Prokaryotic expression of pig nuclear transcription factor-kappaB p65/p50 and its impacts on PRRSV proliferation / 病毒学报
Chinese Journal of Virology
;
(6): 621-631, 2013.
Article
in Chinese
| WPRIM
| ID: wpr-356656
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Physiology
/
Swine
/
Virus Replication
/
Recombinant Fusion Proteins
/
Gene Expression
/
Cell Line
/
Porcine respiratory and reproductive syndrome virus
/
Porcine Reproductive and Respiratory Syndrome
/
Escherichia coli
/
NF-kappa B p50 Subunit
Type of study:
Prognostic study
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Chinese Journal of Virology
Year:
2013
Type:
Article
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