日本血吸虫(中国大陆株)磷酸丙糖异构酶DNA疫苗的研制及其保护性免疫的研究

A STUDY ON PROTECTIVE IMMNNITY OF DNA VACCINE ENCODING JAPONICUM CHINESE STRAIN TRIOSE-PHOSPHATE I SOMERASE IN INFECTED C_(57)BL/6 MICE

目的 研究日本血吸虫中国大陆株磷酸丙糖异构酶 DNA疫苗诱导 C5 7BL /6小鼠的保护性免疫作用。方法 分别以 p U C19- Sj CTPI和 pc DNA1.1- IL - 12为模板设计引物 ,将 PCR扩增到的Sj CTPI基因以及 IL - 12的亚单位 P35 和 P40 基因克隆到真核表达载体 pc DNA .3.1中。大量制备 pc D-NA3.1- Sj CTPI和 pc DNA 3.1- P35 、pc DNA3.1- P40 的质粒 DNA。把 45只 5～ 6周龄 C5 7BL /6小鼠分成 3组 ,对照组 (pc DNA组 )肌肉注射 10 0μg的 pc DNA3.1;实验组 (TPI组 )肌肉注射 10 0μg的pc DNA3. 1- Sj CTPI;加强组 (TPI+ IL - 12组 )肌肉注射 10 0μg的 pc DNA 3.1- Sj CTPI及 10 0μg的pc DNA3.1- P35 和 pc DNA3.1- P40 的混合物 ,分别于第 1、5周各免疫 1次 ,第 9周每鼠用 45条尾蚴攻击感染 ,攻击后 45 d剖杀小鼠 ,计数成虫、肝内虫卵。采用免疫组化法检测 Sj CTPI在局部肌组织内的表达 ,用脾细胞培养法检测特异性抗原刺激后的 IL - 2、IL - 4、IL - 10、IFN-γ在攻击前后的水平。分别于 2次免疫前和攻击前及攻击后 2周经尾静脉采血 ,分别采用 EL ISA和 Western- blot检测血清中抗 TPI抗体。结果 Sj CTPI可在 C5 7BL /6小鼠骨骼肌细胞膜、细胞浆中得到表达。细胞因子 IL -2的水平在攻击前、后 。

Objectives To develop SjCTPI DN A vaccine for schistosomiasis japonica, and test the immune responses and the pr otective efficacy after immunization and challenge in C 57 BL/6 mice.Methods According to the gene sequences of SjCTPI and murine IL 12, the primers were designed. The full length cDNA encoding SjCTPI and P 35 ,P 40 amplified from pUC19 SjCTPI and murine IL 12 by PCR we re subcloned into an eukaryotic expression vector (pcDNA3.1). Forty five of fem ale C 57 BL/6 mice were devided into three groups. Each mouse of control gr oup was injected with 100μg of pcDNA3.1 by intramuscle. TPI group were injected with 100μg of pcDNA3.1 SjCTPI, TPI+IL 12 group were injected with 100μg of pcDNA3.1 SjCTPI and 100μg of mixture of pcDNA3.1 P 35 and pcDNA3.1 P 40 .Each mouse was immunized at week 1 and week 5 and challenged with 45 cerc ariae of Schistosoma japonicum Chinese strain at week 9. Then the mice were killed and perfused 45 days after challenge. The num bers of recovered worms and hepatic eggs were counted. The expression of SjCTPI in muscle tissue was observed by immunohistochemical method. By the culture of s pleen cells, the production of IL 2,IL 4,IL 10 and IFN γ with the stimulati on of specific antigen was observed before and after challenge. Sera were collecte d from each group before each immunization, challenge and two weeks post challen ge. ELISA and Western blot were performed for detection of anti rTPI antibodie s. Results The antigen of SjCTPI could be expre ssed in the membrane and plasma o f the muscle cells of C 57 BL/6 mice. The obvious rising of IL 2 in TPI gro up and TPI+IL 12 group could be observed before and after challenge. The anti rTPI antibody detection with Western blot showed that before challenge, ten s erum samples from the control group were negative, nine of ten serum samples fro m TPI group were weak positive, eight of ten from TPI+IL 12 group were weak pos itive too. The worm, egg reduction rates of TPI group and TPI+IL 12 group were 27 9%,13 7% and 31 9%,18 6% respectively in comparison with the control gro up. Conclusion pcDNA3.1 TPI DNA vaccine could indu ce partial protection against Schistosoma japonicum in C 57 BL/6 mice and might serve as a candidate of DNA vaccine.