关键词: 旋毛虫, 排泄分泌抗原, 制备方法, Western blotting

Abstract:

Objective In order to get a better protocol of excretory-secretory(ES) antigen preparation, the yield products and components of Trichinella spiralis muscle larvae antigens in conventional RPMI 1640 tissue culture medium and in other host mimic culture media, and the temporal appearance of ES antigen-specific antibodies in the early stage of infection in mice were analyzed. Methods Culture media was divided into different groups according to different combination of ultrafiltrated mouse sera, reduced glutathione and bile powder. Under the same conditions such as worm number, volume of culture media, incubation time and temperature, concentration of ES antigen solution etc, ES antigens in different culture media were prepared and collected separately. SDSPAGE was performed with ES products of all groups with the same loading volume. The gel was scanned with Odyssey infrared laser image system, and the signal intensity of the major bands was detected to perform quantitative comparison; the activity and variety of ES antigens in different groups were detected by Western blotting. Western blotting was also performed with sera of mice infected with different time points(15, 18, 20, 21, 22, 24, 27, 30, 40, 50 days postinfection) to determine the temporal appearance of ES antigenspecific antibodies. Results Quantitative analysis of SDS-PAGE gel showed that the antigen yield of group ultrafiltrated mouse sera plus reduced glutathione, bile powder and ultrafiltrated mouse sera plus bile powder groups, were much higher than that of RPMI 1640 group. The ultrafiltrated mouse sera plus bile powder group had the best yield. Western blotting showed that ES antigens of all groups could be recognized by infected mouse sera(40 days postinfection), the fluorescent signal of a band with the probable molecular weight 87000 of ultrafiltrated mouse sera plus reduced glutathione group was evidently stronger than the other groups; Western blotting also showed that in the major components of ES antigens, the proteins molecular weight if 45 000, 53 000, 41 000 induced specific antibodies one after another. The earliest antibodies were detected with mouse sera of 15, 21 and 24 days postinfection respectively. Conclusion Yield of T. spiralis muscle larvae ES antigens with good activity could be highly increased by adding bile powder into culture media. Moreover adding bile powder and ultrafiltrated mouse sera together into culture media could further increase the yield. In the early stage of infection different components of ES antigens induced predominant specific antibodies asynchronously. These might ameliorate the protocol of ES antigen preparation and provide experimental evidence for searching early diagnostic antigens of trichinellosis.

Key words: Trichinella spiralis, excretory secretory antigens, preparation, Western blotting