Study on generation of glycoprotein by recombinant technology.

众所周知，糖蛋白的低聚糖有助于蛋白质的生理特性，包括亲水性，电荷性，抗下垂性以及pH和温度稳定性。本研究旨在利用基因技术、生物化学和有机合成等技术，建立具有生物活性的糖蛋白生产体系。采用化学酶固相合成技术合成了具有重要生物学意义的携带傻Lex抗原的肽片段。此外，我们还合成了o -糖硅化片段（唾液糖蛋白，糖蛋白A）， n -连接的核心五糖，以及与细胞表面蛋白聚糖的糖-蛋白连接区域对应的糖肽。我们利用哺乳动物细胞成功制备了高糖硅化重组马绒毛膜促性腺激素（eCG）、大鼠胎盘乳原和大鼠白血病抑制因子（LIF）。有趣的是，eCG由高度糖基化的a-和b亚基组成，其引发促卵泡激素（FSH）和黄体生成素（LH）的反应特征。我们发现eCG的双重活性，lh样活性和fsh样活性，可以通过去除a亚基Asn 56上的n -连接寡糖或羧基末端肽相关的o -连接寡糖来分离。此外，我们还建立了杆状病毒/昆虫细胞系统来生产糖化蛋白，包括人丙型肝炎病毒包膜糖蛋白和人卵泡刺激素。

It is known that the oligosaccharides of glycoproteins contributes to the physiological characteristics of the protein, including hydrophilicity, electric charge, resistance to ptotease, and pH and temperature stability. This study was designed to establish the production system of glycoproteins bearing biological activity by using the techniques including gene technology, biochemistry and organic synthesis. We have synthesized biologically important peptide fragment carrying silly Lex antigen by employing chemoenzymatic solid-phase synthesis. In addition, we synthesized O-glycosilated fragments (sialoglycoprotein, glycophorin A), N-linked core pentasaccharide, and glycopeptides corresponding to carbohydrate-protein linkage region of cell-surface proteoglycans. We have succeeded to produce highly glycosilated recombinant Equine chorionic gonadotropin (eCG), rat placental lactogen, and rat leukemia inhibitory factor (LIF) by using mammalian cells. Interestingly, eCG consists of highly glycosilated a- and b-subunits, and its elicits response characteristic of both follicle stimulating hormone (FSH) and luteinizing hormone (LH). We found that the dual activity of eCG,LH-like and FSH-like activities, could be separated by removal of the N-linked oligosaccharides on the a-subunit Asn 56 or carboxy-terminal peptide associated O-linked oligosaccharides. In addition, we have established Baculovirus/insect cell system to produce glycosilated proteins including envelop glycoproteins of human hepatitis C virus and human FSH.