中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (7): 1280-1284.doi: 10.3969/j.issn.1673-8225.2012.07.033

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

柯萨奇病毒B3VP1重组腺病毒载体疫苗rAd/VP22-L-VP1的构建及表达**★

李  剑，刘贵霞，米立国，蓝佳明，张永红，金玉怀   

  1. 河北医科大学病原生物学教研室，河北省石家庄市 050017
  • 收稿日期:2011-09-07 修回日期:2011-12-25 出版日期:2012-02-12 发布日期:2012-02-12
  • 通讯作者: 金玉怀，博士，教授，硕士研究生导师，河北医科大学病原生物学教研室，河北省石家庄市 050017 jinyuhuai@sina.com
  • 作者简介:李剑★，女，1982年生，河北省石家庄市人，汉族，2008年河北医科大学毕业，硕士，助教。lijian_amy@sina.com
  • 基金资助:

    河北省科技支撑计划(09276418D-5)；河北省自然科学基金(C2009001087)。

Construction and expression of adenovirus-based coxsackievirus B3 VP1 recombinant adenovirus vector vaccine rAd/VP22-L-VP1 

Li Jian, Liu Gui-xia, Mi Li-guo, Lan Jia-ming, Zhang Yong-hong, Jin Yu-huai   

  1. Department of Pathogenic Biology, Hebei Medical University, Shijiazhuang  050017, Hebei Province, China
  • Received:2011-09-07 Revised:2011-12-25 Online:2012-02-12 Published:2012-02-12
  • Contact: Jin Yu-huai, Doctor, Professor, Master’s supervisor, Department of Pathogenic Biology, Hebei Medical University, Shijiazhuang 050017, Hebei Province, China jinyuhuai@sina.com
  • About author:Li Jian★, Master, Teaching assistant, Department of Pathogenic Biology, Hebei Medical University, Shijiazhuang 050017, Hebei Province, China lijian_amy@sina.com
  • Supported by:

     the Science & Technology Pillar Program of Hebei Province, No. 09276418D-5*; Natural Science Foundation of Hebei Province, No. C2009001087*

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摘要：

背景：VP22是单纯疱疹病毒1型(Herpes simplex virus type 1，HSV-1)UL49基因编码的碱性蛋白质，具有蛋白转导结构域(protein transduction domain, PTD)，能够把与之融合的蛋白或与之结合的DNA 等大分子跨膜送递到邻近细胞，在基因靶向预防中表现出优势。
目的：构建表达单纯疱疹病毒1型VP22与柯萨奇病毒B3主要中和抗原VP1融合蛋白的重组腺病毒载体疫苗，观察外源基因在HEK293细胞中的良好表达。
方法：PCR法扩增目的基因HSV-1 VP22和CVB3 VP1，经Linker连接，将VP22-L-VP1插入腺病毒穿梭载体pAdTrack-CMV，构建重组穿梭质粒AdTrack-CMV/VP22-L-VP1。再将此载体与腺病毒骨架载体pAdEasy-1在大肠杆菌BJ5183中进行同源重组，生成重组腺病毒质粒pAd/VP22-L-VP1，脂质体介导pAd/VP22-L-VP1转染HEK293细胞包装重组腺病毒rAd/VP22-L-VP1。HEK293细胞上进行病毒扩增和滴定并检测外源基因的表达。
结果与结论：构建的重组腺病毒载体pAd/VP22-L-VP1经过第4轮扩增，其滴度达到6.77×107 pfu/mL，体外感染293细胞可见VP22和VP1融合蛋白的表达。说明实验成功构建并包装重组腺病毒rAd/VP22-L-VP1。

关键词: 柯萨奇病毒B3, 腺病毒载体, 基因疫苗, 单纯疱疹病毒1型VP22, 佐剂, 组织工程

Abstract:

BACKGROUND: Encoded by the UL49 gene of herpes simplex virus type 1 (HSV-1), VP22 is an alkaline protein. With the protein transduction domain (PTD), VP22 can mediate a transmembrane transduction of VP22-linked protein or DNA from the cells in which it is synthesized endogenously to adjacent cells, which shows advantage in gene targeting prevention.
OBJECTIVE: To construct a recombinant adenovirus based vaccine in order to express Vp22 of HSV-1 and the VP1, the main neutralizing antigen of coxsackievirus B3 (CV B3), and to observe the expression of exogenous gene in HEK293 cells. 
METHODS: The DNA fragments of HSV-1 VP22 and CVB3 VP1 were amplified by PCR and linked by linker to produce VP22-L-VP1. The VP22-L-VP1 coding sequence was cloned to the pAdTrack-CMV plasmid to construct AdTrack-CMV/VP22-L-VP1. Then AdTrack-CMV/VP22-L-VP1 was transformed into Ecoli.Bj5183 carrying backbone plasmid pAdEasy-1 to produce the recombinant adenovirus plasmid pAd/VP22-L-VP1. HEK293 cells were transfected with pAd/VP22-L-VP1 to produce recombinant adenovirus rAd/VP22-L-VP1. The virus amplification and titration was preformed on HEK293 cells and the exogenous gene expression was detected by Western blot.
RESULTS AND CONCLUSION: The titer of recombinant adenovirus rAd/VP22-L-VP1 of passage 4 was 6.77×107 pfu/ml. And the expression of VP22-L-VP1 was verified on HEK293 cells by Western blotting. Recombinant adenovirus rAd/VP22-L-VP1 was generated successfully, which laid a foundation for further study on CVB3VP1 gene vaccine.
 

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