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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。
Objective To construct prokaryotic expression vector of human testis-specific gene hT279 (GenBank accession number BC016750), purify the fusion protein and immunize Balb / c mice with it as antigen to prepare anti-human testis-specific hT279 monoclonal antibody (mAb) Characterization. Methods The testis-specific new gene hT279 was obtained by PCR and cloned into pET32a prokaryotic expression vector. The fusion protein was expressed in E. coli BL21. The soluble protein was purified by affinity chromatography and identified by SDS-PAGE. Balb / c The anti-hT279 mAb was prepared by lymphocyte hybridoma technique and the characteristics of mAb were identified by indirect ELISA, Western blot and immunohistochemistry. Results The prokaryotic expression of hT279 was achieved and a hybridoma cell line 4B2 stably secreting anti-hT279 mAb was obtained. The antibody subtype was IgG2b (κ) and its titer reached 1 × 104. Western blot analysis showed that this mAb detected a specific band at a molecular weight of about 22 000 in human normal testicular protein. Immunohistochemistry showed that hT279 protein was mainly expressed in normal human testicular spermatocytes and round spermatids, but not in testicular tissue of male infertility patients. Conclusion A mAb 4B2 against hT279 was successfully prepared, which provided a specific tool for the further study on the function of hT279 in the diagnosis of human spermatogenesis and male infertility.