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目的:制备高灵敏度的抗人HPPCn单克隆抗体(mAb),以用于HPPCn的功能研究及其疾病相关性研究。方法:用重组蛋白免疫雌性BALB/c小鼠,采用常规杂交瘤技术进行细胞融合,经ELISA法进行阳性克隆筛选、通过有限稀释法进行亚克隆,获得稳定分泌抗人HPPCn mAb的细胞株。采用间接ELISA、Western blot、Ig亚类快速定性试纸分析法等鉴定抗体的生物学特性;通过细胞免疫荧光实验观察了HPPCn蛋白的细胞定位。通过噬菌体肽库技术分析抗体所识别的抗原表位。结果:得到了1株稳定分泌抗人HPPCn抗体的杂交瘤细胞株,命名为W2-D5。经Ig亚类分析确定,该细胞株分泌的抗体属于IgG1亚类。间接ELISA检测表明,该抗体检测HPPCn的极限为0.1μg/L;Western blot结果显示,该抗体能特异性识别HPPCn。细胞免疫荧光实验,证实了HPPCn蛋白定位在细胞核。通过肽库筛选及表位分析认为,该抗体识别的表位可能为HPPCn7-13(IHLELRN)。结论:成功地获得了抗人HPPCn的特异性mAb。
OBJECTIVE: To prepare high-sensitivity anti-human HPPCn monoclonal antibody (mAb) for the functional study of HPPCn and its disease-related research. Methods: Female BALB / c mice were immunized with recombinant protein. The cells were fused by conventional hybridoma technique. Positive clones were screened by ELISA and subcloned by limiting dilution method. The stable cell line secreting anti-human HPPCn mAb was obtained. The biological characteristics of the antibody were identified by indirect ELISA, Western blot and rapid qualitative analysis of Ig subclasses. The cellular localization of HPPCn protein was observed by immunofluorescence assay. The epitope recognized by the antibody was analyzed by phage peptide library technology. Results: A hybridoma cell line stably secreting anti-human HPPCn antibody was obtained and named W2-D5. The Ig subclass analysis confirmed that the antibodies secreted by the cell line belong to the IgGl subclass. Indirect ELISA showed that the limit of detection of HPPCn by this antibody was 0.1μg / L. The result of Western blot showed that this antibody could specifically recognize HPPCn. Cell immunofluorescence experiments confirmed that HPPCn protein localized in the nucleus. Peptide library screening and epitope analysis suggested that the epitope recognized by this antibody might be HPPCn7-13 (IHLELRN). Conclusion: The specific anti-human HPPCn mAb was successfully obtained.