Construction of Recombinant Adenovirus Vectors Using the DNA Assembly Method

doi:10.13523/j.cb.2102031

China Biotechnology

2021, Vol. 41

Issue (6): 23-26 DOI: 10.13523/j.cb.2102031

Construction of Recombinant Adenovirus Vectors Using the DNA Assembly Method

HUANG Lei,WAN Chang-qing,LIU Mei-qin,ZHAO Min,ZHENG Yan-peng,PENG Xiang-lei,YU Jie-mei,FU Yuan-hui(

),HE Jin-sheng(

)

College of Life Sciences & Bioengineering, Beijing Jiaotong University, Beijing 100044, China

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Abstract

Objective: To establish a recombinant adenovirus vector construction method based on the DNA Assembly method. Methods: First, by designing suitable restriction sites and homology arms, the backbone plasmid pChAd63 of Chimpanzee adenovirus serotype 63 (ChAd63) was obtained using traditional restriction endonuclease ligation methods and the DNA Assembly method. Subsequently, the shuttle plasmid pShuttle63/EGFP carrying the EGFP gene was digested with Sca I, and pChAd63 was digested with Hpa I. The recombinant adenovirus vector pChAd63/EGFP was obtained by the DNA Assembly method. Finally, the recombinant adenovirus rChAd63/EGFP was rescued in 293 cells. Results: The recombinant adenovirus pChAd63/EGFP was successfully constructed by the DNA Assembly method, and the recombinant adenovirus rChAd63/EGFP was rescued. Conclusion: The DNA Assembly method can be widely used in the construction of recombinant adenovirus vectors, and is beneficial to improveing the construction efficiency of recombinant adenovirus vectors.

Key words： Recombinant adenovirus vector DNA Assembly

Received: 24 February 2021 Published: 06 July 2021

ZTFLH:

Q812

Corresponding Authors: Yuan-hui FU,Jin-sheng HE E-mail: yhfu@bjtu.edu.cn;jshhe@bjtu.edu.cn

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	Lei HUANG
	Chang-qing WAN
	Mei-qin LIU
	Min ZHAO
	Yan-peng ZHENG
	Xiang-lei PENG
	Jie-mei YU
	Yuan-hui FU
	Jin-sheng HE

Cite this article:

HUANG Lei,WAN Chang-qing,LIU Mei-qin,ZHAO Min,ZHENG Yan-peng,PENG Xiang-lei,YU Jie-mei,FU Yuan-hui,HE Jin-sheng. Construction of Recombinant Adenovirus Vectors Using the DNA Assembly Method. China Biotechnology, 2021, 41(6): 23-26.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2102031 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I6/23

Fig.1 pShuttle63 identified by restriction endonuclease analysis M: DL 15000 Marker; pShuttle63 digested byPac I and Pme I, respectively

Fig.2 pShuttle63/EGFP identified by restriction endonuclease analysis M: DL 15000 Marker; pShuttle63/EGFP digested by Kpn Ⅰ and Sal Ⅰ

Fig. 3 pChAd63 identified by restriction endonuclease analysis M: DL 15000 Marker; pChAd63 digested by Nde Ⅰ and Avr ⅠI, 1, Xba I and Spe Ⅰ, 2, EcoRV, 3, respectively

Fig.4 pChAd63/EGFP identified by restriction endonuclease analysis M: DL 15000 Marker; pChAd63 digested by MluⅠ andEcoRⅠ, 1,NheⅠ, 2,NdeⅠ, 3, respectively

Fig. 5 EGFP expression after pChAd63/EGFP transfection of 293 cells

Fig.6 Transfection of pChAd63/EGFP into 293 cells and passage of recombinant adenovirus in 293 cells


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