[Comparison of the relative luciferase activity in secondary CEF by different heterogenous strong promoters, MDV gB promoter and the composed promoters].
To improve the protection efficiency of the recombinant Marek' s disease viruses (MDV) in chickens with or without maternal antibodies,the work of selecting the optimal promoters for the construction of recombinant MDV was carried out. Combined with the efficient genetic manipulation, the composed promoters was constructed by use of the MDV gB core promoter with the regulatory elements from the early immediately promoter and enhancer of hCMV, the promoter and enhancer of SV40 or the partial enhancer of hCMV. And these composed promoters were ligased to the luciferase to construct the eukaryotic expressing vectors and named PhCMV-gB, Psv-gB and Pen-gB, respectively. In vitro, these vectors and internal standard plasmid (pSV-beta-LacZ) were transiently co-transfected into secondary CEF by FuGene 6 Transfection Reagent. Furthermore, cells were harvested 48 hours after transfection. Then the luciferase activity was detected by a luciferase assay kit, at the same time, the beta-galactosidase enzyme activity was detected by a beta-galactosidase enzyme assay kit, and the luciferase activity was corrected by the beta-galactosidase enzyme activity to get the relative luciferase activity. The relative luciferase activity was used as the transcriptional activity. By comparison of the relative luciferase activity of every promoter, it was found that these composed promoters could more effectively drive the reporter gene expression than the full legth of gB promoter did. Among them, PhCMV-gB robustly drove the reporter gene expression. On the other hand, PSV-gB and Pen-gB appeared to have the same strength; But compared with the commercial strong promoters, the transcriptional activity of the composed promoter were less than as or the same as that of the strong promoters. Therefore, at a sense, it can be proposed that these composed promoters have not only the characteristic of MDV gB promoter, but also that of the commercial strong promoters. These provide the choices for further developing the new-type recombinant MDV vaccine.
Qiu
Ya-Feng
YF
Key Laboratory of Animal Disease Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China. gffqyf@njau.edu.cn
Ge
Fei-Fei
FF
Xu
Xue-Qing
XQ
Chen
Pu-Yan
PY
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D003587:Cytomegalovirus / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism*; D017930:Genes, Reporter; D005818:Genetic Engineering; D008381:Herpesvirus 2, Gallid / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*
D000956:Antigens, Viral; D014759:Viral Envelope Proteins; C072811:glycoprotein B, Marek's disease virus; D008156:Luciferases