恶性疟原虫裂殖子表面蛋白3功能区片段的制备及其免疫原性分析

The Production and Immunogenicity of the Functional Fragment of Plasmodium falciparum Merozoite Surface Protein 3

目的构建恶性疟原虫裂殖子表面蛋白3功能片段MSP3(69)与谷胱甘肽(GST)的融合蛋白,并在大肠杆菌中进行表达,分析其免疫原性。方法采用大肠杆菌系统表达融合蛋白GST-MSP3(69),以疟疾病人血清进行Western-blot,并以ISA720、佛氏、氢氧化铝与CpG联合3种佐剂与GST-MSP3(69)融合蛋白乳化,免疫Balb/ca小鼠,分析其免疫原性。结果在大肠杆菌系统中成功表达GST-MSP3(69)融合蛋白,疟疾病人血清能与融合蛋白反应。3种佐剂免疫Balb/ca小鼠均能诱发Balb/ca小鼠产生较高水平的特异抗体,3次免疫后的抗体滴度分别为4.5×105,4.1×105和3.5×105。3次免疫后血清抗体亚型分析显示该蛋白能够诱导Balb/ca小鼠产生较高滴度的亲细胞亚型IgG1和IgG2b,ISA720、佛氏、氢氧化铝与CpG联合佐剂组IgG1分别为8×104、7.8×104、6.8×104,IgG2b分别为1.2×104,1.25×104、1.5×104。统计学分析显示不同佐剂组的抗体滴度及抗体亚型滴度之间差异均无统计学意义(P>0.05)。结论制备了在大肠杆菌表达的GST-MSP3(69)融合蛋白,该蛋白具有高免疫原性,并产生亲细胞亚型抗体,这些为研究MSP3蛋白功能区的免疫保护作用打下了基础。

Objective To produce the functional fragment （184-252） of Merozoite Surface Protein 3 （MSP3） of Plasmodiumfalciparum and assess its immunogenicity in Balb/c mice. Method The functional fragment （184-252） of Merozoite Surface Protein 3 （MSP3） of Plasrnodiumfalciparum was fused with GST and the resulting fusion protein, designated GST-MSP3 （69）, was expressed in E.coli. The recombinant GST-MSP3 （69） protein was detected with the sera by Western-blot. Balb/ca mice were immunized with the protein formulated by three adjuvants ISA720, Freund＇s and Alum combined with CPG1826. Result The recombinant fusion protein was recognized by the sera from the malaria patients. The antigen formulated with all of the three adujvants were highly immunogenic in mice with 4.5×10^5 in ISA 720, 4.1×10^5 in the Freund and 3.5×10^5 in the alum combined with CPG1826. Analysis of antibody isotypes showed that the predominant antibody were eytophilous IgG1 and IgG2b. The titer of IgG1 was 8×10^4,7.8×10^4 and 6.8×10^4 in ISA720, the Freund and the Alum combined with CPG1826, respectively, while the IgG2b was 1.2×10^4,1.25×10^4 and 1.5×10^4, in the three adjuvants, respectively. There was no statistically significant difference in the ELISA titer of the adjuvant. Conclusion The GST-MSP3（69） protein was successfully expressed in E.coli and the protein formulated with the three adjuvants were highly immunogenic with predominant IgG isotypes of IgG1 and IgG2b in mice.