PMID- 24520768
OWN - NLM
STAT- MEDLINE
DCOM- 20140401
LR  - 20201209
IS  - 1000-8721 (Print)
IS  - 1000-8721 (Linking)
VI  - 29
IP  - 6
DP  - 2013 Nov
TI  - [Prokaryotic expression of pig nuclear transcription factor-kappaB p65/p50 and 
      its impacts on PRRSV proliferation].
PG  - 621-31
AB  - This study aims to express pig nuclear transcription factor-kappaB (NF-kappaB) 
      p65/p50 fusion protein in E. coli Rosetta, and study its impacts on PRRSV 
      proliferation in vitro. The p65 ORF and mature p50 encoding gene were amplified 
      by RT-PCR, the products were cloned into the pET-21a(+) vector, then transformed 
      into Escherichia coli Rosetta, recombinant fusion protein was expressed by IPTG 
      induction, the expressed product was identified by SDS-PAGE and Western-Blot. The 
      purified and re-folded p65/p50 was added to the 2% FBS DMEM, and the cytotoxicity 
      on Marc145 was observed to select the optimum concentration. The effects of 
      optimum concentration of p65/p50 on PRRSV proliferation activity were 
      investigated by detecting PRRSV infection phase in the culture supernatant using 
      real-time FQ-PCR method and drawing PRRSV one-step growth curve. The results 
      showed the p65/p50-pET21a(+) prokaryotic expression vector were successfully 
      constructed , recombinant p50 and p65 fusion protein was expressed abundantly in 
      the form of inclusion body with molecular weight of 70kD, Western-Blot results 
      showed that the rabbit anti-human p50 polyclonal serum, rabbit anti-human p65 
      purified antibody could bind specifically to p50 and p65 respectively. The 
      optimum concentration of p65/p50 was 0.4 microg/mL. The real-time FQ-PCR results 
      indicated that NF-kappaB p65/p50 could promote CPE appearance and PRRSV 
      proliferation before CPE appeared, and suppress PRRSV proliferation after CPE 
      appeared, and lower the virus titer levels significantly(P < 0.05). These results 
      will provide some new insight of the pathogenic mechanism and treatment 
      strategies of PRRS.
FAU - Wang, Xiao-Di
AU  - Wang XD
AD  - Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, 
      China. wangxiaodi2350@163.com
FAU - Zhu, Ling
AU  - Zhu L
AD  - Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, 
      China.
FAU - Cai, Yu-Han
AU  - Cai YH
AD  - Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, 
      China.
FAU - Huang, Pu
AU  - Huang P
AD  - Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, 
      China.
FAU - Xu, Zhi-Wen
AU  - Xu ZW
AD  - Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, 
      China.
LA  - chi
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - China
TA  - Bing Du Xue Bao
JT  - Bing du xue bao = Chinese journal of virology
JID - 8803009
RN  - 0 (NF-kappa B p50 Subunit)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Transcription Factor RelA)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Escherichia coli/*genetics/metabolism
MH  - Gene Expression
MH  - Humans
MH  - NF-kappa B p50 Subunit/*genetics/metabolism
MH  - Porcine Reproductive and Respiratory Syndrome/metabolism
MH  - Porcine respiratory and reproductive syndrome virus/genetics/*physiology
MH  - Recombinant Fusion Proteins/genetics/metabolism
MH  - Swine
MH  - Transcription Factor RelA/*genetics/metabolism
MH  - *Virus Replication
EDAT- 2014/02/14 06:00
MHDA- 2014/04/02 06:00
CRDT- 2014/02/14 06:00
PHST- 2014/02/14 06:00 [entrez]
PHST- 2014/02/14 06:00 [pubmed]
PHST- 2014/04/02 06:00 [medline]
PST - ppublish
SO  - Bing Du Xue Bao. 2013 Nov;29(6):621-31.