Synthesis Of Swansonine Artificial Antigens And Its Immunogenicity

Locoweeds, the poisonous plants of the Astragalus and Oxytropis genera,are the major poisonous plants found on many rangelands worldwide. Locoweed toxicosis causes death, abortion, fetal anomaly, intention tremors, proprioceptive deficits, and obvious behavioral change known as locoism. The common methods were used for prevention and control locoism including artificial excavation, chemical and biological extermination which had great effects on reducing the loss caused by locoism. Nevertheless, the locolweeds'growing prosperity during the semi-arid years and seasons make them the dominant forage grass, and become the potential component in keeping solid and biomass. Moreover, the finding that the main poisonous compound----swainsonine(SW), an inhibitor of a-mannosidases, can reduce the growth rate of some tumour cells and have potential use in other bio-medical research caused great attention, which cause the between control, elimination and protection and conservation. Fortunately, the immunologic method used on prevention of nosotoxicosis caused by plant toxin revealed a promising future for tackling the contradiction. However, the swainsonine is a hapten of low molecular weight without the necessary chemical group to allow the carrier conjugation. The synthesis procedure of SW artificial antigen was revealed to be more complicated. In order to simplify the procedure, potentize the immunogenicity of the antigen, a new method of synthesis for SW-BSA was completed and the immunogenicity was examined in this thesis. The results lay a solid foundation for the use of SW vaccine and the establishment of immunologic detection for SW. The results are as follow:1. Swainsonine (1,2,8-trihydroxyoctahydroindolizine) was isolated in gram quantities from O.kansuensis Bunge with the ultrasonic wave, followed by a continuous liquid/liquid extraction procedure and sublimation. The extraction rate reached above 45 mg/kg.2. Based on the characteristics that aryl reveals fluorescence and that the immuno- genicity of antigen with aryl in structure is high than linear chain fatty acid, 2 new synthetic routes, in which the aryl was introduced in the"bridge"for the conjugation of SW with carrier protein, were designed for efficient methods to synthesize the SW artificial antigen. RouteⅠ: Treat the 4-(Chloromethyl)benzoic acid with sodium carbonate gave the sodium 4-(Chloromethyl) benzoate, then the reaction of SW with sodium 4-(Chloromethyl) benzoate was given to obtain the key intermediate product in synthesis of SW artificial antigen, N-(4-carboxybenzyl) swainsonine chloride. RouteⅡ: Started from reaction of 4-(Chloromethyl)benzoic acid with methanol under acidic condition to make N-(4-carboxymethylesterbenzyl) swainsonine chloride, then added NaOH to ungrease, followed by adjusting the reaction system to be acidic with hydrochloric acid, finally the product N-(4-carboxybenzyl) swainsonine chloride was then isolated and purified through column chromatographic separation. The structures of the products were analyzed via the MS,1H NMR and 13C NMR. The results indicated that the 2 routes used were practically feasible. The process of the routes allowed the immediate detection via thin-layer chromatography (TLC), fluorescence detection and colorable reaction of SW. Compared with the routeⅠ, routeⅡwas suitable for mass production with the characteristic of reduced steps of purification, simplified procedure, increased homogenicity of the key intermediate product and the higher yield that was up to 79%.The SW artificial antigens were prepared by conjunction of SW and BSA via EDC, yield of 95% according to BSA, in which the ratio of SW and BSA was 14.7∶1 and 20∶1 respectively in 2 batches.3. Anti-serum from mice was prepared by inoculating the mice with SW-BSA. An indirect ELISA was established for detection of antibody against SW with high sensitivity and stability. The optimal conditions for the method were as follow: the density of coating antigen was 1.0μg?mL, the impacting medium was gelatine of 15g/L, and the working concentration of enzyme labelled antibody of goat against mouse IgG was 1:1500, thus providing a basic method for the research of SW-BSA.4. The immunogenicity of prepared artificial antigen SW-BSA was verified by inoculating mice and rabbits with SW-BSA. The antibody titre against SW in serum from the mice and rabbits were up to 1:2000 determined with the indirect ELISA. The results above presented a new approach for preparation of SW artificial antigen, and laid a solid foundation for the quantitive detection in bio-samples and further research of SW.