Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene

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China Biotechnology ›› 2013, Vol. 33 ›› Issue (12) : 15-20.

ZHANG Wen-feng^1,2, ZHANG Qiong-yu³, BO Hua-ben^1,2, SHAO Hong-wei^1,2, LI Xiao-cheng^1,2, WANG Teng^1,2, HUANG Shu-lin^1,2

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Abstract

Aim: To construct the prokaryotic expression vector for Ad5 fiber and Ad35 fiber genes, purify proteins and study the activity. Methods: Recombinant plasmid pET28a-Ad5 fiber and pET28a-Ad35 fiber were transformed into E.coli BL21(DE3) and the proteins expression were induced with IPTG. The expressed proteins Ad5 fiber and Ad35 fiber were purified by Ni²⁺ affinity chromatography and identified by SDS-PAGE. The bioactivity of proteins Ad5 fiber and Ad35 fiber were certificated by competitive binding method. Results: The proteins Ad5 fiber and Ad35 fiber can be detected in the supernatant. The proteins Ad5 fiber specifically inhibited the infectivity of the Ad5 virus in a dose-dependent manner, while had no effect on the Ad5F35-mediated gene transfer. The proteins Ad35 fiber blocked the Ad5F35-mediated gene transfer in a dose-dependent manner and had no effect on the infectivity of the Ad5 virus in competition experiments. Conclusion: Recombinant vectors Ad5 fiber and Ad35 fiber were constructed successfully. The proteins Ad5 fiber and Ad35 fiber could specifically bind with different receptors. The study will lay a foundation for the investigation on the mechanism of Ad5F35 infection.

Key words

Adenoviral vector / Adenovirus fiber protein / Prokaryotic expression

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ZHANG Wen-feng, ZHANG Qiong-yu, BO Hua-ben, SHAO Hong-wei, LI Xiao-cheng, WANG Teng, HUANG Shu-lin. Prokaryotic Expression and Activity Detection of Ad5/Ad35 Fiber Gene[J]. China Biotechnology, 2013, 33(12): 15-20

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