恶性疟原虫合子/动合子表面25蛋白自身偶联条件的优化及其聚体蛋白的制备

Optimization of coupling reaction for Pfs25 protein and preparation of relevant conjugation

目的探讨影响恶性疟原虫合子/动合子表面25蛋白(25 k Da Plasmodium falciparum sexual stage protein,Pfs25)偶联的因素,优化Pfs25蛋白的偶联反应条件,制备具有免疫原性的Pfs25纯化聚体蛋白。方法通过对偶联反应时Pfs25蛋白浓度、偶联反应的酸碱度、不同的缓冲液及偶联试剂添加方式的探索,考察不同反应条件对Pfs25蛋白偶联的影响。凝胶过滤层析纯化Pfs25偶联反应产物,双抗体夹心ELISA和SDS-PAGE对凝胶柱洗脱液进行检测,Western blot检测纯化的Pfs25聚体蛋白。结果 Pfs25蛋白偶联反应选择1/15 mol/L Na_2HPO_4-KH_2PO_4为偶联缓冲液,反应体系在pH 5.6～6.0时偶联效果更好,而Pfs25蛋白反应质量浓度为25 mg/m L时可获得最佳的Pfs25偶联效率(68.67%),凝胶过滤层析法获得Pfs25聚体蛋白,经双抗体夹心ELISA和Western blot检测,形成的Pfs25聚体蛋白保持了与Pfs25蛋白相同的抗体结合能力。结论优化了Pfs25蛋白最佳偶联反应条件,获得具有免疫原性的Pfs25纯化聚体蛋白。在形成Pfs25聚体中,以二聚体(Pfs25)2为主要形式,兼有少量三聚体(Pfs25)3和四聚体(Pfs25)4。

Objective To search for an optimal condition for Pfs25 in the coupling reaction,and to prepare the conjugation of Pfs25. Methods Reaction facters were investigated including,concentration of Pfs25,p H,buffer type and adding mode of the coupling reagent,and the resulted influence in coupling process was evaluated. The conjugation and detection of Pfs25 were carried out by means of gel filtration,SDS-PAGE,double-antibody sandwich ELISA and the Western blot.Results The optimal coupling reaction of Pfs25 included the following factors: buffer was 1/15 mol/L Na2HPO4-KH2PO4,pH 5.6-6.0 and Pfs25 with a concentration of 25 mg/m L. Under these conditions,the best coupling rate( 68.67%)was obtained and the resulted Pfs25 conjugation was kept the same antibody binding capacity for Pfs25. Conclusion The optimal conditions for Pfs25 in the coupling reaction were obtained. As a consequence,Pfs25 conjugation could preserve the original antigenicity,and present in a mixture of polymers including dimer( Pfs25)2,trimer( Pfs25)3 and tetramer( Pfs25)4,and the dimer( Pfs25)2 was the primary type.