title: [Prokaryotic expression of pig nuclear transcription factor-kappaB p65/p50 and its impacts on PRRSV proliferation].
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.
authors: Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China. wangxiaodi2350@163.com
authors: Xiao-Di
authors: XD
authors: Wang
authors: Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China.
authors: Ling
authors: L
authors: Zhu
authors: Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China.
authors: Yu-Han
authors: YH
authors: Cai
authors: Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China.
authors: Pu
authors: P
authors: Huang
authors: Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China.
authors: Zhi-Wen
authors: ZW
authors: Xu
mesh_terms: D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000235:genetics* / Q000378:metabolism; D019318:Porcine Reproductive and Respiratory Syndrome / Q000378:metabolism; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D051996:Transcription Factor RelA / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D052002:NF-kappa B p50 Subunit; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA