Optimization Of Expression And Screening Of The RhFSH Engineering Cell Strain

Human follicle stimulating hormone (hFSH) is a glycoprotein hormone of synthesized and secreted from the anterior lope of the pituitary gland, comprising non-covalently attached a andβsubunits. The a subunit 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 the a subunit(Asn52 and 78 ) and two on theβsubunit(Asn 7 and 24 ).Reproductive function in both female and male mammals, including humans, is regulated by FSH. In females, FSH stimulates the development of ovarian follicles carrying the oocyte, and in males it plays a role in spermatogenesis. The synthesis of FSH by gonadotroph cells takes place within the anterior pituitary gland, before secretion into the general circulation. The synthesis and secretion of FSH are regulated by gonadotrophin releasing hormone (GnRH), secreted by specialized neurones within the hypothalamus, and steroidal and nonsteroidal products secreted from the gonads. Through high-affinity binding to its respective receptor, FSH affects the function of specific target cells in the ovaries and testes and triggers intracellular mechanisms that regulate steroidogenesis, cell replication, and the expression of specific proteins and growth factors that control gametogenesis.Inadequate concentrations of FSH, caused either by deficient FSH synthesis or secretion, are a common cause of infertility in men and women. In women, this state is characterized by abnormal 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 used successfully to treat these fertility disorders. Previously, commercial preparations of FSH for therapeutic use were derived from a urinary source (u-hFSH from post-menopausal women). However, the development of recombinant DNA technology to produce recombinant human FSH (r-hFSH) has resulted in significant improvements in the quality and availability of this product for clinical use.In the research, based on gene recombination technique, the coded sequences of the human FSHαandβsubunits are inserted into a high-expression eukaryotic vector. The translated amino acid sequences from recombinant DNA sequences without introns are the same to the natural FSHαandβsubunits. The high-expression vectors of human FSHαandβsubunits are co-transfected into Chinese hamster ovary (CHO) cells to express activeαandβdimmers. A high-expression cell strain (CHO-F) can be obtained after a limiting dilution assay and an ELISA detection, which can express 2mg/L. At the end of optimization, the fitted temperature is 32℃, the suitable cultivate mode is shake-flask culture and the optimal culture medium compounding is EX-CELLTM 302 1:1 HyQ SFM4CHO^TM, in which the highest expression is 9mg/L. The purification strategy, including an immunoaffinity chromatography and a gel filtration chromatography (GFC), has been shown to produce a stock solution of rhFSH that is≥99% pure from the cultivate supernatant. The research establishes a satisfactory foundation to full scale operation in the future.