CAJ | 학술논문

目的建立T7RNA聚合酶真核表达质粒细胞内病毒拯救系统.方法利用分子生物学技术,以大肠埃希菌BL21(DE3)的T7RNA聚合酶基因为目的基因,构建T7RNA聚合酶真核表达质粒VR-1a并鉴定,然后将VR-1a和EV71病毒感染性克隆质粒共转染Vero细胞并传代,观察其细胞病变,同时用RT-PCR检测EV71病毒核酸和用ELISA检测EV71病毒抗原.结果酶切和测序显示成功构建T7RNA聚合酶真核表达质粒VR-1a,和EV71病毒感染性克隆共转染Vero细胞后出现明显的病变,RT-PCR检测EV71病毒核酸阳性并测序证实,ELISA检测显示有EV71病毒抗原.结论此方法可以用于细胞内拯救EV71病毒,有望应用于EV71病毒的核酸疫苗免疫研究.
목적 건립T7RNA취합매진핵표체질립세포내병독증구계통.방법 이용분자생물학기술,이대장애희균BL21(DE3)적T7RNA취합매기인위목적 기인,구건T7RNA취합매진핵표체질립VR-1a병감정,연후장VR-1a화EV71병독감염성극륭질립공전염Vero세포병전대,관찰기세포병변,동시용RT-PCR검측EV71병독핵산화용ELISA검측EV71병독항원.결과 매절화측서현시성공구건T7RNA취합매진핵표체질립VR-1a,화EV71병독감염성극륭공전염Vero세포후출현명현적병변,RT-PCR검측EV71병독핵산양성병측서증실,ELISA검측현시유EV71병독항원.결론 차방법가이용우세포내증구EV71병독,유망응용우EV71병독적핵산역묘면역연구.
Objective To develop a system to rescue virus by intracellular expression of T7 RNA Polymerase. Methods The gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-1a was then identified. VR-1a and EV71 infectious plasmid were co-transfected in Veto cell. CPE was observed and viral gene viral antigen were detected.Results The gene of T7 RNA Polymerase was successfully cloned into vector VR1012. Vere cell developed to CPE after being transfected VR-1a and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA. Conclusion The method can be used to rescue virus. It could apply to immunologic research of EV71 DNA vaccine.