日本血吸虫重组SjTsp2/Sj29ku蛋白疫苗对小鼠免疫效果的研究

Investigation of the anti-schistosomiasis effect of SjTsp2 /Sj29 ku surface membrane protein from Schistosoma japonicum

目的探讨日本血吸虫重组Tsp2/Sj29 ku表膜蛋白疫苗对小鼠的抗病免疫效果。方法 pet32a/SjTsp2/Sj29 ku重组菌经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,制备纯化的重组蛋白。29只6周龄雌性昆明鼠随机分为蛋白疫苗组、硫氧还蛋白组和空白对照组,前两组各10只,空白对照组9只。蛋白疫苗组,第一次免疫每鼠经背部皮下多点注射用等体积弗氏完全佐剂乳化的50μg SjTsp2/Sj29 ku重组表膜蛋白,第12、24天每鼠皮下多点再次注射用等体积弗氏不完全佐剂乳化的50μg该重组蛋白。硫氧还蛋白组的免疫方法同蛋白疫苗组。空白对照组用生理盐水代替蛋白,免疫方法同前。末次免疫后第10天,每鼠经腹部皮肤感染日本血吸虫尾蚴(30±2)条。于感染45 d后剖杀全部实验小鼠,计算减虫率和减卵率。眼眶取血,分离血清,间接性ELISA法检测IgG1、IgG2a、IgG2b、IgG3和IgM抗体亚型,同时检测血清细胞因子γ干扰素(IFN-γ)、白介素4(IL-4)水平。结果 pet32a/SjTsp2/Sj29 ku菌经IPTG诱导可大量表达,经变性、复性获得纯化的日本血吸虫Tsp2/Sj29 ku重组表膜蛋白。蛋白疫苗组与空白对照组比较,减虫率和减卵率显著升高(P<0.05)。硫氧还蛋白组与空白对照组之间比较,减虫率和减卵率差异均无统计学意义(P>0.05)。蛋白疫苗组小鼠血清中IgG1、IgG2a、IgG2b显著高于空白对照组(P<0.05),而IgG3和IgM较空白对照组差异无统计学意义(P>0.05)。蛋白疫苗组小鼠血清IFN-γ、IL-4细胞因子显著高于空白对照组(P<0.01)。结论日本血吸虫Tsp2/Sj29 ku重组表膜蛋白疫苗对小鼠抗感染有一定的保护性;该重组蛋白免疫引起的是Th1/Th2混合型免疫反应。

Objective To investigate the anti-schistosomiasis effect of Tsp2/Sj29 ku protein from Schistosoma japonicum. Methods The recombinant protein pet32a/SjTsp2/Sj29 ku induced and expressed by IPTG was puri-fied. Twenty nine female 6-week old mice were randomly divided into 3 groups （ Protein vaccine Group, Thioredox-in Group and Control Group）. In Protein vaccine Group （n=10）, each mouse was immunized subcutaneously with 50 μg SjTsp2/Sj29 ku plus equal volume Freund＇s complete adjuvant in 0 day. 12 and 24 days later, each mouse was re-immunized subcutaneously with 50 μg SjTsp2/Sj29 ku plus equal volume Freund＇s incomplete adjuvant. In Thioredoxin Group （ n=10 ） and Control Group （ n=9 ） , each mouse was immunized with thioredoxin and saline respectively. Ten days after last immunization, each mouse was challenged with 30±2 cercaria of Schistosoma ja-ponicum. On the 45th day post-infection, the mice were exeuted. The worm and egg reduction rates were calculat-ed. The sera was collected from orbital blood. The levels of IgG1, IgG2a, IgG2b, IgG3 and IgM antibodies in mouse sera were detected by enzyme-linked immunosorbentassay （ ELISA） . The level of IFN-γ and IL-4 was also detected by ELISA. Results Pet32a/SjTsp2/Sj29 ku was obtained as expected. The worm reduction rates and egg reduction rates in Protein vaccine Group were 23. 95% （P〈0.05）and 55. 57% （P〈0.05） respectively, com-pared with the Control Group. There was no statistical significance （ P 〉0.05 ） between Thioredoxin Group and Control Group in worm reduction rates and egg reduction rates. In Protein vaccine Group, IgG1, IgG2a and IgG2b were remarkably higher than that of Control Group, while IgG3 and IgM did not remarkably change. In comparison with the Control Group, the levels of IFN-γand IL-4 of the mice in Protein vaccine Group were obviously augmen-ted （P〈0.01）. Conclusion The mice, which have been immunized with Tsp2/Sj29 ku show a partial immune protection;the protein vaccine of SjTsp2/Sj29 ku could induce the mixed immune responses of Th1 and Th2 .