伯氏疟原虫顶端膜抗原1胞外区及其亚区的表达与免疫保护作用分析

Expression and Immunogenicity Evaluation of Ectodomain and Subdomains of Plasmodium berghei Apical Membrane Antigen 1

目的表达伯氏疟原虫顶端膜抗原1(PbAMA-1)胞外区E及其各亚区,分析其免疫原性和免疫保护作用。方法抽提伯氏疟原虫基因组,对PbAMA-1基因测序,依据该序列采用毕赤酵母密码子使用频率重新设计PbA-MA-1基因序列。将其胞外区E分为3个彼此重叠的基因片段DⅠ、DⅡ和DⅢ并进行优化,人工合成后在大肠埃希菌中诱导表达。表达产物经纯化和重折叠,分别与福氏佐剂乳化后免疫小鼠和家兔,均免疫3次,间隔2周。每次免疫剂量,小鼠为20$g,家兔为100$g。ELISA检测抗体效价,并以疟原虫攻击实验确定各抗原的免疫保护效果。结果测得的PbAMA-1基因序列与Sanger测序中心公布的序列完全一致。密码子优化并人工合成的DⅠ、DⅡ、DⅢ和E目的基因片段在大肠埃希菌表达载体pET32a均获得诱导表达,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,各目的基因表达产物大小与预计的相对分子质量Mr 44 300、Mr 33 300、Mr 29 900和Mr 67 200相一致。表达产物经镍离子螯合柱(Ni-NTA柱)纯化和谷胱甘肽(GSH)氧化还原法体外重折叠,蛋白纯度达90%以上,各纯化的重组蛋白在小鼠体内均激发产生高滴度抗体。其中,完整的胞外区E第3次免疫后抗体滴度为(34.4±0.15)×10-4,与其他组相比,免疫原性较其他各亚区更强(t=5.66,P<0.01;t=2.27,P<0.05和t=5.05,P<0.01)。取家兔免疫血清与感染伯氏疟原虫ANKA株的小鼠血膜抗原片进行间接荧光抗体试验(IFAT),均为阳性,其中抗E的抗血清免疫荧光强度最高,与ELISA检测的抗体水平一致。取家兔抗血清对小鼠伯氏疟原虫粗提抗原进行蛋白质印迹(Western blot)分析,产生特异性反应条带,表明能够识别天然的PbAMA-1蛋白。免疫小鼠在以伯氏疟原虫攻击实验中得到部分保护,DⅠ、DⅡ、DⅢ和E各免疫组小鼠与佐剂对照组相比,存活时间明显延长(t=2.78,P<0.05;t=2.67,P<0.05;t=3.46,P<0.01和t=3.50,P<0.01)。结论人工合成的PbAMA-1具有良好的免疫原性和免疫保护作用,完整的胞外区E较其各亚区表现出更好的免疫原性。

Objective To express and evaluate the immunogenicity of ectodomain and its subdomains of Plasmodium berghei apical membrane antigen 1 （PbAMA-1）. Methods Sequence of PbAMA-1 gene was isolated from the genome of P. berghei, and was redesigned and divided into three overlapped fragments according to its subdomain structure. The codon-optimized DNA fragments of PbAMA-1 were synthesized and inserted into vector pET32a for expression in E. coli and the recombinant proteins were purified by Ni-NTA column, followed by refolding in vitro, Mice and rabbits were immunized with the recombinant proteins formulated with Freund adjuvant. Titer of the specific antibodies was detected by ELISA and IFA. The immunized mice were challenged by P, berghei to evaluate protective efficacy in vivo. Results The sequence of the PbAMA-1 gene was shown to be identical to that published before. PbAMA-1 sequence was redesigned via codon optimization and synthesized. Both ectodomain and its subdomains of PbAMA-1 were successfully expressed in E.coli after induction. The proteins were isolated with the purity of more than 90% after Ni column purification and refolding in vitro. Immunization of mice with the recombinant proteins induced high level of specific antibodies. The antibody titer to ectodomain E after the 3rd immunization showed a strong immunogenicity at （34.4±0.15）×10^-4. The antibodies interacted with the parasites by indirect fluorescence. The immunized mice were partially protected from the challenge of P. berghei. Conclusion The recombinant PbAMA-1 is highly immunogenic and induces protective immunity against the challenge of P. berghei.