The Preparation And Chacterization Of Arctium Lappa L. Oligosaccharide Liposome And Preliminary Study Of Human α-1, 6 Fucosyltransferease

The first part of our study is the preparation and characterization of Arctium lappa L. oligosaccharide liposome. It contains the establishment of a method to determine the content of Arctium lappa L. oligosaccharide, the process and prescription study of Arctium lappa L. oligosaccharide liposome preparation and the study of its characterization.Objective: To calculate the entrapment efficiency of Arctium lappa L. oligosaccharide liposome by the content determination of the free Arctium lappa L. oligosaccharide, optimize the process and prescription conditions of its preparation and study its physical and chemical properties and stability.Method: Establish a standard curve for glucose concentration determination. Determine the free Arctium lappa L. oligosaccharide content by the conversion factor between glucose and Arctium lappa L. oligosaccharide. Separate the free Arctium lappa L. oligosaccharide from liposome by moline ultrafiltration and make an feasibility analysis. Then discuss the superiority of rotary-film evaporation method and the reverse evaporation method. Optimize the process and prescription conditions, including the proportion of drug and lipid, chloroform and water, phospholipids and cholesterol and the high-pressure homogeneous conditions, mostly in accordance with entrapment efficiency by orthogonal experimental design. Finally analyze the major characteristics of liposomes, such as morphology, entrapment efficiency, zeta potential, size distribution and stability, as well as the study of its stability in different storage conditions.Result: The glucose determination standard curve can be set as y=12.4051x+0.01428(R=0.99962) with a linear range from 0.005 mg/ml to 0.15mg/ml. The conversion factor (f) is 1.28. The moline centrifugal tube with 30KD retention molecular weight can be used to separate free Arctium lappa L. oligosaccharide from liposome. The rotary-film evaporation method is more suitable for the preparation of Arctium lappa L. oligosaccharide liposome. The preparation conditions are optimized as:drug:lipid=1:6, chloroform: water=l:2, phospholipids: cholesterol=4:1. The best high-pressure homogeneous conditions are: pressure: 800bar with 3 circulations. The prepared Arctium lappa L.Oligosaccharide liposome is spherical capsule bubbles in uniform size, About 45.55% of initial Arctium lappa L.oligosaccharide was entrapped into liposomes, and the result of transmission electron micrograph showed that the treated Arctium lappa L.oligosaccharide liposome was evenly distributed small spherical vesicles, with a mean diameter of 75 nm and a zeta potential of-12.56mv after treating with high pressure homogenizer, and the liposome remained more stable at 4℃, which remains to be improved.Conclusion: We successfully prepared the Arctium lappa L.oligosaccharide liposome, optimized its process and prescription conditions and characterized its physical and chemical properties and stability. The obtained liposome is stable, uniform and has high entrapment efficiency; it could be used in cell culture and animal experiments and lays a foundation for its clinical and other future application.The second part of study is the preparation of soluble a 1,6-fucosyltransferase (a-1,6 FucT, FUT8) with three different expression methods and the preliminary study of anti-FUT8 polyclonal antibody.Objective:Express soluble FUT8 with three different expression methods for the future downstream glycoprotein synthesis and the study of enzyme catalysis and reaction mechanism. Prepare anti-FUT8 polyclonal antibody for the future development of cancer diagnosis serological reagent.Method: The cDNA of FUT8 was firstly amplified from human cancer cells, MKN45 lines. The pET28a-sFUT8 plasmid was constructed and transferred into Escherichia coli (E. coli) for mass expression. The obtained inclusion body protein was then purified, degenerated and renatured as antigen for anti-FUT8 polyclonal antibody preparation by four times immunization. The titer of prepared antibody was determined by ELISA. Pichia pastoris expression system and in vitro expression system were attempted to prepare solublea 1,6-fucosyltransferase as only the inclusion bodies can be obtained through prokaryotic expression. The FUT8 was cloned into pPIC9k plasmid, and the constructed recombinant plasmid was transferred into Pichia pastoris GS115 for eukaryotic expression of FUT8 by methanol induction. In addition, the pET28a-sFUT8 plasmid was used to be in vitro transcripted and translated for sFUT8 expression. The expression results were determined by SDS-PAGE and Western Blot.Result: The cDNA of FUT8 was successfully amplified from human cancer cells, MKN45 lines. The pET28a-sFUT8 prokaryotic plasmid was successfully constructed and FUT8 inclusion bodies were obtained by prokaryotic expression. The anti-FUT8 antibody with title from 1:5000 to 1:10000 were prepared, which lays foundation for the future development of cancer diagnosis serological reagent. Moreover, pPIC9k-FUT8 eukaryotic plasmid was successfully constructed and soluble FUT8 protein was expressed in Pichia pastoris. In addition, the in vitro expression of FUT8 with pET28a-sFUT8 was accomplished by SDS-PAGE and Western Blot determination.Conclusion: We successfully constructed pET28a-sFUT8 prokaryotic plasmid and pPIC9k-FUT8 eukaryotic plasmid, which then were transferred into E. coli and Pichia pastoris for expression in order to obtain FUT8 protein. The inclusion bodies were obtained by prokaryotic expression system while a little soluble FUT8 obtained by eukaryotic expression system and in vitro expression system. The obtained soluble FUT8 could be used in future downstream glycoprotein synthesis and the study of enzyme catalysis and reaction mechanism. Moreover, we make an initial study of anti-FUT8 polyclonal antibody, which would lay foundation for the future development of cancer diagnosis serological reagent.