Identification and functional analysis of the GTPV bidirectional promoter region

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• 作者：Hui Zhang ; Zhihua Sun ; Na Zhang ; Zhiqiang Li ; Pengyan Wang…
• 关键词：Goat pox virus ; Bidirectional promoter ; Vector ; Omp31
• 刊名：Archives of Microbiology
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：199
• 期：2
• 页码：357-364
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Microbiology; Microbial Ecology; Biochemistry, general; Cell Biology; Biotechnology; Ecology;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-072X
• 卷排序：199

文摘

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 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.