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目的探讨重组日本血吸虫P14蛋白(SjP14)对血吸虫感染小鼠的免疫保护效果。方法构建SjP14原核表达质粒pET28a(+)SjP14,异丙基βD硫代半乳糖苷诱导表达重组蛋白rSjP14,纯化蛋白后,进行十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS PAGE)和Western blotting鉴定。将6周龄雌性BALB/c小鼠随机分为SjP14重组蛋白实验组(A组)、佐剂对照组(B组)、生理盐水对照组(C组),每组10只。每鼠每次注射抗原100μg,免疫3次,每次间隔2周,末次免疫后2周,血吸虫尾蚴攻击感染。分别于免疫前、免疫后6周和感染后6周经眶静脉采血,分离血清,采用酶联免疫吸附试验(ELISA)检测血清总IgG、IL 4和IFNγ。血吸虫尾蚴攻击感染后6周,剖杀小鼠,计算各组的减虫率和肝脏减卵率。结果SjP14原核表达产物相对分子质量(Mr)约为38 000;rSjP14能被SjP14多克隆抗体识别。血吸虫尾蚴攻击感染后6周,A组特异性IgG抗体水平升高,为(25.52±1.91)μg/ml,与C组[(18.65±3.16)μg/ml]和B组[(22.44±2.83)μg/ml]相比差异均有统计学意义(P均<0.05)。IFNγ浓度升高,为(171.30±70.12)ng/L,与C组[(136.89±37.62)ng/L]和B组[(153.64±43.44)ng/L]相比差异均有统计学意义(P均<0.05)。A组免疫后6周IL 4先略有升高,感染后6周下降,为(112.05±15.02)ng/L,与C组[(102.82±27.46)ng/L]相比差异有统计学意义(P<0.05)。A组小鼠减虫率和减卵率分别为29.2%和41.3%,与B、C组相比差异均有统计学意义(P均<0.05)。结论SjP14蛋白疫苗具有一定的抗血吸虫感染免疫保护作用。
Objective To investigate the immunoprotective effect of recombinant Schistosoma japonicum P14 protein (SjP14) on Schistosoma japonicum infected mice. Methods The SjP14 prokaryotic expression plasmid pET28a (+) SjP14 was constructed and the recombinant protein rSjP14 was induced by isopropyl βD thiogalactoside. The purified protein was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting identification. Six-week-old female BALB / c mice were randomly divided into experimental group (A group), adjuvant control group (B group) and saline control group (C group). Each mouse injected antigen 100μg, immunized three times, each interval of two weeks, two weeks after the last immunization, Schistosoma cercariae challenge infection. Blood samples were collected from the orbital venous before immunization, 6 weeks after immunization and 6 weeks after immunization. Serum total IgG, IL-4 and IFNγ were detected by enzyme-linked immunosorbent assay (ELISA). Six weeks after challenge with Schistosoma japonicum cercariae, the mice were sacrificed and the worm reduction and liver reduction rates were calculated for each group. Results The relative molecular mass (Mr) of prokaryotic expression product SjP14 was about 38 000; rSjP14 was recognized by SjP14 polyclonal antibody. The level of specific IgG antibody in group A was (25.52 ± 1.91) μg / ml at 6 weeks after challenge with cercariae of Schistosoma japonicum. Compared with group C [(18.65 ± 3.16) μg / ml] and group B [(22.44 ± 2.83) μg / ml], the differences were statistically significant (all P <0.05). Compared with C group [(136.89 ± 37.62) ng / L] and B group [(153.64 ± 43.44) ng / L], IFNγ concentration was significantly higher (171.30 ± 70.12 ng / L, P <0.05). IL-4 increased slightly at 6 weeks after immunization in group A and decreased to (112.05 ± 15.02) ng / L at 6 weeks after infection, which was significantly different from that in group C [(102.82 ± 27.46) ng / L] (P <0.05). The worm reduction rate and the egg reduction rate in group A were 29.2% and 41.3%, respectively, which were significantly different from those in group B and C (all P <0.05). Conclusion SjP14 protein vaccine has some anti-immune protection against Schistosoma japonicum infection.