Identification and functional analysis of the GTPV bidirectional promoter region
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ORIGINAL PAPER
Identification and functional analysis of the GTPV bidirectional promoter region Hui Zhang1 · Zhihua Sun1 · Na Zhang1 · Zhiqiang Li2 · Pengyan Wang1 · Qiang Fu1 · Yan Ren3 · Xuehua Shao1 · Yu Zhang1 · Zhiru Guo1 · Chuangfu Chen1
Received: 6 July 2016 / Revised: 7 October 2016 / Accepted: 16 October 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract The goat pox chick embryo-attenuated virus (GTPV) has been developed as an effective vaccine that can elicit protective immune responses. It possesses a large genome and a robust ability to express exogenous genes. Thus, this virus is an ideal vector for recombinant live vaccines for infectious diseases in ruminant animals. In this study, we identified a novel bidirectional promoter region of GTPV through screening named PbVV(±). PbVV(±) is located between ETF-l and VITF-3, which are transcribed in opposite directions. A new recombinant goat pox virus (rGTPV) was constructed, in which duplicate PbVV(+) was used as a promoter element to enhance Brucella OMP31 expression, and duplicate PbVV(−) was used as a promoter element to regulate enhanced green fluorescent protein (EGFP) at the same time as the selection marker. PbVV(−) promoter activity was compared to that of the P7.5 promoter of vaccinia virus, as measured by EGFP expression; the fluorescence intensity of EGFP expressed in cells was confirmed by fluorescence microscopy and flow cytometry. PbVV(+) promoter activity was
Communicated by Erko Stackebrandt. Hui Zhang and Zhihua Sun have contributed equally to this work. * Chuangfu Chen [email protected] 1
College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang Province 832000, People’s Republic of China
2
School of Life Sciences, Shangqiu Normal University, Shangqiu, Henan Province 476000, People’s Republic of China
3
School of Medicine, Shihezi University, Shihezi, Xinjiang Province 832000, People’s Republic of China
measured by Brucella OMP31 expression. Interaction with the anti-Brucella-OMP31 monoclonal antibody was confirmed by western blotting, and OMP31 mRNA expression was assessed by qRT-PCR. The results of this study will be useful for the further study of effective multivalent vaccines based on rGTPV. This study also provides a theoretical basis for overcoming the problem of low expression of exogenous genes. Keywords Goat pox virus · Bidirectional promoter · Vector · Omp31
Introduction Goat pox is a disease of goats that is caused by goat pox virus (GTPV) (Venkatesan et al. 2012). The virus belongs to the Capripoxvirus (CaPV) genus of the Poxviridae family and is closely related to sheep pox virus (SPPV) and lumpy skin disease virus (LSDV) (Tulman et al. 2002). Both GTPV and SPPV are responsible for some of the most economically significant diseases of domestic ruminants in Central and Northern Africa, the Middle East, the Indian subcontinent, Central Asia, and parts of China (Zhou et al. 2012). CaPVs are generally considered to be host-specific because disease outbreaks tend to occur preferential
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