Wang Xiao-Di, Zhu Ling, Cai Yu-Han, Huang Pu, Xu Zhi-Wen
Animal Biotechnology Center, Sichuan Agricultural University, Ya'an 625014, China.
Bing Du Xue Bao. 2013 Nov;29(6):621-31.
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.
本研究旨在在大肠杆菌Rosetta中表达猪核转录因子-κB（NF-κB）p65/p50融合蛋白，并研究其对猪繁殖与呼吸综合征病毒（PRRSV）体外增殖的影响。通过RT-PCR扩增p65开放阅读框（ORF）和成熟p50编码基因，将产物克隆到pET-21a(+)载体中，然后转化到大肠杆菌Rosetta中，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达重组融合蛋白，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western-Blot）鉴定表达产物。将纯化复性后的p65/p50加入到含2%胎牛血清（FBS）的 Dulbecco's改良 Eagle培养基（DMEM）中，观察其对Marc145细胞的细胞毒性以选择最佳浓度。采用实时荧光定量聚合酶链反应（FQ-PCR）法检测培养上清中PRRSV感染阶段，并绘制PRRSV一步生长曲线，研究最佳浓度的p65/p50对PRRSV增殖活性的影响。结果显示成功构建了p65/p50-pET21a(+)原核表达载体，重组p50和p65融合蛋白以包涵体形式大量表达，分子量为70kD，Western-Blot结果表明兔抗人p50多克隆血清、兔抗人p65纯化抗体分别能与p50和p65特异性结合。p65/p50的最佳浓度为0.4μg/mL。实时FQ-PCR结果表明，NF-κB p65/p50在细胞病变效应（CPE）出现前可促进CPE出现和PRRSV增殖，在CPE出现后抑制PRRSV增殖，并显著降低病毒滴度水平（P<0.05）。这些结果将为PRRS的致病机制和治疗策略提供一些新的见解。
