title: Construction of recombinant Marek's disease virus (rMDV) co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.
abstract: 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.
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Zhenjie
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Chengtai
authors.initials: C
authors.lastname: Ma
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Luntao
authors.initials: L
authors.lastname: Duan
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Wenqing
authors.initials: W
authors.lastname: Chen
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Fushou
authors.initials: F
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Shandong Agricultural University, Taian, China; Animal Disease Prevention Technology and Research Center of Shandong Province, Taian, China.
authors.forename: Zhizhong
authors.initials: Z
authors.lastname: Cui
mesh_terms: D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D005347:Fibroblasts / Q000821:virology; D019084:Fluorescent Antibody Technique, Indirect; D005814:Genes, Viral / Q000235:genetics*; D053127:Influenza A Virus, H9N2 Subtype / Q000201:enzymology / Q000235:genetics*; D025922:Mardivirus / Q000235:genetics*; D008380:Marek Disease / Q000821:virology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D009439:Neuraminidase / Q000235:genetics; D009522:Newcastle disease virus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D060888:Real-Time Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D009439:Neuraminidase