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不同脊髓灰质炎病毒株3CD蛋白酶在昆虫细胞中的表达及其对P1前体蛋白的剪切 预览

Expression of 3CD proteases from different poliovirus strains in insect cells and their cleavage efficiency of the P1 protein from the Mahoney strain
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摘要 目的获得在昆虫细胞中有效表达脊髓灰质炎病毒P1基因和3CD基因的重组杆状病毒,为制备脊髓灰质炎病毒样颗粒疫苗提供了科学依据。方法将I型脊髓灰质炎病毒(Mahoney株)的P1基因和3CD基因构建到供体质粒中,通过flash BAC ULTRATM系统制备重组杆状病毒;将Mahoney株的P1基因与Sabin株Ⅲ型的3CD基因组合,用同样方法构建重组杆状病毒。通过接种昆虫细胞草地贪夜蛾细胞(sf-9细胞)对两种病毒进行扩增,再接种昆虫细胞粉纹夜蛾细胞(High five细胞)扩大培养,并通过定量PCR对P1和3CD基因的表达进行验证,利用Western blot检测P1蛋白的表达及被3CD蛋白酶剪切的情况。结果获得了两株稳定表达脊髓灰质炎病毒P1和3CD基因的重组杆状病毒。其中,重组杆状病毒(Bac U-Mahoney-P1-3CD)在感染细胞后,3CD蛋白酶表达量较低,不能有效剪切P1前体蛋白;而重组杆状病毒(Bac U-Mahoney-P1-Sabin PV3 3CD)感染细胞后,3CD蛋白酶的表达量和对P1前体蛋白的剪切效力都有明显提高(P〈0.05)。结论将Mahoney株P1基因和Sabin株Ⅲ型的3CD基因的组合构建重组杆状病毒,可有效地在昆虫细胞中表达P1和3CD基因,并且Sabin株Ⅲ型的3CD蛋白酶可有效地剪切Mahoney株的P1前体蛋白。 Objective To generate recombinant baculoviruses which can be used to express both poliovirus P1 and 3 CD genes in insect cells,so as to provide a scientific basis in preparation of virus-like particles( VLPs) vaccine in control of poliomyelitis. Methods Two recombinant baculoviruses were constructed using the flash BAC ULTRATMvector system. The viruses carried either the Mahoney P1/3 CD genes or the Mahoney P1/Sabin type Ⅲ 3 CD genes. Transcription levels of the P1 and 3 CD genes were determined by quantitative real-time reverse transcription PCR. Expression of the P1 precursor protein and the cleavage of the P1 precursor protein by the 3 CD protease were confirmed by Western blot. Results Two recombinant baculoviruses,Bac U-Mahoney-P1-3 CD and Bac UMahoney-P1-Sabin type Ⅲ 3 CD,were successfully generated.The insect cells infected with Bac U-Mahoney-P1-Sabin PV3 3 CD showed good expression of both P1 precursor protein and 3 CD protease,resulting in efficient cleavage of P1 precursor protein by the 3 CD protease. Conclusion Mahoney P1 gene and Sabin type Ⅲ 3 CD gene were co-expressed in insect cells. The 3 CD protease of Sabin type Ⅲ strain could efficiently cleave the P1 precursor protein from the Mahoney strain.
作者 马金 李军强 莘春林 段静留 朱涛 MA Jin, LI Jun-qiang, XIN Chun-lin, Duan Jing-liu, ZHU Tao(Post Doctoral Research Station of Tianjin Economic and Technological Development Area Cansino Biologics lnc sub station, Tianjin Key Laboratory of Respiratory Bacteria Recombination and Conjugate Vaccine Enterprise, Cansino Biologics Inc, Tianjin 300457, China)
出处 《微生物学免疫学进展》 2018年第1期1-6,共6页 Progress In Microbiology and Immunology
基金 国家“重大新药创制”科技重大专项资助项目(2015ZX09102009) 天津市科技计划项目(16PTWYHZ00070)
关键词 脊髓灰质炎病毒 草地贪夜蛾细胞 3CD蛋白酶 P1前体蛋白 重组杆状病毒 类病毒颗粒 Poliovirus Spodoptera frugiperda cell 3CD protease P1 precursor protein Recombinant baculovirus Viruslike particles(VLPs)
作者简介 马金(1985-),男,博士后,主要从事类病毒颗粒疫苗的研发工作。;通信作者:朱涛,教授,E-mail:tao.zhu@cansinotech.com
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  • 1张延龄,张晖主编..疫苗学[M].北京:科学出版社,2004:1500.

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