两种血吸虫病DNA疫苗的候选抗原基因研究

Study of Two Candidates of Schistosoma japonicum Vaccine

目的:以日本血吸虫基因SjFABP和SjGST原核表达产物检测二价DNA疫苗pVIVO2-SjFABP-SjGST在体内诱发的特异性抗体。方法:克隆日本血吸虫抗原基因SjFABP和SjGST,构建重组原核表达载体pET30a-SjFABP、pET30a-SjGST及真核表达载体pVIVO2-SjFABP-SjGST;将pET30a-SjFABP和pET30a-SjGST进行原核表达,并将表达产物用镍亲和柱分离纯化;采用Western印迹对日本血吸虫DNA疫苗pVIVO2-SjFABP-SjGST免疫4周后的BALB/c小鼠血清进行特异性抗体检测。结果:克隆了日本血吸虫抗原基因SjFABP(399bp)和SjGST(657bp),并构建了pET30a-SjFABP、pET30a-SjGST及pVIVO2-SjFABP-SjGST重组质粒;经Western印迹检测,pET30a-SjFABP及pET30a-SjGST原核表达的抗原蛋白均能够与经日本血吸虫二价DNA疫苗pVIVO2-SjFABP-SjGST免疫的小鼠的血清产生特异性免疫反应。结论:日本血吸虫SjFABP和SjGST基因的原核表达系统成功建立;原核表达的抗原蛋白具有免疫原性;以原核表达产物可检测日本血吸虫DNA疫苗pVIVO2-SjFABP-SjGST在体内诱发的特异性抗体。

Objective： To test the expression of DNA vaccine of Schistosoma japonicum using prokaryotic expressed proteins SjFABP and SjGST of S.japonicum. Methods： Specific primers were designed, SjFABP and SjGST genes were amplified by RT-PCR and were cloned to pGEM-T Easy vector and sequenced, then were subeloned to build prokaryotie expression vectors pET30a-SjFABP, pET30a-SjGST and eukaryotie expression vector pVIVO2-SjFABP-SjGST. The E.Coli BL21 （DE3） cells were transformed with the recombinant plasmids pET30a-SjFABP and pET30a-SjGST respectively, then induced by IPTG to express the recombinant proteins which were purified through His-Bind Ni-Agarose. The purified recombinant proteins were used to recognize sera from mice induced by pVIVO2-SjFABP-SjGST. Results： The SjFABP（399 bp） and SjGST（657 bp） genes PCR products were obtained, and the recombinant plasmids pET30a-SjFABP, pET30a-SjGST and pVIVO2-SjFABP-SjGST were constructed, the prokaryotie expressed proteins were detected by SDS-PAGE. The rSjFABP and rSjGST can be recognized by the sera from mice induced by pVIVO2-SjFABP-SjGST using Western blotting. Conclusion： The SjFABP and SjGST genes were effectively expressed in prokaryotie cells, and can be recognized by antibody induced by DNA vaccine pVIVO2-SjFABP-SjGST.