides to prepare multiple antiserum, and to identify its function. Methods Bioinformatics methods were used to determine the immunoglobulin G (Tg) CDPK5 sequence, and immunized New Zealand white rabbits with the synthesized peptides to prepare polyclonal antibodies. Multi-antiserum was collected, multiple anti-titers were detected by enzyme-linked immunosorbent assay (ELISA), immunological activity was identified by Western blot, and subcellular localization of Tg CDPK5 was analyzed by immunofluorescence. Results By bioinformatics analysis, a peptide sequence of 17bp at the N-terminus of Tg CDPK5 sequence was selected as the immune polypeptide. The rabbit polyclonal antibody was successfully immunized with the synthesized polypeptide. The titer of multi-antiserum was 1: 640 000 by ELISA. Western blot showed that the polyclonal antiserum specifically recognized the band of Tg CDPK5 (75.4 × 103). The result of immunofluorescence showed that this polyclonal antibody could specifically recognize Toxoplasma gondii Source Tg CDPK5 protein. Conclusions Based on the analysis of the sequence information of Tg CDPK5, the Tg CDPK5 sequence was obtained and the rabbit polyclonal antibody was prepared.