Prokaryotic expression of pig nuclear transcription factor-kappaB p65/p50 and its impacts on PRRSV proliferation.
- Author:
Xiao-Di WANG
1
;
Ling ZHU
2
;
Yu-Han CAI
2
;
Pu HUANG
2
;
Zhi-Wen XU
2
Author Information
1. Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China. wangxiaodi2350@163.com
2. Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cell Line;
Escherichia coli;
genetics;
metabolism;
Gene Expression;
Humans;
NF-kappa B p50 Subunit;
genetics;
metabolism;
Porcine Reproductive and Respiratory Syndrome;
metabolism;
Porcine respiratory and reproductive syndrome virus;
genetics;
physiology;
Recombinant Fusion Proteins;
genetics;
metabolism;
Swine;
Transcription Factor RelA;
genetics;
metabolism;
Virus Replication
- From:
Chinese Journal of Virology
2013;29(6):621-631
- CountryChina
- Language:Chinese
-
Abstract:
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.
