人雌激素受体α真核表达载体构建及质粒免疫制备多克隆抗体

目的：克隆构建表达人雌激素受体仅（ERα）的真核表达质粒，并探讨通过质粒免疫的方法制备ERa抗体的可行性。方法：利用RT—PCR方法从MCF一7细胞中克隆出ERα基因，并将其构建在pCDNA3．1载体中，质粒经鉴定为正确后，大量提取重组质粒，利用脂质体转染试剂包裹，肌肉注射免疫BALB／c小鼠，通过测定血清抗ERα效价，观察重组质粒携带ER仪在小鼠体内的表达情况。结果：成功克隆构建了表达ERα的真核载体，细胞内瞬时转染实验证实重组质粒成功表达目的蛋白；通过采用该质粒免疫小鼠，获得了高效价的小鼠多克隆抗体，经验证抗体可识别内源性ERα。结论：采用真核表达质粒进行免疫制备ER仅抗体具有可行性，为进一步通过质粒免疫制备ERα单克隆抗体奠定基础。

Cloning and construction of eukaryotic expression plasmid of human estrogen receptor alpha and preparation of antibodies by DNA immunization

Objective： To clone and construct eukaryotic expression plasmid of human estrogen receptor alpha ERα, with which to investigate the possibility of preparation of ERα antibody by DNA immunization. Methods：The ERa gene was cloned from MCF-7 cells by RT-PCR. The ERa gene was then cloned into plasmid pCDNA3.1. The verified plasmid pCMV-ERc~ was mixed with lipofectamine 2000 and intramuscularly injected into mice. The expression of ERc~ in vivo was examined through measuring the serum titers by ELISA. Results： The eukaryotic expression plasmid pCMV-ERc~ was successfully constructed. 293T cells transiently transfected with the recombin- ant plasmid expressed the target protein successfully. Mice immunized with the plasmid produced polyclonal antibodies of high titers. And the polyclonal antibody was proven to recognize endogenous ERcL by Western blotting. Conclusion： It is feasible to prepare antibodies to ERa by DNA immunization. These provide the foundation to further prepare monoclonal antibodies to ERα by plasmid immunization.