日本血吸虫重组抗原SjPGAM-SjEnol的保护性免疫效果评价

Evaluation on the Immuno-protective Efficacy of the Recombinant Antigen SjPGAM-SjEnol against Schistosoma japonicum in Mice

目的构建日本血吸虫重组表达质粒pET32a-SjPGAM-SjEnol并在大肠埃希菌（E.coli BL21）中表达,观察重组抗原在小鼠抗血吸虫感染中的免疫保护作用。方法利用生物信息学技术筛选SjPGAM和SjEnol富含人源（HLA）Ⅱ类、鼠源（H2-d）Ⅱ细胞结合表位且与宿主同源性较小的肽段,将对应编码的核苷酸序列进行拼接,构建重组质粒pET32a（＋）-SjPGAM-SjEnol,并在E.coli BL21中表达。用蛋白质印迹（Western blotting）分析该重组抗原的抗原性。用小鼠实验评估重组抗原的免疫保护效果,即将55只雄性BALB/c小鼠均分成5组,其中3个试验组分别用重组抗原pET32a-SjPGAM-SjEnol（A组）、pET28a-SjPGAM（B组）、pET28a-SjEnol（C组）（各27μg）与206佐剂混合后免疫小鼠,每次间隔2周,共免疫3次。同时设佐剂对照组（D组）和空白对照组（E组）。末次免疫后2周,每鼠经腹部皮肤分别感染日本血吸虫尾蚴40±2条,感染后6周,经肝门静脉灌注法收集成虫和检测每克肝虫卵数（EPG）,计算减虫率和肝脏减卵率。各组小鼠分别于免疫前、各次免疫后1周尾部取血和剖杀时收集血清,应用ELISA检测血清特异性IgG抗体水平。结果确定SjPGAM的96~147、SjEnol的233~312肽段为重组片段。PCR扩增出一条含编码这两个肽段的核苷酸序列的重组DNA序列,大小447bp。获得的重组蛋白pET32a-SjPGAM-SjEnol相对分子质量（Mr）为33000。Westernblotting结果显示,该重组蛋白可被日本血吸虫成虫抗原免疫兔血清识别,具有良好的抗原性。小鼠免疫实验结果显示,与空白组相比,A组获得39.7%的减虫率和64.9%的肝减卵率,其减虫率与B组（18.5%）、C组（14.7%）比较,差异有统计学意义（均P〈0.05）;肝减卵率与B组（47.5%）、C组（30.5%）比较,差异也有统计学意义（P〈0.05,P〈0.01）。ELISA结果显示,第3次免疫后A组的特异性IgG抗体达到较高水平（2.372±0.268）,与D组（0.490±0.138）、E组（0.220±0.088）间的差异有统计学意义（P〈0.01）。结论成功构建了重组表达质粒pET32a-SjPGAM-SjEnol,多表位重组蛋白pET32a-SjPGAM-SjEnol在小鼠抗血吸虫感染中比单一重组抗原pET28a-SjPGAM和pET28a-SjEnol诱导了更高的免疫保护作用。

Objective To construct and express the recombinant plasmid pET32a-SjPGAM-SjEnol and evaluate its immuno-protective efficacy against the infection of Schistosoma japonicum in mice.Methods The peptides of SjPGAM and SjEnol containing the multivalent epitopes with higher binding capacity of human MHCⅡand mouse H2-dⅡ but low homology with the host were analyzed and screened through bioinfomatics.The corresponding nucleotide sequence of selected epitopes was spliced and the recombinant plasmid pET32a-SjPGAM-SjEnol was constructed and expressed in Escherichia coli BL21 cells.The antigenicity of the recombinant protein was detected by Western blotting and the protective effect in-duced with the recombinant was evaluated in mice.55 BALB/c mice were randomly divided into 5 groups each with 11.Mice from groups A,B and C were injected with a mixture of recombinant protein（27 μg）pET32a-SjPGAM-SjEnol（A）,pET28a-SjPGAM（B）and pET28a-SjEnol（C）respectively together with 206 adjuvant,mice from groups D and E re-ceived adjuvant or PBS only,all injected for three times at two-week intervals.Mice were then challenged with 40±2 cercariae of S.japonicum at two weeks after the last vaccination,and sacrificed for perfusion by 6 weeks post infection.Adult worms were collected,the number of eggs in a gram of liver tissue was counted,and the rates of worm reduction and egg reduction were calculated.Serum samples were collected before vaccination,every one week after each inoculation and be-fore sacrifice,and specific IgG was detected by ELISA.Results The sequences encoding the 96-147 aa of SjPGAM and 233-312 aa of SjEonl were chosen for constructing the recombinant plasmid,a cDNA fragment with the length of 447 bp was amplified by PCR.The recombinant plasmid was expressed in E.coli with a molecular weight of Mr 33 000.Western blotting revealed that the fusion protein was recognized by the rabbit serum specific to SjSWAP,and showed an adequate antigenicity.Vaccination experiment showed that when compared with those of the blank control,the worm re-duction rate in group A was 39.7%,significantly higher than that of groups B（18.5%）and C（14.7%）（P〈0.05）.The liver egg reduction rate in group A was 64.9%,also higher than that of groups B（47.5%,P〈0.05）and C（30.5%,P 〈0.01）.ELISA showed that the serum specific IgG in group A（2.372±0.268）was much higher than that of groups D（0.490±0.138）（P0.01）and E（0.220±0.088）（P0.01）.Conclusion The recombinant plasmid pET32a-SjPGAM-SjEnol has been constructed,and recombinant protein pET32a-SjPGAM-SjEnol induces higher immuno-protection against S.japonicum than that of SjPGAM and SjEonl.