| Objective:To analyze the protein components and the immunoreactivity of three kinds of antigens of Trichinella spiralis pre-encysted larva in Dali:the pre-encysted larva excretor-secretory antigens(PELESA). the pre-encysted larva surface antigen(PELSA) and the pre-encysted larva body soluble antigens(PELBSA). And to observe the protein components'diffenrence of the three kinds of antigens and to analyze, screen some protein components with strong immunogenicity and immunoreactivity, Trichinella spiralis pre-encysted larvae than encysted larva appeared about two weeks early in hosts.The host produces anti-becomes the pouch preliminary larva immune body to appear also early, it May use the damaging effect of Trichinella spiralis pre-encysted larva, It will be the development direction of the immunization and prevention research about Define to look for protective antigen from Trichinella spiralis pre-encysted larva.Methods:(1) Collected Trichinella spiralis pre-encysted larva. Twenty adult rats were inoculated with 3000 Trichinella spiralis encysted larva per rat by the oral route. In 14, 15,16,17,18,19,20days, rats were killed, Artificial digestion muscle and collect the Trichinella spiralis. (2) Prepared the antigens of Trichinella spiralis pre-encysted larva Trichinella spiralis pre-encysted larva was cultured in culture flask with serum-free RPMI-1640 medium in 37℃5%CO2 incubator for 24h, and culture medium was centrifuged. Supernatants was collected, dialysed, and concentrated, and obtained ESA; Trichinella spiralis pre-encysted larva was cultured in culture flask with serum-free RPMI-1640 medium with 0.25%CTAB and 2%Deoxycholic acid Sodium Salt in 37℃ incubator for 2.5h, and shook once during the period, and culture medium was centrifuged. supernatants was collected,dialysed, and concentrated, and obtained SA; Trichinella spiralis pre-encysted larva was frozen and thawed repeatedly, then ultrasonicated and macerated coldly, centrifugated,and obtained the pre-encysted larva body soluble antigen (BSA). (3) Prepared the immune serum.Prepared the negative serum.Before the SD rat infected by Trichinella spiralis encysted larvae, collected blood from the SD rat's caudal veil and got the negative serum.conserved in-20℃refrigeratory.Prepared the the serum of rat anti- Trichinella spiralis.Infected the SD rats with T.spiralis larvae and each rat infected with 8×103 T.spiralis larvae, after 17 d, collected blood from the SD rat's caudal veil and got the negative serum,then examined it's antibody effect with the IgG which anti- IgG in the serum of anti-T.spiralis. after 20 d, when the antibody effect was 1:10240, and collect the blood from the rat's femoral by anaesthetizing with aether, Standing 30min at room temperature and centrifugated at 3×103 r/min for 5 minutes and got the liquid supernatant which was the serum anti-T spiralis.subpackaged and conserved in-20℃refrigeratory. (4) Analyzed the protective immunity of Trichinella spiralis pre-encysted larva antigens.The mice were immunized with Trichinella spiralis pre-encysted larva somatic antigen, encapsulated larva excretory-secretory antigen and encapsulated larva surface antigen,3 times at 7day interval, and the adjuvant control and normal control were set up. Seven days after the final immunization, each mouse was orally challenged with 200 Trichinella spiralis larva. The intestinal adult worms and muscle larvae of Trichinella spiralis of each group were recoveried and examined on Day 7 and Day 30 post-challenge, respectively. The level of serum IgG to antigens of Trichinella spiralis muscle larvae was detected by ELISA. (5) Analyzed the three kinds antigens of T.spiralis pre-encysted larva.Regarded the three kinds of antigens of Trichinella spiralis muscle larva in Dali as contrast, adopting sodium dodecyl sulfate-polyacryl amine gel electrophoresis (SDS-PAGE) to analyze protein components of the kinds of antigens of pre-encysted larva; adopting immunoblotting test(Western-blot) to analyze the immunoreactivity of the three kinds of antigens of pre-encysted larva.Results:(1) Collected Trichinella spiralis pre-encysted larva.14,15,16 days, can't saw Trichinella spiralis in diaphragm,17,18,19,20,21 days, can saw Trichinella spiralis in diaphragm and increase with time; 14,15,16 days.unable to gather the Trichinella spiralis.17,18,19.20,21 days, were collected Trichinella spiralis 5000,8000,10000, 30000,30000 perrat. (2) Protective immunity of Trichinella spiralis pre-encysted larva antigens.The intestinal adult worms were reduced by 84.89%,89.73%,85.65%,2.57% in the encapsulated larva somatic, excretory-secretory and surface antigen groups, respectively. The muscle larvae were reduced by 71.71%,80.98%,73.66%,5.60%, respectively. Adult worm reduction rates of the encapsulated larva excretory-secretory antigen group and surface antigen group were higher than those of encapsulated larva somatic antigen group (P<0.05). Muscle larva reduction rates of the encapsulated larva excretory-secretory antigen group and surface antigen group were higher than those of encapsulated larva somatic antigen group (P<0.01). The antibody titers in all the immunized groups increased significantly, and the GMRT values of the encapsulated larva somatic, excretory-secretory and surface antigen groups were 2798.89,3474.51,2 984.83, respectively, and were 6.09,7.56,6.50 times higher than those of the control (459.32). (3) The result of SDS-PAGE analysis for Trichinela spiralis pre-encysted larva antigens.The results acquired by SDS-PAGE analysis displayed:the body soluble antigens of Trichinella spiralis pre-encysted larva displayed 20 protein dyeing bands and the range of the molecular weight is between 120kDa and 14kDa, in which have ten main bands and the molecular weights are 100,67,55,47.45.43,39,37,35,30kDa respectively; the body soluble antigens of Trichinella spiralis encysted larva displayed 22 protein dyeing bands and the range of the molecular weight is between 120kDa and 10kDa, in which have fourteen main bands and the molecular weights are 90,88,67,66, 55,45,43,39,30,20,16,14,12, 10kDa respectively; the surface antigens antigens of Trichinella spiralis pre-encysted larva displayed 9 protein dyeing bands and the range of the molecular weight is between 120kDa and 20kDa, in which have four main bands and the molecular weights are 119,40,39,35kDa respectively; the surface antigens antigens of Trichinella spiralis encysted larva displayed 11 protein dyeing bands and the range of the molecular weight is between 120kDa and 10kDa, in which have seven main bands and the molecular weights are 119,63,55,45,39,20,13kDa respectively;the excretory-secretory antigens of Trichinella spiralis pre-encysted larva displayed 6 protein dyeing bands and the range of the molecular weight is between 45kDa and 18kDa, in which have three main bands and the molecular weights are39,28,22kDa respectively; the excretory-secretory antigens of Trichinella spiralis encysted larva displayed 10protein dyeing bands and the range of the molecular weight is between 70kDa and 14kDa, in which have eight main bands and the molecular weights are53,45,43,35,33,28,25, 18kDa respectively. (4) The results of We stern-blot detection for Trichinella spiralis pre-encysted larva antigens.The results acquired by Western-blot detection displayed:when detected by the serum of rats anti-Trichinella spiralis, the body soluble antigens of Trichinella spiralis pre-encysted larva displayed 5 reaction bands and the molecular weights are100,67,45,43.39kDa respectively; the body soluble antigens of Trichinella spiralis encysted larva displayed 5 reaction bands and the molecular weights are 90,67, 45,43,39kDa respectively; the surface antigens of Trichinella spiralis pre-encysted larva displayed 2 reaction bands and the molecular weights are 40,39kDa respectively; the surface antigens of Trichinella spiralis encysted larva displayed 3 reaction bands and the molecular weights are 63,55,45kDa respectively; the excretory-secretory antigens of Trichinella spiralis pre-encysted larva displayed 1 reaction bands and the molecular weights are 39kDa, the excretory-secretory antigens of Trichinella spiralis encysted larva displayed 2 reaction bands and the molecular weights are 45,43kDa. While the negative serum has no specific reaction band on the corresponding position.Conclusions:(1) At 20 days of Trichinella spiralis infect, is the best time to collect the pre-encysted larvae of Trichinella spiralis. The method of artificial digestion muscle can collect the pre-encysted larvae of Trichinella spiralis. (2) Trichinella spiralis pre-encysted larva antigens can induce strong resistance of host to a subsequent challenge infection. Among these antigens, excretory-secretory antigen is more immunogenic. (3) The molecular weights of Trichinella spiralis pre-encysted larva body soluble antigens are100, 67,45,43,39kDa protein components. They have stronger specific recognition to the IgG in the serum of rat anti-Trichinella spiralis. (4) And the molecular weights of Trichinella spiralis pre-encysted larva surface antigens are 40,39kDa protein components have stronger specific recognition to the IgG in the serum of rat anti-Trichinella spiralis. (5) And the molecular weights of Trichinella spiralis pre-encysted larva excretory-secretory antigens is 39kDa protein components has stronger specific recognition to the IgG in the serum of rat anti-Trichinella spiralis. |
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