人孕激素受体在大肠杆菌中的高效表达及应用

HIGH EXPRESSION OF HUMAN PROGESTERONE RECEPTOR IN E.coli AND ITS APPLICATION

为了制备抗重组人孕激素受体单克隆抗体，用聚合酶链反应（ＰＣＲ）扩增人孕激素受体氨基端编码区段，定位克隆连入高效表达载体ＰＭＳ－３１ｂ，构建重组质粒ＰＭＳ－ＰＲａ，将其转入大肠杆菌ＰＯＰ２１３６，经３０℃扩增和４２℃温度诱导，表达出分子量为３３ｋＤ的ＭＳ２－ＰＲａ融合蛋白。摇菌液的蛋白表达量约为３０ｍｇ／Ｌ。用这种蛋白免疫ＢＡＬＢ／ｃ小鼠，制备出４株抗人孕激素受体的单克隆抗体。它们用于乳腺癌石蜡和冰冻切片的免疫组织化学染色。

In order to prepare the monoclonal antibodies (mAbs) against human progesterone receptor(PR) using recombinant PR, the part coding fragment of PR N terminal domain was obtained by PCR method and ligated to the expression vector PMS 31b. Recombinant plasmid PMS PRa was then transfered into E.coli POP 2136 and the MS2 PRa fusion protein was expressed by amplifing bacteria at 30℃ and inducting protein expression at 42℃. The relative molecular weight of the fusion protein was calculated about 33kD and the average 30mg fusion protein can be recovered from 1L E.coli cell culture. Four mAbs were produced using spleen cells from BALB/c mice immunized with the fusion protein. The activities of these four mAbs were further tested on some frozen and paraffin sections of human breast cancer tissue, using immunohistochemical(IHC) method. The results showed that these four mAbs are substitute and even better than the improted ones as the IHC method is used for determination of the PR status of breast carcinoma.