【作者】 陈宁；

【导师】 金磊；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2007， 硕士

【摘要】 人促卵泡激素(human follicle stimulating hormone,hFSH)是垂体前叶嗜碱性细胞合成和分泌的一种糖蛋白类促性腺激素。在不孕不育的诊断和治疗中,FSH是发挥着重要作用的有生物活性的分子,在促使卵泡正常生长、成熟和性腺甾体类固醇的产生中不可缺少。对于女性,FSH的浓度对卵泡发育的启动和周期,及随后使卵泡达到成熟的时间和卵泡数目均至关重要。对男性来说,能与LH控制的睾丸素结合激活并保持正常精子的数目和质量。应用重组DNA技术生产的重组人FSH (rhFSH)具有标准化的分子量和生物学活性,避免了以前用于治疗的FSH由尿源提取在糖基结构上的差异,从而造成的FSH异构体生物活性上的差异。rhFSH还有优良的安全特性,这一点与尿源性的FSH相似,但却避免了尿源FSH给药时报道中一些病人可能出现的抗免疫反应。重组人促卵泡激素(rhFSH)为rhFSH基因转染到dhf-缺陷型的中国仓鼠卵巢细胞(CHO)后,经过细胞培养,分离和高度纯化后得到。本文就重组人促卵泡激素(rhFSH)的理化性质和生物学活性进行了较为全面系统的研究。SDS-PAGE及MALDI-TOF-MS测定分子量,Edman降解法分别测定两个亚基的N末端15个氨基酸残基的序列,同时结合供试品的紫外光谱图,等电点及免疫印迹对蛋白,肽图进行定性分析,从而达到对重组人促卵泡激素(rhFSH)的结构确证及定性分析的目的。纯度用HPLC和非还原SDS-PAGE两种方法测定,其纯度达到95%以上;利用SDS-PAGE(银染)检测聚合体,样品中没有发现聚合体;蛋白浓度用Lowry法和SEC-HPLC,结果能够较好的吻合;RP-HPLC测氧化亚基的含量,氧化亚基小于5%;在唾液酸的含量检测方面我们采取了药典酸水解UV法及RP-HPLC;参考药典测了外源性DNA的残留量,CHO细胞蛋白残留及鼠IgG残留,符合规定。采用Wister大鼠测量体内生物活性,我们对多批样品进行测定,rhFSH的生物比活在12453IU/mg,12855U/mg,13403IU/mg,比文献报道7700-11300IU/mg高。同时我们考察了不同pH值、温度对rhFSH活性的影响,发现在pH7.0,-20℃时最稳定。更多还原

【Abstract】 Human follicle stimulating hormone(hFSH) is a glycoprotein hormone of synthesized and secreted from the anterior lope of the pituitary gland. FSH is a heterodimeric glycoprotein comprising non-covalently attachedαandβsubunits. Theαsubunit contains 92 amino acid residues,with five disulphide bonds contributing to its tertiary structure. Theβsubunit contains 111 amino acid residues, with six disulphide bonds. Of the four Asn-linked glycosylation sites in FSH ,two are located on theαsubunit(Asn52 and 78 ) and two on theβsubunit(Asn 7 and 24 ).Inadequate concentrations of FSH,caused either by deficient FSH sythesis or secretion, are a common cause of infertility in men and women. In women ,this state is characterized by abnomal or the absence of ovulation. In men, it may lead to infertility because of the production of inadequate numbers of viable spermatozoa. Administration of FSH ,either alone or in combination with LH, has been successfully to treat these fertility disordes.The genes of the human FSHαandβsubunit are co-transfected in the DHR-deficient CHO cell line.The recombinant human FSH is highly purified from the cell culture supernatant .The dissertation mainly concentrates on its structure confirmation and identification, biological activity in vivo.First is about recombinant human follicle stimulating hormone structure confirmation and identification. The electrophoretic mobility in SDS-PAGE showed an apparent molecular weight of the rhFSH (estimated using molecular weight markers in the range of 14.4-97.6KDa),of ~23KDa.Glycoproteins are known to behave anomalously, probably because they bind SDS only to the protein part of the molecule.The reduced net charge resulting from reduced SDS binding lowers the polypeptide mobility during electrophoresis, yielding artefactually high molecular massestimates .Thus accurate determination of the molecular weights of the FSH was also performed using Matrix-Assisted Laser Desorption Ionization time of fly Mass Spectrometer(MALDI-TOF-MS). With this method , the r-hFSHαandβsubunits were found to have molecular weights of ~14.1 and ~17.2KDa, respectively, and the molecular weight of the rhFSH is ~31.3 KDa. The results are as same as the theoretical values.In order to get the N-terminal amino acid sequence of rhFSHαandβsubunits ,we use the RP-HPLC method to separate the subunits ,and then the Endman degradation to get the amino acid residues. Theαsubunit is A-P-D-V-Q-D-C-P-E-C-T-L-Q-E-N,βsubunit is N-S-C-E-L-T-X-I-T-I-A-I-E-K-E. A lack of readable (X of theβsubunit) signals at specific location in primary amino acid residue sequence confirmed that the Asn 7 on theβsubunit was glycosylated.The RP-HPLC method has been adopted in the separation of peptide fragments produced by V8 protease. We find that the rhFSH molecule must be deoxidized ,or the rhFSH can’t be digested by V8 protease.The results of isoelectric focusing ,Western Blotting and Ultraviolet spectrum are accord with the theoretical values.Second, rhFSH related substances and the purity were studied.SDS-PAGE,the size exclusion chromatography-high press liquid chromatography( SEC-HPLC ) and a rever-phase high pressure liquid chromatography (RP-HPLC) were employed to measure protein contaminants in the bulk material of rhFSH.The results shown that the rhFSH contain very low concentrations(<5%) of the degraded forms , also a little oxidated forms,only trace quantitaies of aggregated FSH .DNA contamination, IgG contamination and CHO host cell proteins were determined according to the Pharmacopoeia of the People’s Republic of China. The results were well below the accepted limit .To determination of sialic acid in rhFSH we establish a method .Sialic acid in rhFSH was released by weak acid hydrolysis and derivatised with the o-phenylenediamine. The derivatives were then separated by reversed-phase HPLC.The results of RP-HPLC wereclose to the result of the literature and results of the UV method (Pharmacopoeia of the People’s Republic of China).The potency of rh FSH was assessed by determining wister rat ovarian weight gain in response to FSH administration. The result was 12000~13000IU/mg protein,which was higher than that of literature.The bulk of the rhFSH is stored at pH 7.0 ,-20℃.更多还原