摘要
目的 对从抗华支睾吸虫噬菌体抗体库中筛选到的阳性克隆进行诱导表达和鉴定。方法 对阳性克隆株B0 5、C10和E38用异丙基 - β -D -硫代半乳糖苷 (IPTG)诱导表达 ,经SDS聚丙烯酰胺凝胶电泳 (SDS -PAGE)和蛋白印迹试验 (Westernblot)观察有无Fab抗体条带。然后 ,用卫氏并殖吸虫 (Pw)、肝片吸虫 (Fh)、姜片吸虫 (Fb)、华支睾吸虫 (Cs)、日本血吸虫 (Sj)脱脂全虫粗抗原和华支睾吸虫代谢抗原 (Cs-MAg)进行SDS -PAGE ,并分别和B0 5、C10和E38噬菌体抗体进行Westernblot,鉴定抗体活性。结果 Westernblot结果表明 ,3株阳性克隆的培养上清和细菌冻融上清在约 5 0kD和 2 7kD处均有特异抗体条带出现。B0 5噬菌体抗体与Cs-MAg和Cs-Ag在约 2 5kD处出现特异性反应条带 ,而与其它 4种吸虫抗原无反应条带的出现。C10除与Cs-MAg和华支睾吸虫全虫抗原 (Cs -Ag)在约 38kD处出现反应条带外 ,还与Sj-Ag在该处有弱阳性反应 ;而与其它 3种吸虫抗原无反应条带出现。E38与Cs-MAg和Cs-Ag在约 16kD处出现特异性反应条带 ,而与其它 4种吸虫抗原无反应条带出现。 结论 3株阳性克隆均可表达抗华支睾吸虫循环抗原 (CAg)噬菌体抗体 ,其中B0 5和E38具有特异结合Cs-CAg活性。
Objective To express and identify the phage antibodies against circulating antigen of Clonorchis sinensis (Cs -CAg).Methods B05、C10 and E38 positive clones were incubated with IPTG respectively,Fab antibodies were observed by SDS-PAGE and Western blot.Moreover,Ag of Paragonimus westermani(Pw),Fasciola hepatica(Fh),Fasciolopsis buski(Fb),clonorchis sinensis(Cs),schistosoma japonicum(Sj) and Cs -CAg were separated in SDS-PAGE,then bound with B05、C10 and E38 phage antibodies respectively in Western blot.Results By Western blot analysis of 3 positive clones,two molecular masses of 50kDa and 27kDa could be found under reducing condition in supernatant and lysed cell pellet of 3 positive clones.The former was an intact Fab molecular,the latter was heavy chain Fd fragment and light chain.The results of immunoblotting showed that both B05 and E38 could bind to Cs -MAg specifically,former at a molecular weight of 25kDa and latter at 16kDa respectively,both of them did not react with any other four trematode antigens.While C10 bound to not only Cs -MAg,but also Sj -Ag at a molecular weight of 38kDa,and did not react with any other three trematode antigens.Conclusion Three positive clones can express the phage antibodies against clonorchis sinensis CAg.B05 and E38 can bind Cs -CAg specifically.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2004年第11期1320-1322,共3页
Chinese Journal of Public Health
基金
卫生部 (98 - 1 - 85)
广东省高教厅"2 1 1工程"学科建设科研基金
