[Cloning, prokaryotic expression and subcellular localization in the infected host cells of the duck plague virus DPV UL35 gene].
Based on the duck plague virus (DPV) UL35 gene sequence that our laboratory obtained (GenBank accession number EF643558), a pair of primers was designed using Oligo6.0 and primer5.0, then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion, the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a(+). The resultant recombinant plasmid pET-32a(+)-UL35 was then transformed into E. coli BL21 (DE3) strain and optimally-expressed under the induction of 1.0 mmol/L IPTG at 34 degrees C for 5 hours. SDS-PAGE analysis showed the recombinant protein (VP26) had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni(2+)-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1:32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography, Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the duck embryo fibroblast (DEF) were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.
Cai
Ming-Sheng
MS
Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Yaan 625014, China. caimingsheng@163.com
Cheng
An-Chun
AC
Wang
Ming-Shu
MS
Zhu
De-Kang
DK
Luo
Qi-Hui
QH
Zhao
Li-Chan
LC
Jia
Ren-Yong
RY
Liu
Fei
F
Chen
Xiao-Yue
XY
D000818:Animals; D015153:Blotting, Western; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004372:Ducks / Q000821:virology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000821:virology; D006564:Herpesviridae / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D036022:Capsid Proteins; D011994:Recombinant Proteins