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  

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

作　　者：朱路[1,2] 龙全科[1,2] 刘海峰[1,2] 鲁明波[1,2] 胡媛[3] 石佑恩[3] 余龙江[1,2] 

机构地区：[1]分子生物物理教育部重点实验室,武汉430074 [2]华中科技大学生命科学与技术学院,武汉430074 [3]华中科技大学同济医学院,武汉430030

出　　处：《中国生物工程杂志》2010年第5期36-42,共7页China Biotechnology

基　　金：国家"863"计划(2004AA2Z3212);教育部博士点基金(20070487059);教育部新世纪优秀人才计划(NCET-06-0646)资助项目

摘　　要：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 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.

关 键 词：日本血吸虫 脂肪酸结合蛋白 3-磷酸甘油醛脱氢酶 26kDa谷胱甘肽S转移酶 三价DNA疫苗 

分 类 号：Q78[生物学—分子生物学]