日本血吸虫多价DNA疫苗的构建及鉴定  

Construction of multivalent DNA vaccine of Schistosoma japo-nicum and its identification

作　　者：王晓婷[1] 朱荫昌[1] 管晓虹[2] 司进[1] 赵松[1] D.A.Harn 殷旭仁[1] 何伟[1] 梁幼生[1] 徐明[1] 

机构地区：[1]江苏省血吸虫病防治研究所,江苏省寄生虫分子生物学重点实验室,江苏省寄生虫病学重点学科,无锡214064 [2]南京医科大学分子生物学研究所 [3]美国哈佛大学公共卫生学院

出　　处：《中国血吸虫病防治杂志》2005年第3期166-171,F003,共7页Chinese Journal of Schistosomiasis Control

基　　金：国家高技术研究发展计划(863计划)(2002AA214181)联合国开发署/世界银行/世界卫生组织热带病研究与培训特别规划署(TDR)(No.991051)

摘　　要：目的构建含日本血吸虫中国大陆株磷酸丙糖异构酶(TPI)基因和日本血吸虫单克隆抗独特型抗体NP30H链CDR3区6倍重复表位基因(CDR3)6的多价DNA疫苗,并观察该融合基因在真核细胞中表达。方法设计合成连接肽(Gly4Ser)3基因的两条互补单链,退火成双链后克隆到真核表达载体pcDNA3.1,改建为pcDNA3.1-linker。分别将SjCTPIDNA片段及NP30的(CDR3)6DNA片段克隆到连接肽的上游或下游,构建pcDNA3.1-TPI-linker-(CDR3)6和pcD-NA3.1-(CDR3)6-linker-TPI2种多价疫苗。设计引物分别扩增TPI-linker-(CDR3)6和(CDR3)6-linker-TPI,再分别插入真核表达载体pEGFP-N1多克隆位点,绿色荧光蛋白基因的上游,重组质粒用脂质体导入真核细胞COS-7,观察插入基因在真核细胞的表达。结果经双酶切及测序鉴定证实多价疫苗pcDNA3.1-TPI-linker-(CDR3)6和pcDNA3.1-(CDR3)6-linker-TPI构建成功;构建的pEGFP-N1-TPI-linker-(CDR3)6和pEGFP-N1-(CDR3)6-linker-TPI在导入COS-7细胞后能成功表达。结论日本血吸虫TPI和NP30-(CDR3)6的多价DNA疫苗构建成功,且该融合基因能在真核细胞中表达。Objective To construct multivalent DNA vaccine including the Triosephosphate iso-merase(TPI) gene and the NP30 6×VHCDR3 gene of Schistosoma japonicum. Methods The gene of (Gly4Ser)3 linker was cloned into pcDNA3.1(+),then either the TPI gene or the (CDR3)6 gene of Schistosoma japonicum was inserted before or behind the linker gene to construct the multivalent DNA vaccines: pcDNA3.1-TPI-linker-(CDR3)6 and pcDNA3.1-(CDR3)6-linker-TPI. The genes of TPI-linker-(CDR3)6 and (CDR3)6-linker-TPI(stop codon was removed) were cloned into pEGFP-N1 at upstream of the green fluorescent protein (GFP) respectively to construct the eukaryotic expression vector which could express the GFP. The recombinants were transfected into COS-7 cells by Lipofectamine?000. The expression of recombinants was observed under a fluorescent microscope. Results The multivalent DNA vaccines, pcDNA3. 1-TPI-linker-(CDR3)6 and pcDNA3.1-(CDR3)6-linker-TPI were successfully constructed. The two target genes both TPI-linker-(CDR3)6 and (CDR3)6-linker-TPI were expressed in COS-7 cells after transfection. Conclusion The multivalent DNA vaccines are successfully constructed and the target genes can be expressed in eukaryot- ic cells.

关 键 词：日本血吸虫 磷酸丙糖异构酶 NP30-(CDR3) 多价DNA疫苗 

分 类 号：R383.24[医药卫生—医学寄生虫学]