日本血吸虫三价DNA疫苗pVIVO2 SjFABP/Sj26.SjGAPDH的构建及其免疫保护作用评价

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。

Construction and Immuno-protection Evaluation of Trivalent-epitope DNA Vaccine pVIVO2-SjFABP/Sj26. SjGAPDH

Objective: To construct trivalent-epitope DNA vaccine encoding SjFABP, SjGAPDH and Sj26, and evaluate the protective immunity efficacy against Schistosoma japanicum by pharmacological trials. Methods: The monovalent antigen gene SjFABP, SjGAPDH and Sj26 were obtained by RT-PCR. Sj26 and SjGAPDH are spliced into covalent fusion gene of Sj26.SjGAPDH by recombinant PCR. pVIVO2-SjFABP/Sj26.SjGAPDH is constructed by subcloning the SjFABP and Sj26.SjGAPDH into mcs1 and mcs2 of pVIVO2-mcs respectively. The expression of fusion antigen protein in MCF-7 cell line transfected with pVIVO2-SjFABP/Sj26.GAPDH was determined by indirect immunofluorescence assay (IIF) and reverse transcriptase-polymerase chain reaction (RT-PCR). The evaluation of vaccine’s immuno-protection efficacy is tested by pharmacological trials. Results: The trivalent-epitope DNA vaccine pVIVO2-SjFABP/Sj26.SjGAPDH was successfully constructed. In the trivalent-epitope DNA vaccine group, the worm reduction rate and the egg reduction rate were 58.6% and 59.8% compared with control group. Conclusion: The trivalent-epitope DNA vaccine pVIVO2-SjFABP/Sj26.SjGAPDH could induce higher protection efficiency than both monovalent and bivalent vaccine, and paves the way for development of DNA vaccine against Schistosoma japanicum.