人睾丸特异表达基因TDRG1单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against human testis development related gene 1

目的:构建原核质粒表达TDRG1重组蛋白,制备其单克隆抗体(mAb)并鉴定其生物学特性。方法:用逆转录聚合酶链反应法从成人睾丸组织中扩增TDRG1编码序列,将其克隆到pET21原核表达载体,在大肠杆菌BL21(DE3)内进行诱导表达TDRG1重组蛋白。以纯化后的重组蛋白免疫BALB/C小鼠,利用杂交瘤细胞融合技术制备单克隆抗体(mAb)。ELISA法检测杂交瘤细胞分泌mAb的效价和类型,Western印迹和免疫组织化学染色鉴定mAb的特异性。结果:成功构建了pET21/TDRG1原核表达质粒并获得纯度较高的重组蛋白。筛选出2株能稳定分泌抗TDRG1的单克隆抗体杂交瘤细胞系,其效价比高达1∶1.6×106,免疫球蛋白类型均为IgG1类。Western印迹和免疫组织化学染色显示该mAb能特异结合成人睾丸组织中的TDRG1蛋白。结论:所获得的重组TDRG1蛋白纯度高,免疫抗原性强,并成功制备了抗TDRG1的单克隆抗体。

Objective To construct a prokaryotic plasmid to express the testis development related gene 1（TDRG1）recombinant protein and obtain anti-TDRG1 mAb by immunizing mice,and to identify the biological properties of the mAb.Methods The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21,and then was expressed in the E.coli BL 21（DE3）to get TDRG1 recombinant protein.The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb.Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells.The hybridoma cells that secreted anti-TDRG1 mAb were subcloned with limited dilution.Enzyme-linked immunosorbent assay（ELISA）was used to evaluate titers and subtypes of mAb.Western blot and immunohistochemistry were used to detect specificity of mAb.Results The prokaryotic plasmid expressing the recombinant protein was constructed,and the TDRG1 recombinant protein was expressed and purified.Two hybridoma cell lines that secreted anti-TDRG1 mAb were obtained.The titer of the mAb in ascites was 1∶1.6×106,and the subtype of the mAb was IgG1.Westem blot and immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes.Conclusion The TDRG1 recombinant protein is highly purified and has strong antigenicity.The anti-TDRG1 mAb is produced successfully.