人睾丸特异性新基因hT279单克隆抗体制备及其应用

Preparation and application of monoclonal antibody for human testis-specific conserved gene hT279

目的构建人睾丸特异性新基因hT279(GenBank登录号BC016750)的原核表达载体,纯化融合蛋白并以其为抗原免疫Balb/c小鼠制备抗人睾丸特异性hT279蛋白单克隆抗体(mAb)并进行特性鉴定。方法用PCR方法得到睾丸特异性新基因hT279并克隆至pET32a原核表达载体中,转化大肠杆菌BL21诱导融合蛋白表达;获得的可溶性蛋白经亲和层析纯化、SDS-PAGE鉴定后,免疫Balb/c小鼠,应用淋巴细胞杂交瘤技术制备抗hT279 mAb,并通过间接ELISA、Western blot和免疫组织化学法对mAb进行特性鉴定。结果实现了hT279的原核表达,获得了1株稳定分泌抗hT279 mAb的杂交瘤细胞株4B2,抗体亚型为IgG2b(κ),效价达到1×104。Western blot鉴定表明,该mAb在人正常睾丸蛋白中相对分子质量约为22 000处检测到特异条带。免疫组织化学显示hT279蛋白主要在正常人睾丸的精母细胞及圆形精子细胞中表达,而在男性不育患者睾丸组织中表达消失。结论成功制备出1株抗hT279的mAb 4B2,为进一步研究hT279在人类精子发生中的功能和男性不育症的诊断提供了特异的检测工具。

This study designed to construct expression vectors of human testis-specific conserved gene （hT279）, prepare monoclonal antibody （mAb） against hT279, and characterize the properties of the mAb. The hT279 gene was amplified by PCR; the target fragment digested by the enzyme was cloned into pET32a expression vector and then transformed into E. coli BL21 for fusion protein expression, which subsequently used as immunogen for preparing antiserum. The mAb against hT279 protein was produced using the routine hybridoma technique and identified by ELISA, Western blot and immunohistochemistry staining. Results showed that the relative molecular mass of the expressed product matched the predicted value. After cell fusion and subeloning, one hybridoma cell line secreting specific mAb against hT279 protein, designated 4B2, was obtained, and the Ig subclass of the mAb was IgG2b （K）. ELISA detection showed that the titer of the mAb in culture supernatant was 1 ~104; Western blot analysis proved that the mAb could specifically recognize a protein of 22 kDa （hT279） in human testis total protein. Immunohistochemistry staining indicated that the hT279 protein mainly expressed in spermatocytes and round spermatids, while no hT279 protein appeared in the testes of patients with male infertility. Together, hT279 proteinand its corresponding mAb has been prepared, which provides an efficient tool for the functional studies of hT279 expression in spermatogenesis and the diagnosis of male infertility.