不同培养条件对旋毛虫肌幼虫产生排泄分泌抗原的影响

目的对比在常规1640培养基中和在模拟宿主生理环境的培养液的刺激下旋毛虫肌幼虫排泄分泌(excretory-secretory,ES)抗原产量及成分的变化,同时观察在感染初期ES抗原的不同组分在小鼠体内诱导产生主要抗体的时间,以寻找获得更高产量的肌幼虫ES抗原的制备方法。方法在虫数、培养液体积、培养时间、温度、ES抗原体积浓缩倍数等条件相同的情况下,按培养液中小鼠血清超滤液(简称血超)、还原型谷胱甘肽(简称谷胱甘肽)和胆汁粉的不同组合分组,分别培养肌幼虫并收集ES抗原,再以相同上样体积作SDS-PAGE电泳,Odyssey双色红外激光成像系统扫描凝胶并测定条带信号强度进行定量比较。用Western blotting对比观察不同组ES抗原的活性及成分变化。以旋毛虫人工感染小鼠不同时期收集的血清(感染后15、18、20、21、22、24、27、30、40、50 d)作为一抗进行Western blotting,观察ES抗原主要成分在小鼠体内产生抗体的先后顺序。结果S DS-PAGE电泳凝胶定量分析结果显示,血超+谷胱甘肽组、胆汁组和胆汁+血超组的抗原产量明显高于1640培养基组,其中胆汁+血超组的抗原产量最高。Western blotting结果显示,各组抗原均能被感染血清(感染后40 d)识别产生多个条带,其中血超+谷胱甘肽组的条带中相对分子质量约为87 000的条带荧光信号明显强于其他组;感染初期ES抗原不同组分在小鼠体内产生抗体的先后顺序为:45 000、53 000、41 000,其抗体可被检测到的最早时间分别为感染后15、21、24 d。结论培养液中添加胆汁粉可大大提高旋毛虫肌幼虫ES抗原产量,胆汁粉与血超共用后ES抗原的增产效果更佳;感染初期肌幼虫ES抗原的不同组分诱导小鼠产生抗体的时间不同。此研究将为改进ES抗原制备方法,寻找旋毛虫病早期诊断抗原提供实验依据。

Effects of Different Culture Conditions on Output of Excretory-secretory Antigens Produced by Tichinella spiralis Muscle Larvae

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.