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. 2017 Dec 14;4:221. doi: 10.3389/fvets.2017.00221

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Copyright © 2017 Liu, Sun, Huang, Zhang, Jia, Luo, Shen, El-Ashram and He.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Confirmation of PmpD-N protein expression in herpesvirus of turkeys (HVT) vector by immunoblotting assay and indirect immunofluorescence. (A) The PmpD-N expression in rHVT-EF-pmpD was detected by immunoblot using Chlamydia psittaci strain 6BC-specific polyclonal antibodies. Lane M, pre-stained protein ladder; Lane 1, cell lysate post inoculation with rHVT-EF-pmpD; Lane 2, cell lysate post inoculation with rHVT-CMV-pmpD; Lane 3, cell lysate post inoculation with parental HVT. The black arrow indicates the approximate size of 43 kDa. (B) Indirect immunofluorescence analysis of PmpD-N expression in chicken embryo fibroblast (CEF) cells. CEF cells on glass coverslips were infected with rHVT-EF-pmpD, then incubated with mouse anti-PmpD-N polyclonal antibody of C. psittaci and chicken anti-HVT polyclonal serum, and then subsequently reacted with the goat anti-mouse IgG conjugated with Alexa Fluor 488 (green fluorescence, shown in the lower left panel) and goat anti-chicken IgY labeled with Alexa Fluor 568 (red fluorescence, shown in the lower right panel), respectively. Finally, cell nuclei were stained with diamidino-2-phenylindole (blue fluorescence, shown in the top right panel). The merged image is shown in the top left panel. The expression of the targeted protein is indicated by white arrows in the top left panel. (C) rHVT-CMV-pmpD control. CEF cells on glass coverslips were infected with rHVT-CMV-pmpD, and then the process of test and the panel meaning are the same as those shown in panel (B).