人精子蛋白17单克隆抗体的制备及特性鉴定

Preparation and characterization of the monoclonal antibody against human sp-erm protein 17

目的:制备抗精子蛋白17(Sp17)的单克隆抗体(mAb)并鉴定其特性。方法:克隆人Sp17cDNA,表达带有6His标记的重组Sp17,用纯化的重组Sp17免疫BALB/c小鼠制备mAb。用ELISA筛选抗体阳性的细胞克隆。用免疫组化染色法及阻断试验鉴定mAb的特异性。结果:获得2株杂交瘤细胞系3C12和3D6,其分泌的mAb的Ig亚类(型)分别为IgG1和IgM(κ),杂交瘤细胞培养上清的ELISA效价分别为1∶64和1∶32;腹水mAb的效价分别为1∶1×105和1∶5×104。用人和大鼠睾丸组织以及人精液精子免疫组化染色及阻断试验证明,抗Sp17mAb具有良好的特异性。抗Sp17mAb也可识别卵巢癌组织中异常表达的Sp17。结论:成功地制备特异性的抗Sp17mAb,为研究该蛋白的功能、天然分布及异常表达奠定了基础。

AIM： To prepare and characterize the monoclonal antibody （mAb） against sperm protein 17 （Spl7）. METHODS： Human Spl7 cDNA was cloned and recombinant Spl7 protein was expressed as a fusion protein with an N-terminal 6-His tag. The purified recombinant Spl7 protein was used to immunize BALB/c mice for preparing mAb. mAb-produced hybridoma cells were screened by ELISA and the specificity of mAb was analyzed by immunohistochemical staining and blocking test. RESULTS： Two hybridoma cells （3C12 and 3D6） secreting mAb against Spl7 were developed, The isotypes of these two mAbs, 3C12 and 3D6, were IgGt and IgM, respectively. ELISA detection showed that titers of mAbs, 3C12 and 3D6, were 1： 64, 1：32 in cultured supernatant and 1 ： 1×10^5 , 1 ： 5×10^4 in ascites, respectively. The results of immunohistochemical staining and blocking test indicated that 2 mAb specifically bound to Spl7 in human and rat testis and human ejaculated spermatozoa. Anti-Spl7 mAb also detected Spl7 in ovarian cancer. CONCLUSION： The successful preparation of anti-Spl7 mAb will be useful for assessing the native distribution and aberrant expression of Spl7 protein.