生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (6): 7-.doi: 10.3969/j.issn.2095-1736.2023.06.007

• 研究报告 • 上一篇    下一篇

表达G Ⅱ .4型诺如病毒VP1复制缺陷型腺病毒构建与鉴定

施　鹏， 林晓晨， 周　艳， 李娇春， 宋泽鑫， 鲁辰星， 谭银珍， 胡晓青， 陈　蓉， 李鸿钧   

  1. 中国医学科学院北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发
    重点实验室， 昆明 650118
  • 出版日期:2023-12-18 发布日期:2023-12-18
  • 通讯作者: 李鸿钧，博士，研究员，研究方向为病毒载体及干细胞等对神经系统退行性疾病、糖尿病等的基因治疗及病毒疫苗、基因工程药物研究开发，E-mail:lihj6912@hotmail.com
  • 作者简介:施鹏，硕士，主要从事生物化学与分子生物学相关研究工作，E-mail:1506249032@qq.com
  • 基金资助:
    云南省应用基础研究计划项目 (202201AT070236)； 云南省重大科技专项 (202202AA100006)； 中国医学科学院医学与健康科技创新工程重大协同创新项目 (2021-I2M-1-043)

Construction and identification of replication defective adenovirus expressing VP1 of G Ⅱ .4 norovirus

SHI Peng, LIN Xiaochen, ZHOU Yan, LI Jiaochun, SONG Zexin, LU Chenxing, TAN Yinzhen, HU Xiaoqing, CHEN Rong, LI Hongjun   

  1. Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Disease, Institute of
    Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College, Kunming 650118, China
  • Online:2023-12-18 Published:2023-12-18

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摘要： 人诺如病毒（norovirus , NoV）在体外难以培养，不宜采用经典疫苗的方式进行疫苗研发，以腺病毒为载体的重组腺病毒疫苗为诺如病毒疫苗的研发提供另一种路线选择。构建表达人G Ⅱ .4型诺如病毒VP1蛋白的重组腺病毒为该技术路线的疫苗研发奠定基础。首先使用PCR扩增诺如病毒VP1片段，通过酶切、连接、转化，克隆含VP1基因的pshuttle-CMV-VP1穿梭质粒，再与腺病毒骨架质粒进行同源重组，重组质粒鉴定正确后使用PacⅠ酶切线性化，转染至HEK293细胞进行病毒包装，包装成复制缺陷型的完整腺病毒颗粒命名为rAd-VP1，病毒传代后进行滴度测定，并感染HEK293细胞，通过Western Blot方法鉴定重组腺病毒外源蛋白VP1的表达情况。结果表明，包装传代后的重组腺病毒浓缩后滴度达到CCID50=5×109/mL，并且能在HEK293细胞成功表达VP1蛋白。研究为rAd-VP1的动物免疫评价和诺如病毒的疫苗研发提供基础。

关键词: 诺如病毒, 主要结构蛋白VP1, 腺病毒载体, 同源重组, 病毒包装

Abstract: Human norovirus (norovirus,NoV) is difficult to be cultured in vitro, so it is not suitable to use the classical vaccine method for vaccine development. The recombinant live virus vaccine based on adenovirus provides an alternative route for the development of norovirus vaccine. The construction of recombinant adenovirus expressing VP1 protein of humanG Ⅱ .4norovirus may lay a foundation for the research and development of vaccine of this technical route. Firstly, the VP1 fragment of norovirus was amplified by PCR, and the pshuttle-CMV-VP1 shuttle plasmid containing VP1 gene was cloned by enzyme digestion, ligation and transformation, and then homologous recombination was performed with adenovirus skeleton plasmid. After correct identification, the recombinant plasmid was digested and linearized byPacⅠenzyme, and transfected into HEK293 cells for virus packaging. The intact adenovirus particles packaged into replication-deficient type were named rAd-VP1. The titer of the virus was determined after passage, and the recombinant adenovirus was infected with HEK293 cells. The expression of recombinant adenovirus VP1 was identified by Western Blot. The results showed that the concentrated titer of recombinant adenovirus after packaging and passage reached CCID50=5×109/mL, and VP1 protein could be successfully expressed in HEK293 cells. It provided a basis for the evaluation of rAd-VP1 in animal immunization and the development of norovirus vaccine.

Key words: norovirus, major structural protein VP1, adenoviral vector, homologous recombination, virus packaging

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