人睾丸特异性新基因hTSC29合成肽单克隆抗体制备与鉴定

摘要

目的：制备和初步鉴定人睾丸特异性新基因hTSC29（ GenBank 登录号： BC032957）合成肽的单克隆抗体（ mAb）。方法：利用生物信息学方法对hTSC29的蛋白质序列进行分析和预测，根据免疫原性、表露性、亲疏水性及柔韧性4个参数，合成一段由18个氨基酸组成的多肽，并以此为抗原免疫BALB／c小鼠，取其脾细胞和小鼠骨髓瘤细胞融合后，通过有限稀释法筛选阳性克隆，采用ELISA法、Western blot、免疫组化和免疫荧光法鉴定hTSC29 mAb的特异性。结果：获得了1株稳定分泌hTSC29 mAb的杂交瘤细胞株，分泌的抗体属IgG2b（κ）亚型，杂交瘤细胞培养上清效价达到1∶104；Western blot鉴定表明，制备hTSC29 mAb可特异性地识别人睾丸总蛋白中Mr约为60000处的蛋白；免疫组织化学和免疫荧光结果显示hTSC29蛋白主要定位在正常人睾丸组织中的精母细胞和精子细胞。结论：成功建立了1株稳定分泌抗hTSC29 mAb的杂交瘤细胞株，其分泌的抗体效价高、特异性强，为进一步研究hTSC29的功能提供了特异的检测工具。%Objective:To prepare monoclonal antibody ( mAb ) against human testis-specific conserved gene ( hTSC29 ) peptides and characterize its immunological and biological features.Methods:According to bioinformatics analysis and prediction of the antigenicity, surface property, hydrophilicity and flexibility of hTSC29, a 18-amino acid residue partial peptide of hTSC29 was synthesized,then immunized the BALB/c mice for preparing antiserum.The mAb against hTSC29 was produced using the routine hybridoma technique.The properties of the mAb against hTSC29 were identified by ELISA, Western blot and immunohistochemistry staining.Results:After cell fusion and subcloning, one hybridoma cell lines secreting specific mAb against hTSC29 protein were obtaind.The Ig subclass of the mAb was IgG2b(κ).ELISA detection showed that the titer of mAbs in cultured was 1∶104.Western blot analysis proved that the mAb could specifically recognize Mr 60 000 protein in human testis total protein.The hTSC29 protein main located at circumference of spermatocyte and spermatid in human testis tissue by immunohistochemistry staining and immunofluorescence assay.Conclusion:One hybridoma cell lines which can secrete specific mAb against hTSC29 protein with high titers and specificity have been established successfully.The mAb will provide efficient tools for functional studies of hTSC29 expressed in spermatogenesis.