两类猪ADP-核糖基化因子的克隆分析与原核表达载体构建

Molecular Cloning of two ADP Ribosylation Factors and Construction of Their Prokaryotic Expression Vector in Sus Sacrofa

哺乳动物小G结合蛋白——二磷酸腺苷-核糖基化因子(ADP-ribosylation factors,Arfs)除在囊泡运输、细胞骨架重排和调节细胞吞噬等方面起重要作用外,还与病毒的感染有关.前期研究发现猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)感染早期ARFs基因表达明显上调,然而在猪只中ARFs与PRRSV的感染至今知之甚少.本研究中,猪ARF1和ARF4分别被克隆,序列分析发现:ARF1包含一个长度为546bp的开放阅读框(Open reading frame,ORF),预测编码181个氨基酸,理论分子质量为20.70kDa,pI为6.62;ARF4包含一个长度为543bp的开放阅读框,预测编码180个氨基酸,理论分子质量为20.50kDa,pI为5.46.蛋白序列结构分析显示猪ARF1和ARF4蛋白含有典型的p loop,switch区,interswitch区和N端疏水区的Hasp,在第二位都含有可被肉豆蔻酰基化的甘氨酸.猪ARF1和ARF4的原核表达载体也被构建,为进一步研究其在PRRSV感染过程中的调控提供基础.

ADP-ribosylation factors （Arfs） which play an essential role in intracellular trafficking, remodeling of the cy- toskeleton and mediating phagoeytosis are small GTP-binding protein, which have been identified recently to be involved in vi- rus infection. The results of our lab indicated expression of ARFs gene of pig were up-regulated in early infectioo of Porcine re- productive and respiratory syndrome virus （PRRSV）. However, little is known about their relationships with PRRSV infection in pig to date. In the present study, ARF1 and ARF4 gene were cloned from the pig Sus sacrofa. Sequences analysis showed ARF1 gene contains a length of 546 bp of ORF, which encodes a predicted protein of 181 deduced amino acids with a calculated molecular weight of 20.70kDa and a pI of 6.62. For ARF4, the corresponding parameters were 543 bp, 180 deduced amino acids, 20.50 kDa and 5.46 respectively. The predicted structure indicated that ARF1 and ARF4 possessed the property of Arfs, including the site of myristoylation at position 2, ploop, switch1, switch2 and interswitch regions and N-terminal Hasp. To provide a basis for the future research of their functions during infection of PRRSV, the prokaryotic expression vector of ARF1 and ARF4 were constructed.