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为了研究经原核表达的猪细胞核转录因子NF-κB p65/p50融合蛋白对猪繁殖与呼吸综合病毒(PRRSV)体外增殖影响,本文采用RT-PCR技术扩增出编码成熟p50区和p65开放阅读框(ORF),将其克隆至pET21a(+)中,转化至Rosetta感受态细胞,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,采用聚丙烯酰胺凝胶电泳(SDSPAGE)和蛋白免疫印迹法(Western-blot)对表达产物进行鉴定;纯化复性后添加到DMEM细胞维持液中,观察p65/p50对Marc145细胞毒性,确定最佳使用浓度;探讨最佳浓度的p65/p50对PRRSV体外感染增殖活力的影响,通过实时荧光定量PCR方法研究PRRSV感染时相,绘制PRRSV增殖曲线。成功构建了大量以包涵体形式表达Mr均约为70kD的p65/p50融合蛋白的p65/p50-pET21a(+)原核表达载体;Western-blot显示p50和p65重组蛋白可分别与兔抗人p50多克隆血清、兔抗人p65纯化抗体反应。确定p65/p50最佳添加浓度为0.4μg/mL。通过实时荧光定量PCR技术研究PRRSV感染时相,结果表明NF-κB p65/p50可促进PRRSV感染Marc-145CPE出现及病毒早期增殖;但CPE出现后,病毒增殖受到抑制,病毒滴度水平显著降低(P<0.05)。该结果为进一步研究PRRS的发病机理及治疗策略提供了新思路。
Abstract：This study aims to express pig nuclear transcription factor-kappaB(NF-κB)p65/p50fusion protein in E.coli Rosetta,and study its impacts on PRRSV proliferation in vitro.The p65ORF and mature p50encoding 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/p50was added to the 2%FBS DMEM,and the cytotoxicity on Marc145was observed to select the optimum concentration.The effects of optimum concentration of p65/p50on 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 p50and p65fusion protein was expressed abundantly in the form of inclusion body with molecular weight of 70kD,Western-Blot results showed that the rabbit anti-human p50polyclonal serum,rabbit anti-human p65purified antibody could bind specifically to p50and p65respectively.The optimum concentration of p65/p50was 0.4μg/mL.The real-time FQ-PCR results indicated thatNF-κB p65/p50could 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.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002428
中图分类号:S852.65
引用信息:
[1]王潇娣,朱玲,蔡雨函,等.猪NF-κB p65/p50亚基的原核表达及其对猪繁殖与呼吸综合征病毒增殖的影响[J].病毒学报,2013,29(06):621-631.DOI:10.13242/j.cnki.bingduxuebao.002428.
基金信息:
四川省科技支撑计划项目资助(2011FZ0065);; “四川省科技支撑计划项目-生猪现代产业链关键技术研究集成与产业化示范”资助
2013-11-19
2013-11-19
2013-11-19