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目的:构建含有日本血吸虫中国大陆株脂肪酸结合蛋白(SjFABP),3-磷酸甘油醛脱氢酶(SjGAPDH),26kDa谷胱甘肽S转移酶(Sj26)的三价DNA疫苗,并通过药理实验评价其抗血吸虫感染的免疫保护作用。方法:以血吸虫成虫RNA为模板制备cDNA第一链,RT-PCR扩增得到抗原基因片段,再通过重组PCR技术,将Sj26和SjGAPDH融合为Sj26.SjGAPDH基因。将SjFABP和Sj26.SjGAPDH分别克隆入pVIVO2的mcs1和mcs2,获得重组质粒pVIVO2-SjFABP/Sj26.SjGAPDH。瞬时转染MCF-7细胞,通过逆转录PCR(RT-PCR)检测抗原基因在mRNA水平的表达,通过间接荧光免疫(IIF)检测抗原基因在小鼠体内抗原水平表达,验证了DNA疫苗的有效性;并免疫小鼠后进行免疫保护性初步试验。结果:经过酶切、测序及体内外表达验证,该三价DNA疫苗pVIVO2-SjFABP/Sj26.SjGAPDH构建成功;该三价DNA疫苗在小鼠抗血吸虫感染中诱发减虫率和减卵率达到58.6%和59.8%。结论:该三价DNA疫苗组具有比单价和双价DNA疫苗组更加良好的免疫保护作用,为日本血吸虫DNA疫苗的研制奠定了基础。
OBJECTIVE: To construct a trivalent DNA vaccine containing SjFABP, SjGAPDH and 26kDa glutathione S-transferase (Sj26) from Chinese mainland strains of Schistosoma japonicum, and to evaluate their pharmacological effects Its anti-schistosome infection of the immune protection. Methods: The first strand cDNA of Schistosoma japonicum was used as a template to amplify the antigen gene fragment by RT-PCR. Then Sj26 and SjGAPDH were fused into Sj26.SjGAPDH gene by recombinant PCR. SjFABP and Sj26.SjGAPDH were cloned into mcs1 and mcs2 of pVIVO2 respectively to obtain the recombinant plasmid pVIVO2-SjFABP / Sj26.SjGAPDH. MCF-7 cells were transiently transfected, and the expression of antigen gene at mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of antigen gene in mice was detected by indirect fluorescent immunofluorescence (IIF) ; And immunization of mice after initial immunoprotection assay. Results: The trivalent DNA vaccine pVIVO2-SjFABP / Sj26.SjGAPDH was successfully constructed by restriction enzyme digestion, sequencing and in vitro and in vivo expression. The trivalent DNA vaccine induced worm reduction rate and oviposition rate in mouse anti-schistosome infection reached 58.6 % And 59.8%. Conclusion: The trivalent DNA vaccine group has better immunoprotection than monovalent and bivalent DNA vaccine group, which lays a foundation for the development of DNA vaccine of Schistosoma japonicum.