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Generation of a replication-deficient recombinant human adenovirus type 35 vector using bacteria-mediated homologous recombination.

Abstract：

:The use of adenovirus type 35 (Ad35) as a vector in vaccine and gene therapy studies is promising due to its broad cell tropism and low seroprevalence in humans. However, to date, a simple and effective system for producing recombinant Ad35 (rAd35) has not been well developed. This report describes a two-plasmid Ad35-Easy system to facilitate the production of recombinant Ad35 (rAd35). The system employed the pAd35-shuttle vector for foreign gene transfer and the pAd35-backbone vector to provide the Ad35 genomic backbone. A 293-Ad35E1B cell line was used to trans-complement rAd35 replication. rAd35 plasmids were obtained through homologous recombination following co-transformation of E. coli BJ5183 cells with recombinant pAd35-shuttle vectors harboring foreign genes. rAd35 viruses were obtained directly by transfecting 293-Ad35E1B cells with foreign gene-containing rAd35 plasmids and the pAd35-backbone vector. The production of E1 deficient rAd35 was evaluated by transfecting the 293-Ad35E1B cells with the rAd35 plasmid containing the enhanced green fluorescent protein (EGFP) gene. The virus grew effectively at a yield comparable to that of wild type Ad35 in HEp2 cells, indicating that the Ad35-Easy system is an efficient method for rapid production of rAd35 in sufficient quantities for vaccine development or gene therapy.

journal_name

J Virol Methods

journal_title

Journal of virological methods

authors

Wu C,Lei X,Wang J,Hung T

doi

10.1016/j.jviromet.2011.06.016

subject

Has Abstract

pub_date

2011-10-01 00:00:00

pages

55-63

issue

1

eissn

0166-0934

issn

1879-0984

pii

S0166-0934(11)00280-1

journal_volume

177

pub_type

杂志文章

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