重组日本血吸虫信号蛋白14-3-3不同剂量对免疫保护性的影响

Influence of immunization dose schemes on immunoprotective response to recombinant signaling protein 14-3-3 of Schistosoma japonicum

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 dicuss the optimal immunization dose by observing the immunoprotective effects of different doses of recombinant Schistosoma japonicum（ Chinese strain） signaling protein 14-3-3 （rSj14-3-3）. Methods Sj14-3-3 gene was amplified by reverse transcriptase PCR （RT-PCR）, subcloned into prokaryotic expression vector pET28a, then transformed into E. coli to express by inducing. Purified rSj14-3-3 was prepared through SDS polyacrylamide gel electrophoresis （SDS-PAGE）, eleetroelution, dialysis, then BALB/c mice were divided into 5 groups and immunized in rSj14-3-3 protein followed by challenging infection （the 1st, 2nd, and 3rd groups were immunized in 50 μg, 100 μg and 300 μg antigen, respectively. The 4th, 5th groups were immunized in Freund＇s adjuvant and normal saline controls）. After 6 weeks of challenging infection, the mice were killed and the worm and egg reduction rates were calculated. And the mice sera in different time were taken to examine the specific anti-Sj14-3-3 IgG. Results rSj14-3- 3 protein was expressed successfully. After immunizing and challenging , worm reduction was found to be 28.20% in the 1 st group, 43.10% in the 2nd group, 40. 00% in the 3rd group, respectively. Number of eggs in liver tissue was reduced by 41.80%, 57.50% ,55.70% ,respectively. Compared the results of the tested groups to the controls, the differences were of significance by t-test （worm reduction rate： t = 6. 8 in the 1 st group, t =8. 7 in the 2nd group, t =7.3 in the 3rd group, P 〈0. 01 in all tested groups. Egg reduction rate at the group＇s number above： t = 11.23, t = 11.54, t =7.99, P 〈0. 01 in all tested groups）. As compared the results between the tested groups by x^2, the differences were of significance between the 1st and the 2nd groups （worm reduction rate： x^2 = 8.96, P 〈 0. 05 ; egg reduction rate ： x^2 = 15. 69, P 〈 0. 05 ）, between the 1st and the 3rd roups, the differences were also of significance （worm reduction rate：x^2 =6. 52,P 〈0. 05 ; egg reduction rate：x^2 = 12. 52, P 〈0. 05）. The difference was not of significance between the 2nd and the 3rd groups （worm reduction rate： x^2 = 1.20, P 〉 0. 05 ; egg reduction rate ： x^2 = 0. 93, P 〉 0. 05 ）. In all tested groups, total anti-Sj14-3-3 specific IgG rised markedly. IgG1 and IgG2a subtypes were high, but IgG2b and IgG3 were near the background in four subtypes tested. Condusion Immunoprotection of rSj14-3-3 should have some relations with immunization dose, and the protection obtained from immunizing mice by using 100 μg antigen was the best.