马尔堡病毒GP蛋白高抗原性片段筛选及免疫性研究

Screening of high antigenic fragments of Marburg virus GP protein and their immunogenicity

目的从马尔堡病毒GP蛋白上寻找与GP免疫原性相当的小片段用于疫苗设计,以克服全长蛋白缺陷。方法基于GP(aa:1~681)结构及功能分区,构建3个片段[GP1△(aa:25~239)、GPM(aa:250~520)、GP2△(aa:436~648)],经原核表达后免疫小鼠,不同时间检测血清特异抗体滴度,检测脾淋巴细胞增殖指数及测定细胞因子浓度。结果 ELISA检测结果显示,GP2△组与GP混合组(GP1△+GPM+GP2△)产生体液免疫能力相当,明显高于GP1△与GPM组(P<0.05);Con A非特异刺激后,GP2△组脾淋巴细胞的SI值高于GP混合组(P<0.05),GP混合组特异刺激后,GP2△组脾淋巴细胞的刺激指数(SI)值低于GP混合组(P<0.05);细胞因子检测中,GP2△组的白细胞介素2(IL-2)及干扰素γ(IFN-γ)浓度均高于对照组,低于GP混合组(P<0.05)。结论马尔堡病毒GP2△片段免疫小鼠能诱导与完整GP蛋白相当的体液免疫,同时也能诱导一定的细胞免疫,可用来替代完整GP用于疫苗设计。

Objective To screen small fragments with high antigenicity in order to overcome the defects of the fulllength Marburg virus GP as a vaccine antigen. Methods Based on the structure and function of GP sequence [amino acids（ aa） 1-681],three small fragments,including GP1△（ aa 25-239）,GPM（ aa 250-520） and GP2△（ aa 436-648）,were expressed by prokaryotic cells and immunized into mice,and the serum specific antibodies were detected after different immunization time. The proliferation of spleen lymphocytes and the concentration of cytokines of immunized mice were also measured. Results ELISA test results showed that high humoral immunity of GP2 △ was produced as the full-length GP group did,and was higher than that of GP1 △ and GPM（ P 〈0. 05）. GP2 △ immunization groups exhibited a higher SI value in mouse splenic lymphocytes stimulated by Con A than the mixed GP immunization groups did（ P〈 0. 05）,but the effect was the opposite when mouse splenic lymphocytes were stimulated by the mixed GP. In addition,the amount of cytokines IL-2 and IFN-γ of mouse splenic lymphocytes in the GP2△ group was larger than that of the saline group（ P〈 0. 05）,but smaller than that of the mixed GP group. Conclusion The fragment GP2 △ can induce not only the high humoral immunity as GP does,but also moderate cellular immunity,which can be used for vaccine design.