Construction and expression of adenovirus expressing ribosomal protein RPS3a

WU Hong-yuan; LI Jian-zhong; ZHANG Jun-ping

doi:10.3969/j.issn.1006-0111.2013.05.007

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Objective To construct the recombinant adenovirus expression vector containing ribosomal protein RPS3a and detect its expression in NIH3T3 cells. Method The RPS3a gene was obtained by RT-PCR.,total RNA was extracted from human HEK293 cell as the template. The cDNA fragments were cloned into the shuttle vector pAdTrack-CMV. After identified by enzyme digested and DNA sequence analysis,the product pAdTrack-RPS3a was lineared by PmeI and transformed into competent E.Coli BJ5183 carrying backbone plasmid pAdeasy-1 for homologous recombination. The identified positive recombinant adenoviral plasmid was then lineared by PacI and transfected into 293 cells for packaging and amplifying. Western blotting was used to detect the expression of RPS3a protein in the NIH3T3 cells infected with adenovirus. Results The pAdTrack-RPS3a and pAd-RPS3a plasmids had been successfully constructed. Protein expression was markedly increased in the NIH3T3 cells infected with Ad-RPS3a compared to that with control Ad-GFP. Conclusion The recombinant adenovirus harboring the RPS3a gene was successfully constructed, which could provide a useful tool for further investigating its biological function.

Construction and expression of adenovirus expressing ribosomal protein RPS3a

1. Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China;Department of Biochemical Pharmacy, School of Pharmacy, the Second Military Medical University, Shanghai 200433, China

2. Department of Biochemical Pharmacy, School of Pharmacy, the Second Military Medical University, Shanghai 200433, China

Received Date: 2012-12-31

Rev Recd Date: 2013-05-20

Key words:

RPS3a /
adenovirus /
construction /
expression

Abstract: Objective To construct the recombinant adenovirus expression vector containing ribosomal protein RPS3a and detect its expression in NIH3T3 cells. Method The RPS3a gene was obtained by RT-PCR.,total RNA was extracted from human HEK293 cell as the template. The cDNA fragments were cloned into the shuttle vector pAdTrack-CMV. After identified by enzyme digested and DNA sequence analysis,the product pAdTrack-RPS3a was lineared by PmeI and transformed into competent E.Coli BJ5183 carrying backbone plasmid pAdeasy-1 for homologous recombination. The identified positive recombinant adenoviral plasmid was then lineared by PacI and transfected into 293 cells for packaging and amplifying. Western blotting was used to detect the expression of RPS3a protein in the NIH3T3 cells infected with adenovirus. Results The pAdTrack-RPS3a and pAd-RPS3a plasmids had been successfully constructed. Protein expression was markedly increased in the NIH3T3 cells infected with Ad-RPS3a compared to that with control Ad-GFP. Conclusion The recombinant adenovirus harboring the RPS3a gene was successfully constructed, which could provide a useful tool for further investigating its biological function.

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Reference (11)

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Construction and expression of adenovirus expressing ribosomal protein RPS3a

doi: 10.3969/j.issn.1006-0111.2013.05.007

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