单链抗体组织穿透力强，可用大肠杆菌快速、大量廉价制备，在疾病治疗方面具有巨大潜在应用价值。血浆半衰期短限制其在临床应用。唾液酸修饰N-糖基化蛋白能显著提高蛋白药物血浆半衰期和理化性质。目前，大肠杆菌体内尚无唾液酸修饰人源化N-糖基化机制。本课题拟通过重新整合不同原核生物寡糖合成机制，在大肠杆菌基因组上重建寡糖合成机制，实现体内高效唾液酸修饰人源化N-糖基化单链抗体，以期提高单链抗体理化性质及血浆半衰期。主要研究内容包括：1.以流感嗜血杆菌寡糖合成机制和空肠弯曲菌寡糖翻转酶和寡糖转移酶为基础，建立人源化N-糖基化机制；2.以空肠弯曲菌唾液酸合成酶和发光杆菌唾液酸转移酶为基础，在大肠杆菌基因组上构建高效唾液酸修饰人源化N-寡糖代谢途径；3.系统研究唾液酸修饰人源N-糖基化单链抗体理化性质及药代动力学，以期获得适合体内治疗的新型重组单链抗体。本研究成果也将为应用大肠杆菌生产糖蛋白药物开辟新途径。

ScFvs (Single-chain fragment variable) could facilitate a potentially unique molecule to be used especially in therapeutic application, as they are versatile and offer many advantages over current therapies using whole immunoglobulines.Because of its smaller size and lower molecular complexity,scFvs can not only be allowed to penetrate more rapidly and evenly to tissue in comparison to the whole antibodies, but also can be inexpensively produced in high yields in E.coli.However,it is limited for therapeutic application by the faster clearance rate through glomerular filtration and the degradation in serum,resulting short serum half-life.N-linked glycoprotein with sialylation modification can increase its biophysical and pharmacokinetic properties. But the machinery for sialylation of humanized N-glycosylated protein in E.coli is currently not avaialbe. The goal of this proposed project is to reconstitute a machinery via engineering E.coli genome DNA with the glycosylation machineries from different prokaryota for producing modified scFvs with sialylated humanized N-glycan at high efficiency, so that an new style scFv with favorable pharmacokinetic properties could be produced in E.coli.Followings are the contents:1.For high efficiency producing humnaized glycosylated therapeutic scFv, the glycosylation machineries from haemophilus influenzea and the transporter pglK ,oligosaccharyltransferase from C.jejuni will be employed as a base synthesis of humanized glycan to build up humanized N-glycosylation machinery in E.coli;2.For the sialylation of humanized N-glycosylated scFv production in E.coli, a research will carry out to engineer a metabolic pathway on genome DNA of E.coli for producing CMP-N-acetylneuraminic acid and α lipooligosaccharide sialyltransferase based on the pathway of C.jejuni CMP-N-acetylneuraminic acid synthetases and the 2,6-sialytransferase from photobacterium;3.The biophysical and pharmacokinetic properties of the sialylated humanized N-glycosylated scFv produced in E.coli will be investigated in vitro and in vivo.Then the sialylated humanized N-glycocylated scFv with suitable pharmacokinetic properties could be produced with the engineered E.coli. This will be also a promising way to prolong the half-life and thus to improve the pharmacokinetic and pharmacodynamic properties of therapeutic protein and will be applicable for the industry of producing thearpuetic N-glycoprotein.