长效人促卵泡激素CHO细胞株的构建及其体内活性

Expression of human long-acting FSH in CHO cell and its bioactivity in vivo

摘要:

促卵泡激素 (FSH) 是具有促进卵泡与睾丸发育作用的一种垂体糖蛋白激素。因其体内半衰期较短，临床上需要连续10 d以上每天注射，病人使用非常不方便。本文旨在通过提高糖基化程度，研制一种长效FSH。通过一段含有两个N-糖基化位点的连接序列, 将人FSH α链与β链cDNA融合，并插入pcDNA3.1(+)表达载体。表达载体转染CHO-K1细胞后，通过G418筛选得到阳性单克隆细胞，并经PCR和Western blotting证实。该重组FSH为单链蛋白，分子量约为49 kDa。经无血清培养，工程细胞株培养上清液中重组FSH的表达量可达3 mg/L。单次注射该重组FSH能够促进大鼠卵巢发育与卵泡成熟，且药效与连续8次注射Folltropin-V的相近。实验结果显示，本研究已成功获得一种长效重组FSH。

Abstract:

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. The relatively short half-life of FSH in vivo requires daily injections for more than 10 days that is inconvenient and possibly contribute to the stress perceived by the patients. The goal of the present study was to increase FSH glycosylation, in order to develop a long-acting recombinant FSH. The cDNA of native α and β subunit of human FSH was linked by a sequence with two N-linked glycosylation sites, and the resulted DNA was inserted into pcDNA3.1 vector to generate a recombinant vector of pcDNA3.1-FSH. The pcDNA3.1-FSH was linearized and transfected into CHO-K1, positive transformants were selected by G418 and confirmed by PCR and Western blotting. A single chain recombinant FSH was expressed, with molecular weight of about 49 kDa. The recombinant FSH expression level in CHO-K1 cell strain in serum-free culture was 3 mg/L. Single injection of this recombinant FSH could induce folliculogenesis and ovulation in rats, the efficacy was similar with the commercially available FSH preparation (Folltropin-V) administrated 8 times consecutively. The results suggested a long-acting FSH was produced successfully.