广州管圆线虫一期幼虫的分离纯化及其抗原分析

Pruification and Characterization of Antigens Isolated from the Sage-1 Larvae of Angiostrongylus cantonensis

目的建立广州管圆线虫一期幼虫的分离纯化方法,分析其抗原特性及血清学诊断价值。方法分别用贝尔曼漏斗法、蔗糖离心浮选法及改良贝尔曼法联合胰酶消化法从大鼠粪便中分离纯化一期幼虫;用SDS-PAGE和Western blotting进行蛋白谱与抗原谱分析;分别用一期幼虫和成虫抗原包被ELISA反应板,间接法检测不同样本中相应抗体。结果3种方法分离效率分别为50.1%、33.6%和19.7%;联合法获得的纯度最高,且90%以上的幼虫有活力;贝尔曼漏斗法分离的幼虫99%以上具有活力但纯度低;蔗糖离心浮选法分离的绝大多数虫体失去活力。一期幼虫104000Mr与广州管圆线虫感染2周的大鼠血清出现强反应,32000Mr和31000Mr与感染6周的大鼠血清、32000Mr与广州管圆线虫病患者血清均出现较强反应,与对照血清均未出现明显的反应;在ELISA检测中,一期幼虫抗原对广州管圆线虫病患者血清和感染大鼠血清的检出率与成虫抗原无统计学差异。结论改良贝尔曼法联合胰酶消化法可从大鼠的粪便中分离高纯度且具有活力的一期幼虫,一期幼虫104000Mr、32000Mr和31000Mr具有潜在的诊断价值,可望从大鼠粪便中分离一期幼虫开发诊断性抗原。

Objective To establish a method for the isolation of stage-1 larvae of A. cantonsis, and to evaluate its diagnostic value for the treatment of angiostrongyliasis. Method Three different purification procedures, namely Baermann funnel method, sucrose density centrifugation method and modified Baermann funnel combined with trypsin digestion, were compared. Stage-1 larval antigens and adult worm antigens were analyzed by SDS-PAGE, Western blotting and enzyme-linked immunosorbent assay （ELISA）. Result The separation efficiency of the three methods was 50.1%, 33.6% and 19.7%, respectively. Highly purified larvae with 90% of viability were obtained by the modified Baermann funnel combined with trypsin digestion method. 99% of larvae obtained by the Baermann funnel method were viable, but the purity was low. Most of the larvae obtained by sucrose centrifugation method were inactive. SDS-PAGE analysis showed that the total number of protein bands of stage-1 larvae was less than that of antigens prepared from adult worm. Western blotting analysis showed that the 104 000 Mr stage-1 larval antigen strongly reacted with the immune sera from rats infected with A. cantonensis for 2 weeks. Both 31 000 Mr and 32 000 Mr antigens were found to react with the immune sera of rats infected with A. cantonensis for 6 weeks. The 32 000 Mr antigen also reacted with the sera of patients with angiostrongyliasis, and immuno-reactivity to normal sera was not observed. Both stage-1 larval antigens and the adult antigens showed the same level of reactivity to sera of patients with angiostrongyliasis and rats infected with A. cctntonensis by the ELISA method. Conclusion Viable and highly purified larvae can be obtained by the modified Baermann funnel combined with trypsin digestion method. The 31 000, 32 000 and 104 000 Mr stage-1 larval antigens can be used in serological diagnosis of angiostrongyliasis.