Expression of the newcastle disease virus fusion glycoprotein in vero cells using attenuated Salmonella typhimurium as transgenic carrier.
The full-length cDNA of fusion protein (F) gene of newcastle disease virus (NDV)strain F48E9 was amplified by RT-PCR and inserted into pcDNA3 under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter. The resulting recombinant plasmid pcDNA3-F was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam(-) and phoP(-)), which was then used to transfect the Vero cells. The DNA and RNA dot blotting revealed that the F gene was transcribed into mRNA in the Vero cells. There was expression of the F protein as shown by indirect immunofluorescent assay. The expression began at 48 h post-infection and increased thereafter, as indicated by ELISA. A 55 kD band of the F protein was identified by SDS-PAGE and Western blotting. These results clearly show that the expressed fusion protein was immuno-reactive with chicken anti-NDV serum.
Fang
Wei-Huan
WH
Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China. whfang@zju.edu.cn
Liang
Xue-Ya
XY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins