摘要
为构建A型塞内卡病毒(SVA)P12A-3C基因的真核表达质粒,提取SVACH-FJ-2017株的RNA,经RT-PCR方法扩增得到衣壳前体蛋白P12A基因和蛋白酶3C基因,利用融合PCR方法获得P12A-3C基因并将其构建到真核表达质粒pc DNATM3.1/myc-His(-)上,获得表达质粒pCD-P12A-3C,用脂质体LipofectamineTM2000将该质粒转染293T细胞后,进行间接免疫荧光(IFA)检测;且用Myc单抗和SVA VP1单抗进行Western-blot分析;大量提取并纯化表达质粒,免疫小鼠后用间接ELISA法检测抗体水平变化情况。结果,SVA真核表达质粒pCD-P12A-3C经酶切及测序鉴定证明构建正确,转染293T细胞后IFA可见明显的绿色荧光,表明P12A-3C基因可在细胞内表达;将转染细胞裂解后进行Western-blot分析,结果显示该质粒可表达3C蛋白,且表达产物可与SVA VP1单抗发生特异性反应;免疫小鼠后抗体检测结果表明,二免后第14天,免疫小鼠抗体水平比对照组明显增高;该结果表明本研究构建的真核表达质粒pCD-P12A-3C可正确表达相应蛋白,且所表达的蛋白具有良好的反应原性和免疫原性。结论:本研究成功构建了A型塞内卡病毒P12A-3C基因真核表达质粒并进行了免疫效力初步验证,为A型塞内卡核酸疫苗的研制奠定了基础。
To construct and identify a eukaryotic expression plasmid for the P12 A-3 C gene of Senecavirus A,the RNA of SVA CH-FJ-2017 strain was extracted,and the capsid precursor protein P12 A gene and protease 3 C gene were amplified by RT-PCR method.The P12 A-3 C gene was obtained by fusion PCR method and constructed into the eukaryotic expression vector pc DNATM3.1/myc-His(-),the expression plasmid p CD-P12 A-3 C was obtained,then the plasmid was transfected into 293 T cells with LipofectamineTM2000,the expression of plasmid was verified by indirect immunofluorescence(IFA)and Western-blot.The expression plasmids were extracted and purified in large quantities,and the changes of antibody levels were detected by indirect ELISA after immunizing mice.In result,the SVA eukaryotic expression plasmid p CD-P12 A-3 C was proved to be constructed correctly by enzyme digestion and sequencing.IFA showed obvious green fluorescence after transfecting 293 T cells,indicating the plasmid can be expressed in cells.Western-blot analysis after lysis of transfected cells shows that the plasmid can express 3 C protein,and the exoression product can specifically react with SVA VP1 monoclonal antibody.The results of antibody test after immunizing mice showed that after 14 days of enhanced immunity,the antibody level of immunized mice was significantly higher than that of the control groups.These results indicated the P12 A-3 C gene was expressed.and the proteins had good reactivity and immunogenicity.In conclusion,the eukaryotic expression plasmid of Seneca virus A P12 A-3 C gene was successfully constructed and the preliminary immunopotency was verified,which laid a foundation for the development of Senecavirus A nucleic acid vaccine.
作者
魏婷
杨帆
张伟
王志芳
孙晓林
郑海学
WEI Ting;YANG Fan;ZHANG Wei;WANG Zhi-fang;SUN Xiao-lin;ZHENG Hai-xue(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 703370,China;Stale Key Laboratory of Veterinary Etiological Biology/National Foot and Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2020年第7期861-868,共8页
Chinese Veterinary Science
基金
国家重点研发计划项目(2017YFD0501103)
甘肃省科技重大专项(19ZDNA001)。
作者简介
魏婷(1993-),女,甘肃兰州人,硕士生,E-mail:1756970984@qq.com;通讯作者:孙晓林,教授,主要从事兽医公共卫生学的教学与研究工作,E-mail:sunxl@gsau.edu.cn;通讯作者:郑海学,研究员,主要从事口蹄疫及动物新发疫病防控技术的研究,E-mail:zhenghaixue@caas.cn。
