【摘要】 目的:制备和初步鉴定人睾丸特异性新基因h TSC29(Gen Bank登录号:BC032957)合成肽的单克隆抗体(m Ab)。方法:利用生物信息学方法对h TSC29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h TSC29 m Ab的特异性。结果:获得了1株稳定分泌h TSC29 m Ab的杂交瘤细胞株,分泌的抗体属Ig G2b(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h TSC29 m Ab可特异性地识别人睾丸总蛋白中Mr约为60 000处的蛋白;免疫组织化学和免疫荧光结果显示h TSC29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h TSC29 m Ab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h TSC29的功能提供了特异的检测工具。更多还原

【Abstract】 Objective: To prepare monoclonal antibody( m Ab) against human testis-specific conserved gene( h TSC29) peptides and characterize its immunological and biological features. Methods: According to bioinformatics analysis and prediction of the antigenicity,surface property,hydrophilicity and flexibility of h TSC29,a 18-amino acid residue partial peptide of h TSC29 was synthesized,then immunized the BALB/c mice for preparing antiserum. The m Ab against h TSC29 was produced using the routine hybridoma technique. The properties of the m Ab against h TSC29 were identified by ELISA,Western blot and immunohistochemistry staining. Results: After cell fusion and subcloning,one hybridoma cell lines secreting specific m Ab against h TSC29 protein were obtaind. The Ig subclass of the m Ab was Ig G2b( κ). ELISA detection showed that the titer of m Abs in cultured was 1 ∶104. Western blot analysis proved that the m Ab could specifically recognize Mr 60 000 protein in human testis total protein. The h TSC29 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 m Ab against h TSC29 protein with high titers and specificity have been established successfully. The m Ab will provide efficient tools for functional studies of h TSC29 expressed in spermatogenesis.更多还原