重组表达的恶性疟原虫顶端膜抗原1片段诱导小鼠保护性抗体应答

Protective Antibody Response to Recombinant Fragments of Plasmodium falciparum Apical Membrane Antigen 1

目的 分析恶性疟原虫顶端膜抗原(apical membrane antigen 1,AMA1)主要结构域在诱导保护性抗体应答中的作用,为正确选择疫苗应用片段提供依据。方法 PCR扩增编码P.fAMA1相应结构域的基因序列,构建pET原核表达载体,用纯化重组蛋白免疫BALB/c小鼠,ELISA测定抗体效价,用免疫荧光和免疫印迹分析抗体的特异性,用小鼠免疫血清进行疟原虫体外生长抑制实验。结果 成功表达并纯化了代表AMA1不同结构域的重组抗原片段,包括完整的胞外域片段E、结构域Ⅰ+Ⅱ、Ⅰ、Ⅱ和Ⅲ,免疫小鼠后分别诱导出不同滴度的IgG抗体,免疫血清可特异地识别天然AMA1抗原,重组蛋白E和Ⅰ+Ⅱ免疫血清可明显抑制疟原虫的体外生长。结论 AMA1胞外结构域的完整性是构成保护性抗体表位的重要基础,保护性抗体表位主要分布在结构域Ⅰ中。

Objective To determine the role of putative apical membrane antigen (AMA)l domains in inducing protective immunity and to provide basis for selection of vaccine applicable segments. Methods Encoding gene segments of AMA1 were amplified and cloned into pET prokaryotic expression vectors. Recombinant proteins were expressed and purified. Groups of BALB/c mice were immunized by using recombinant protein in Freund's adjuvant, and the IgG liter and specificity of the immune sera were analyzed by IFA and Western blotting. Efficiency of the immune sera in inhibiting Plasmodium falciparum in vitro growth was evaluated. Results Recombinant AMA1 fragments including the entire ectodomain E and subdomain Ⅰ + Ⅱ, Ⅰ, Ⅱ and Ⅲ were successfully expressed and purified. Different levels of antibody were induced in mice by individual proteins and all the immune sera recognized native antigen in the parasites. Sera from protein E and Ⅰ + Ⅱ immunized mice inhibited the growth of parasites. Conclusion The integrality of the ectodomain of AMA1 determines the conformation of the protective antibody epitopes, and these protective epitopes distribute mainly in the subdomain I.