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俞慕华, 陈代雄, 詹希美. 抗华支睾吸虫CAg噬菌体抗体表达与活性鉴定[J]. 中国公共卫生, 2004, 20(11): 1320-1322.
引用本文: 俞慕华, 陈代雄, 詹希美. 抗华支睾吸虫CAg噬菌体抗体表达与活性鉴定[J]. 中国公共卫生, 2004, 20(11): 1320-1322.
YU Mu-hua, CHEN Dai-xiong, ZHAN Xi-mei. Expression and identification of phage antibodies against clonorchis sinensis CAg[J]. Chinese Journal of Public Health, 2004, 20(11): 1320-1322.
Citation: YU Mu-hua, CHEN Dai-xiong, ZHAN Xi-mei. Expression and identification of phage antibodies against clonorchis sinensis CAg[J]. Chinese Journal of Public Health, 2004, 20(11): 1320-1322.

抗华支睾吸虫CAg噬菌体抗体表达与活性鉴定

Expression and identification of phage antibodies against clonorchis sinensis CAg

  • 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B05、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B05、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50kD和27kD处均有特异抗体条带出现。B05噬菌体抗体与Cs-MAg和Cs-Ag在约25kD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B05和E38具有特异结合Cs-CAg活性。

     

    Abstract:
      Objective   To express and identify the phage antibodies against circulating antigen ofClonorchis sinensis, (Cs-CAg).
      Methods   B05、C10 and E38 positive clones were incubated with IPTG respectively, Fab antibodies were observed by SDS-PA GE 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-PA GE, 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.

     

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