Studies On Synthesis Of SW-HSA And It's Immunigenicity

Locoweed is a kind of poison plants that endanger the grassland husbandry seriously in worldwide. It distributes in western region of China, the United States, Russia, Australia and Canada extensively, and causes an enormous economy loss. To control locoism and utilize locoweed comprehensively, scholars research it through various ways. Because the content of crude protein of locoweed is equal to alfalfa, so we think locoweed has potential to be a good grass. According to the immunology, the locoweed toxin swainsonine (SW) is a hapten, can be synthesized to a 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 animal, and make the animal acquire resistance to feed the locoweed. The main of this thesis is to couple SW with human serum albumin (HSA) to get different couple ratio 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 HC1, 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 through TLC, MP, IR,MS.2 Synthesis of 4 different ratio swainsonine-HSAAt first, quaternary ammonium salt was prepared by reaction between SW and active ester at 70℃, and then, SW-HSA was obtained by stirring between quaternary ammonium salt and HSA at condition of ice bath for 12 h, and adjusted mole ratio between quaternary ammonium salt and HSA. Dialyzing and freeze drying the conjugate, 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-HSA were 18.6, 12.5, 13.5 and 32.6 respectively.3 The immune experiment of swainsonine-HSA on mice Twenty kunming mice were randomly separated into group A, group B, group C, group D and group E, 4 mice per group, the number were A1～A4,B1～B4,C1～C4,D1～D4 and E1～E4 respectively。Mice in group A, group B, group C and group D were immunized by SW-HSA with 4 kinds of conjugate rates. The first immunization, 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 0.050 mg per mouse. The second immunization was done at 30 d and SW-HSA was emulsified by Freund′s incomplete adjuvant. There was an immunization per 20 d after the second immunization, the dose of antigen was 1.5 times as the dose of antigen before it, and SW-HSA was dissolved in physiologic saline. The immune ways and doses from the second to the fifth were same as the first immunization. The sixth immune ways were sub cutis injection of back and intraperitoneal injection. The seventh immune way was intraperitoneal injection. Group E was control and injected with physiologic saline. Serum SW antibody titers in each group were assayed initially at the third immunization. ELISA assay showed that serum SW antibody titers in group A, group B, group C and group D began to increase after the third immunization. Among the groups, SW antibody titers of group A, group B, group C, group D increased to the peak (28, 29, 29, 29 respectively) at the fifth immunization. Meanwhile, SW antibody titer of group D was up to 29 at the fourth immunization, and kept the same antibody level at the fifth immunization. SW antibody titers of 4 groups began to decrease gradually at the sixth immunization. The results of this experiment confirmed the SW antibody could generate in the body of mice with 4 different SW-HSA, and the high conjugate ratio could induce high SW antibody level.