猪促卵泡激素β亚基基因克隆及表达分析-cloning and expression analysis of porcine follicle stimulating hormone β subunit gene.docxVIP

摘要 促卵泡激素 CFollicle Stimulating hormone ，郎的是动物垂体前叶分泌 的一种促性腺激素，属于糖蛋白激素家族。促卵泡激素在垂体中含量最少，最不 稳定，但却具有极其重要的生理功能。 促卵泡激素出 α 、自两个糖基化亚基组成。一般认为 α 亚基具有负责信弓- 转录的作用?而 6 亚基是功能亚基?参与受体结合 p 激素生理作用的特异性是 由￡亚基所决定的e 本论文是国家自然科学基金项目飞猪促卵泡激素基因表达及调控研究巾的 部分内容，主要工作是从猪脑垂体中提取总 R!!A 后，利用 Smart cDr\A 试剂盒合 成猪促卵泡激素已亚基 cD A，去除自身的信号肤序列后，将其克隆于质粒pPIC9K 中，电转化毕赤酵母， Southern-blot 检测结果表明白亚恭呆囚已经整合主 IJ 酵 母染色体上;工程菌在发酵过程中用甲醇进行诱导表达，将上清浓进行 SDS-PACE 及 Western-blot 分析，结果表明构建的工程菌能够产生有免疫学活性的猪促卵 泡激素自亚基。 国内利用生物技术表达猪促卵泡激素的研究只有我所在进行，并且在以前的 工作中己取得了一定突破，本项目为使猪促卵泡激素的工业化生产奠定基础。 关键词:猪促卵泡激素已亚基， Pichia Pastoris 甲醇酵母，基肉表达 ABSTRCT Follicle stimulating hormone(FSH) belongs to the famiIy of glycoprotein hormones secreted by the anterior pituitary. Though the content of FSH ín pituitary is least and the structure is most unstable，it has very important physiology function. FSH is a heterodimer composed of anαsubunit ，which is in charge oftranscript of signal，and a ? subunit，which is the functional subunit and can integrate with the receptor. The recol1ibinant FSH(rFSI匀，which was produced by gene engineered technique，is total1y as same as the nature FSH. Thís thesis ís a part of the National Nature Science Fund item ‘ Study of Gene Expression and Regulate of Porce FSH. Firstly，the total RNA was extracted fronl porce pituitary. Then the pFSH ? subunit cDNA was synthesis hy the Smart cDNA synthesis kit. The cDNA was then cloned in the chromosome of Pichia Pastoris， which was tested by the Southern-blot. The recombinant was induced to express pFSH ? subunit protein ，which was tested by SDS and Westem-blo t. 1t showed that the recombinant can secrete the ? subunit which had immunological activity. This technique was reported only by our institution in China. Our study will make the production of rpFSH possible. Keywords: pFSH ? subunit ，Pichia Pastoris ，gene expression 南京工业大学，崎士学位 it 文 第 1章 :文献综述 1.1 促卵泡激素研究进展 1..1.1 促卵泡激素 (Follic Stimulating hormone ，FSH) 简介 {足卵j包激 J亲是动物业{斗 I)UII-}- :分泌的斗中侃 fiiH1: 激素=在 l