Isolation and Chaacterization of Human Inhibin in Follicular Fluid and Production of recombinant Inhibin.

免疫亲和层析是目前分离纯化卵泡液中抑制素最有效、最成功的方法。因此，我们尝试用改进的方法大规模纯化32-KDa牛抑制素的多克隆抗体免疫亲和层析。用Matrex凝胶红A、免疫亲和层析、凝胶层析和反相HPLC等层析从10 L人卵泡液中纯化了人抑制素。在非还原条件下的SDS-PAGE上，生物活性抑制素蛋白有两条迁移带，分子量分别为34,000和29,000，免疫反应性抑制素蛋白也有两条带，其迁移量分别为30,000和26,000。将其连接到表达载体上，转入中国仓鼠细胞(CHO细胞)。单独转染BETAB亚单位的细胞和同时含有分泌BETAB二聚体和抑制素B及BETAB同源二聚体的细胞均可激发其对大鼠垂体细胞分泌FSH的生物学作用。

Immunoaffinity chromatography is by far the most useful and successfully applied method for purification of the inhibin in follicular fluid. Therefore, we tried to develop the immunoaffinity chromatography with polyclonal antibody to 32-KDa bovine inhibin purified by the improved methods on large scale. Human inhibins were purified from 10 liter human follicular fluid by various kinds of chromatography including Matrex gel red A, immunoaffinity chromatography, gel chromatography and revesed-phase HPLC.On SDS-PAGE under nonreducing conditions, bioactive inhibin protein had two band migrating at MW 34,000 and 29,000 and similarly, immunoreactive inhibin protein had two bands migrating at MW 30,000 and 26,000, respectively.cDNA clone encoding human inhibin alpha-and betaB subunits were isolated from a human testicular cDNA library. The cDNAs were linked to an expression vector and transfected into Chinease hamsteer cells (CHO cells). The cells transfected with the betaB subunit cDNA alone and with both the alpha and betaB subunit cDNAs secreted betaB dimer and inhibin B and betaB homodimer could evoke their biological actions on the FSH secretion from rat pituitary cultured cells.