鸡柔嫩艾美耳球虫三价DNA疫苗的构建及鉴定

Construction and Identification of Trivalent Eimeria tenella DNA Vaccine

[目的]构建含鸡柔嫩艾美耳球虫3-1E和CDPK抗原基因及鸡IFN-γ基因的三价DNA疫苗。[方法]应用SOE-PCR将3-1E、CD-PK和鸡IFN-γ共3个基因通过2个(GGGGS)3连接子相连,扩增出IFN-γ/3-1E/CDPK三价融合基因,将其克隆入真核表达载体proVAX中构建三价融合真核表达质粒proIEC,后用双酶切和PCR进行鉴定。阳性重组质粒提取纯化后体外转染PK-15细胞,通过间接免疫荧光技术检测目的基因在转染细胞中的表达情况。[结果]经双酶切和PCR鉴定证实鸡柔嫩艾美耳球虫三价DNA疫苗构建成功,且在PK-15细胞中获得了成功表达。[结论]鸡柔嫩艾美耳球虫IFN-γ/3-1E/CDPK三价DNA疫苗构建成功,为进一步探讨其免疫保护性奠定了基础。

[Objective] To construct trivalent DNA vaccine including the 3-1E,CDPK antigen genes of Eimeria tenella and chicken IFN-γ gene.[Method] The IFN-γ/3-1E/CDPK fusion gene which was linked with two hydrophobic polypeptide linkers（Gly4Ser）3 was amplified by SOE-PCR.Then the fusion gene was cloned into the mammalian expression vector proVAX to form the chimeric plasmid proIEC identified by enzyme digestion and PCR.The purified recombinant plasmid was transfected into PK-15 cells in vitro and its expressed protein was detected by indirect immunofluorescence assay.[Result] The trivalent DNA vaccine was successfully constructed and the target fusion gene was expressed in PK-15 cells.[Conclusion] The trivalent DNA vaccine is successfully constructed and the target gene can be expressed in eukaryotic cells,which can provide valuable support for further study of its immunoprotection.