[抗人睾丸发育相关基因1单克隆抗体的制备与鉴定]
[Preparation and identification of monoclonal antibody against human testis development related gene 1].
作者信息
Wen Jiaming, Jiang Xianzhen, Tang Yuxin, Yang Jianfu, Chen Houyang, Liu Zhizhong
机构信息
Department of Andrology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
出版信息
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2010 Mar;35(3):230-5. doi: 10.3969/j.issn.1672-7347.2010.03.007.
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 x 10(6), and the subtype of the mAb was IgG(1). 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.
目的
构建表达睾丸发育相关基因1（TDRG1）重组蛋白的原核质粒，通过免疫小鼠获得抗TDRG1单克隆抗体（mAb），并鉴定该mAb的生物学特性。
方法
采用逆转录-聚合酶链反应（RT-PCR）从正常人睾丸组织中扩增TDRG1的编码序列，克隆至载体pET21，然后在大肠杆菌BL21（DE3）中表达，获得TDRG1重组蛋白。用纯化的TDRG1重组蛋白免疫BALB/C小鼠以制备抗TDRG1 mAb。收集免疫小鼠的脾细胞，与小鼠骨髓瘤细胞系Sp2/0细胞进行融合。对分泌抗TDRG1 mAb的杂交瘤细胞进行有限稀释法亚克隆。采用酶联免疫吸附测定（ELISA）评估mAb的效价和亚型。采用蛋白质印迹法（Western blot）和免疫组织化学法检测mAb的特异性。
结果
构建了表达重组蛋白的原核质粒，表达并纯化了TDRG1重组蛋白。获得了两株分泌抗TDRG1 mAb的杂交瘤细胞系。腹水mAb效价为1∶1.6×10⁶，mAb亚型为IgG₁。蛋白质印迹法和免疫组织化学分析表明，该mAb与人睾丸组织中的TDRG1蛋白呈特异性结合。
结论
TDRG1重组蛋白高度纯化，具有较强的抗原性。成功制备了抗TDRG1 mAb。
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