中国寄生虫学与寄生虫病杂志 ›› 2011, Vol. 29 ›› Issue (4): 3-254-257.

• 论著 • 上一篇    下一篇

不同的化学连接剂偶联恶性疟原虫Pfs25抗原和绿脓杆菌重组去毒外毒素rEPA

 钱锋   

  1. 第二军医大学长征医院风湿免疫科, 上海 200003
  • 出版日期:2011-08-30 发布日期:2012-09-27

Conjugation of Plasmodium falciparum Pfs25 to Pseudomonas aeruginosa ExoProtein A with Different Chemical Linkers

QIAN  Feng   

  1. Department of Rheumatology and Immunology, Changzheng Hospital, The Second Military Medical University,Shanghai 200003, China
  • Online:2011-08-30 Published:2012-09-27

摘要: 目的 偶联恶性疟原虫Pfs25抗原和绿脓杆菌重组去毒外毒素rEPA,并测试不同化学连接剂的偶联效果。 方法  用化学连接剂NAHT(DL-N-acetylhomocysteine thiolactone)在Pfs25蛋白上加自由巯基,化学连接剂Sulfo-EMCS、EMCH、SBAP和Sulfo-SIAB分别修饰载体蛋白rEPA,通过巯基化的Pfs25和经化学基团修饰的rEPA在一定条件下反应,形成Pfs25与rEPA的偶联蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测偶联结果。 结果  成功地将Pfs25偶联到rEPA上,构建了偶联蛋白Pfs25-rEPA。对于蛋白的化学基团修饰,伯胺基与NHS(N-hydroxysuccin-imide)酯之间的反应效率要高于以EDC为桥联剂的羧基与酰肼基之间的反应;对于偶联反应,马来酰亚胺基与巯基之间的反应效率要高于卤素乙酰基与巯基之间的反应。 结论  各化学连接剂都能将Pfs25偶联到rEPA上,但偶联效率各不相同,并形成不同样式的偶联蛋白。

关键词:  , 恶性疟原虫;Pfs25;绿脓杆菌;rEPA;偶联

Abstract: Objective   To conjugate Plasmodium falciparum Pfs25 to recombinant Pseudomonas aeruginosa ExoProtein A (rEPA) and test the effect of conjugation using different chemical linkers.  Methods   The Pfs25 was thiolated by NAHT (DL-N-acetylhomocysteine thiolactone), whereas the carrier protein rEPA was modified by Sulfo-EMCS, EMCH,SBAP and Sulfo-SIAB respectively. The Pfs25-rEPA conjugates were formed by incubating the thiolated Pfs25 with modi-fied rEPA under certain conditions. Coomassie blue stained SDS-PAGE was performed to examine the results of the conjugation.  Results   The Pfs25 was successfully conjugated to the carrier protein rEPA by the linkers used in this study. For the addition of a chemical group onto the protein, the reaction between the groups of primary amine and NHS (N-hy-droxysuccinimide) ester was more efficient than the reaction between the groups of carboxyl and hydrazide with EDC as a crosslinker; for the formation of a conjugate, the reaction between the groups of maleimide and free sulfhydryl was more efficient than the reaction between the groups of haloacetyl and free sulfhydryl.   Conclusion   The Pfs25 can be conjugated to the rEPA by the chemical linkers with different conjugation efficiency and coupling proteins.

Key words: Plasmodium falciparum, Pfs25, Pseudomonas aeruginosa, rEPA, Conjugation