Zhang Zhenjie, Ma Chengtai, Zhao Peng, Duan Luntao, Chen Wenqing, Zhang Fushou, Cui Zhizhong
College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
PLoS One. 2014 Mar 5;9(3):e90677. doi: 10.1371/journal.pone.0090677. eCollection 2014.
To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA) gene from Avian Influenza Virus H9N2 strain and a Fusion (F) gene from the Newcastle disease virus (NDV). The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional promoter. To test whether the expression of pp38/pp24 heterodimers are the required activators for the expression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containing expression cassette for the two foreign genes was co-transfected with a pp38/pp24 expression plasmid, pBud-pp38-pp24, in chicken embryo fibroblast (CEF) cells. Alternatively, plasmid pPpp38-NA/1.8kb-F was transfected in GX0101-infected CEFs where the viral endogenous pp38/pp24 were expressed via virus infection. The expression of both foreign genes was activated by pp38/pp24 dimers either via virus infection, or co-expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had no expression. We chose to insert the expression cassette of Ppp38-NA/1.8kb-F in the non-essential region of GX0101ΔMeq US2 gene, and formed a new rMDV named MZC13NA/F through homologous recombination. Indirect fluorescence antibody (IFA) test, ELISA and Western blot analyses indicated that F and NA genes were expressed simultaneously under control of the bi-directional promoter, but in opposite directions. The data also indicated the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene. The expression of pp38/pp24 dimers either via co-tranfection of the pBud-pp38-pp24 plasmid, or by GX0101 virus infection were critical to activate the bi-directional promoter for expression of two foreign genes in both directions. Therefore, the confirmed function of the bi-directional promoter provides better feasibilities to insert multiple foreign genes in MDV genome based vectors.
为了在瞬时和转基因系统中定性分析和评估双向启动子的转录功能，构建了几种不同的质粒，并开发了重组1型马立克氏病病毒（MDV）株GX0101，以共表达来自H9N2亚型禽流感病毒的神经氨酸酶（NA）基因和来自新城疫病毒（NDV）的融合（F）基因。两个外源基因，即NDV-F基因和AIV-NA基因，被插入到由双向启动子在每个方向驱动的质粒中。为了测试pp38/pp24异二聚体的表达是否是外源基因表达所需的激活剂，将含有两个外源基因表达盒的重组质粒pPpp38-NA/1.8kb-F与pp38/pp24表达质粒pBud-pp38-pp24在鸡胚成纤维细胞（CEF）中共转染。或者，将质粒pPpp38-NA/1.8kb-F转染到被GX0101感染的CEF中，其中病毒内源性pp38/pp24通过病毒感染表达。通过病毒感染或共表达，pp38/pp24二聚体激活了两个外源基因的表达。单独用pPpp38-NA/1.8kb-F转染的CEF没有表达。我们选择将Ppp38-NA/1.8kb-F的表达盒插入GX0101ΔMeq US2基因的非必需区域，并通过同源重组形成了一种新的重组MDV，命名为MZC13NA/F。间接荧光抗体（IFA）试验、ELISA和Western印迹分析表明，F基因和NA基因在双向启动子的控制下同时表达，但方向相反。数据还表明，启动子在1.8kb mRNA转录方向的活性高于pp38基因方向。通过共转染pBud-pp38-pp24质粒或GX0101病毒感染来表达pp38/pp24二聚体，对于激活双向启动子在两个方向上表达两个外源基因至关重要。因此，双向启动子的确认功能为在基于MDV基因组的载体中插入多个外源基因提供了更好的可行性。
