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目的观察不同剂量重组日本血吸虫(中国大陆株)信号蛋白14-3-3(rSj14-3-3)的免疫保护性,以探讨最佳的免疫剂量。方法以逆转录-多聚酶链反应(RT-PCR)扩增Sj14-3-3基因,并将其亚克隆入原核表达载体pET28a,然后转化大肠埃希菌进行诱导表达;用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、电洗脱和透析的方法制备纯化的rSj14-3-3蛋白,分5组(第1、2、3组为rSj14-3-3蛋白50μg、100μg和300μg实验组,第4、5组分别为福氏佐剂和生理盐水对照组)免疫BALB／c小鼠并进行攻击感染实验。在尾蚴攻击感染6周后,剖杀小鼠计算各组的减虫率和减卵率。并在试验过程中留取不同时期小鼠血清,检测抗Sj14-3-3特异性IgG抗体。结果成功表达出rSj14- 3-3蛋白,纯化蛋白免疫小鼠行攻击感染后各实验组的减虫率为第1组28．20％、第2组43．10％、第3组40．00％;各组的减卵率分别为(按以上组序)41．80％、57．50％、55．70％。各实验组与对照组比较,差异有统计学意义(减虫率:第1组t=6．8、第2组t=8．7、第3组t=7．3,各组P值均<0．01;减卵率:第1组t=11．23、第2组t=11．54、第3组t=7．99,各组P值均<0．01);各实验组间两两比较,第1组与第2组间(减虫率:X2=8．96,P<0．05;减卵率:X2=15．69,P<0．05)、第1组与第3组间(减虫率:X2=6．52,P<0．05;减卵率:X2=12．52,P<0．05)差异有统计学意义;第2、3两组间差异无统计学意义(减虫率:X2=1．20,P>0．05;减卵率:X2=0．93,P>0．05)。各实验组的抗Sj14-3-3特异性总IgG水平都有显著升高,所测的4个亚型中以IgG1和IgG2a亚型升高为主,IgG2b和IgG3亚型升高不明显。结论重组Sj14-3-3的免疫保护性与免疫剂量有一定关系,以100μg抗原量免疫小鼠获得的保护性最好。
Objective To observe the immunoprotection of signaling protein 14-3-3 (rSj14-3-3) of different doses of recombinant Schistosoma japonicum (Chinese mainland strain) to explore the best immunization dose. Methods Sj14-3-3 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) and subcloned into prokaryotic expression vector pET28a. The recombinant plasmid was transformed into Escherichia coli for induction of expression. Purified rSj14-3-3 protein was prepared by sodium-polyacrylamide gel electrophoresis (SDS-PAGE), electroelution and dialysis. The purified rSj14-3-3 protein was divided into 5 groups (groups 1, 2 and 3 were rSj14-3-3 protein 50μg , 100μg and 300μg experimental groups, groups 4 and 5 were Freund’s adjuvant and saline control group respectively) were immunized BALB / c mice and challenge infection experiments. After 6 weeks of cercariae challenge infection, mice were sacrificed and the worm reduction and oviposition rates were calculated for each group. And in the course of the experiment to take different periods of mouse sera, anti-Sj14-3-3 specific IgG antibodies. Results The rSj14-3-3 protein was successfully expressed. The worm reduction rates of the experimental groups after challenge infection were 28.20% in group 1, 43.10% in group 2 and 40.00% in group 3 %; The egg reduction rates of each group are (respectively, according to the above sequence) 41.80%, 57.50%, 55.70%. The experimental group and the control group, the difference was statistically significant (worm reduction rate: the first group t = 6.8, the second group t = 8.7, the third group t = 7.3, P values ?? were <0.01; the oviposition rate: the first group t = 11.23, the second group t = 11.54, the third group t = 7.99, each group P <0.01) Between groups 1 and 2 (worm reduction rate: X2 = 8.96, P <0.05; egg reduction rate: X2 = 15.69, P <0.05) And the third group (worm reduction rate: X2 = 6.52, P <0.05; egg reduction rate: X2 = 12.52, P <0.05) The difference was not statistically significant (worm reduction rate: X2 = 1.20, P> 0.05; egg reduction rate: X2 = 0.93, P> 0.05). The levels of total anti-Sj14-3-3 specific IgG in all experimental groups were significantly increased. The four subtypes tested were mainly IgG1 and IgG2a subtypes, and IgG2b and IgG3 subtypes were not significantly increased. Conclusion The protective immunity of recombinant Sj14-3-3 is related to the dose of immunization. The protective effect of immunized mice with 100μg antigen is the best.