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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 the recombinant plasmid pET32a-SjPGAM-SjEnol of Schistosoma japonicum and express it in Escherichia coli (BL21), and to observe the protective effect of the recombinant antigen on the infection of the mouse. Methods The bioinformatics techniques were used to screen the SjPGAM and SjEnol-rich peptide fragments with low homology with human host (HLA) Ⅱ and murine (H2-d) Ⅱ cell-binding epitopes, The acid sequence was spliced to construct the recombinant plasmid pET32a (+) - SjPGAM-SjEnol and expressed in E. coli BL21. The antigenicity of the recombinant antigen was analyzed by Western blotting. Fifty-five male BALB / c mice were equally divided into five groups. The three experimental groups were immunized with the recombinant antigens pET32a-SjPGAM-SjEnol (group A), pET28a-SjPGAM (B Group), pET28a-SjEnol (group C) (27 μg each) and 206 adjuvant. The mice were immunized three times at intervals of 2 weeks. At the same time adjuvant control group (D group) and blank control group (E group). Two weeks after the last immunization, 40 ± 2 of Schistosoma japonicum cercariae were infected in the abdomen skin of each mouse. After 6 weeks of infection, adult mice were collected by hepatic portal vein perfusion method and the number of eggs per gram of testis (EPG) was detected to calculate the worm reduction rate Egg reduction by the liver. The mice in each group were collected before the immunization, one week after the last immunization, and the serum was collected at the time of dissection. Serum specific IgG was detected by ELISA. The results confirmed that SjPGAM 96 ~ 147, SjEnol 233 ~ 312 peptide fragment as a recombinant fragment. PCR amplified a recombinant DNA sequence containing the nucleotide sequence encoding these two peptides, size 447bp. The molecular weight (Mr) of the recombinant protein pET32a-SjPGAM-SjEnol obtained was 33,000. Western blotting showed that the recombinant protein could be recognized by rabbit serum of adult Schistosoma japonicum antigen and had good antigenicity. The results of mouse immunization showed that the worm reduction rate of 39.7% and the liver ejaculation rate of 64.9% in Group A were significantly lower than those in the blank control group, and the worm reduction rates of Group A were significantly lower than those in Group B (18.5%) and Group C (14.7%) (P <0.05). There was also a significant difference in the rate of liver egg reduction between group B (47.5%) and group C (30.5%) (P <0.05, P <0.01). The result of ELISA showed that the specific IgG antibody in group A reached a high level (2.372 ± 0.268) after the third immunization, and there was significant difference with group D (0.490 ± 0.138) and group E (0.220 ± 0.088) P <0.01). Conclusion The recombinant plasmid pET32a-SjPGAM-SjEnol was constructed successfully. The multi-epitope recombinant protein pET32a-SjPGAM-SjEnol induced higher immunoprotection than single recombinant antigens pET28a-SjPGAM and pET28a-SjEnol in mouse anti-schistosome infection.