Studies On Synthesis Of Three Artificial Antigens Of Swainsonine And Their Immunogenicity

Locoweed is a kind of poisonous plants that endanger the grassland husbandry seriously all over the word. It distributes in western region of China, the United States, Russia, Australia and Canada extensively, and causes an enormous economic loss. To control locoism and utilize locoweed comprehensively, scholars research it through various ways. For the content of crude protein of locoweed is equal to alfalfa, we think locoweed has potential to be a good grass. According to the immunology, the locoweed toxin swainsonine (SW) is a hapten, could be synthesized to artificial antigen by the chemical method after coupling with the macromolecular carrier protein, and the artificial antigen can be made as a vaccine to induce the special antibody of animals, and make the animal acquire resistance after feeding locoweed. The main of this thesis is, at first, prepared swainsonine, and then coupled SW with three kinds of macromolecular carrier protein, ovalbumin (OVA), bovine serum albumin (BSA), human serum albumin (HSA) to get SW-OVA, SW-BSA, SW-HSA and vaccinated to mice. The results are as follows.1 Isolation and identification of swainsonine from A.Variabilis BungeA.Variabilis Bunge powder was extracted by hot ethanol at first. After recycling solvent, the residue was acidified with 1 mol/L HCl, and successively extracted several times with chloroform and n-butanol to get crude alkaloid. The crude alkaloid was mixed with silica gel and applied on a silica gel, and then eluted by eluting agent. Detected by TLC, finally the same parts of constituent were collected together for extracting by ammoniated chloroform. The principal in ammoniated chloroform was sublimated at boiling oil ( 100℃) and decompressed to obtain white needle crystal, which was identified as swainsonine by TLC, MP, IR,MS.2 Syntheses of SW-OVA, SW-BSA, SW-HSAAt first, quaternary ammonium salt was prepared by reaction between SW and active ester at 70℃, and then, SW-OVA, SW-BSA, SW-HSA was obtained by stirring between quaternary ammonium salt and OVA, BSA, HSA separately at condition of ice bath for 12 h, and dialyzeing and freeze drying the conjugate. SW-OVA, SW-BSA, SW-HSA was assayed by ultraviolet spectrophotometer to detect the conjugate ratio. The results of ultraviolet spectra assay showed that the conjugate ratio of SW-OVA, SW-BSA, SW-HSA were 13, 10 and 19 respectively. 3 The immune experiment of swainsonine-HSA on miceThirty-five kunming mice were randomly separated into group A, group B, group C, group D, group E, group F and group G, 5 mice per group. Mice in group A, group C, group E were immunized by SW-OVA, SW-BSA, SW-HSA separately in a high dose, and mice in group B, group D and group F were immunized by SW-OVA, SW-BSA, SW-HSA separately in a high dose in a low dose. And setting group G as blank. The first immunization, SW-OVA, SW-BSA, and SW-HSA was emulsified by Freund′s complete adjuvant and injected at two sides of transverse part, sub cutis of back and wall of hind legs. The dose of antigen was 0.01 mg per mouse separately on group A, group C, and group E, and 0.005 mg per mouse separately on group B, group D, and group F. The second immunization was at the 15th day and each group was treated with the same antigen as 1.5 times as the former immunization in dose. The antigens of SW-OVA, SW-BSA, and SW-HSA were separately emulsified by Freund′s incomplete adjuvant. The third immunization was done at the 29th day, the dose of antigen was 1.5 times as former, and SW-OVA, SW-BSA, and SW-HSA were dissolved in physiologic saline. Group G was control and injected with physiologic saline. Serum SW antibody titers in each group were assayed initially at the 28th day, and assay was settled in every 7th day, and it in every 14th day from the 13th times. ELISA assay showed that SW antibody can be found in group A, group B, group C group D, group E, and group F, ELISA assay showed that serum SW antibody titers of group A and group B increased to the peak 13(log2)and 12(log2)at the day 98. However, that of group C~F were lower, and increased to the peak 2 (log2) at the day of 35, then decreased to zero gradually. The results of this experiment showed that SW antibody can be generated in the body of mice with SW-OVA, SW-BSA, and SW-HSA. SW-OVA has a high immunogenicity than SW-BSA and SW-HSA. Mice vaccinated with a high dose produced high titre antibody than those with a low dose.