MDRV P10基因的克隆、分析及蛋白质结构预测  

Cloning,sequence analysis and predicted protein construction of MDRV P10 gene

作　　者：蔡一龙[1] 庄育彬 周五朵 王全溪[1] 邱晓东[1] 吴宝成[1] 

机构地区：[1]福建农林大学动物科学学院,福州350002

出　　处：《福建农林大学学报（自然科学版）》2015年第6期606-611,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家自然科学基金-促进海峡两岸科技合作联合基金项目(U1305212);福建省自然科学基金项目(2013J0169)

摘　　要：参考Gen Bank中番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)S4基因序列,设计合成一对非结构蛋白P10基因的特异性引物,以MDRV-YB株病毒RNA为模板进行RT-PCR扩增,并将目的基因克隆到PGEM-T载体,提取质粒测序.对测序结果进行遗传进化树分析和生物信息学软件预测、分析蛋白质结构和功能.结果表明,MDRV-YB株病毒的P10蛋白基因开放阅读框(ORF)全长为288 bp,编码95个氨基酸.核苷酸比对结果表明,与标准株法国MDRV-89026株同源性为95%,而与ARV-S1133株氨基酸同源性仅为21.9%.运用Prot Param tool软件对番鸭呼肠孤病毒的P10蛋白分析结果表明,该蛋白分子质量为10.7 ku,不稳定系数为52.37,不存在信号肽和糖基化位点,有4个丝氨酸和1个络氨酸的磷酸化位点.此外,MDRV的P10蛋白为疏水性蛋白,但不存在跨膜区,这与禽呼肠孤病毒(Avian reo virus,ARV)的P10蛋白具有较大差异.二级结构分析表明,MDRV-YB株的P10蛋白与ARV-S1133株P10蛋白相比,α-螺旋和无规则卷曲减少,β-折叠则增加.因此,MDRV-YB株的非结构蛋白P10不仅在基因水平上发生了较大的变异,而且在蛋白质性质与结构上也发生了较大的变异,其与该病毒毒力的增强可能存在一定的关系.Referring to the S4 gene sequences of Muscovy duck reovirus（ MDRV）,a pair of specific primer about the non-structural protein P10 gene was designed for RT-PCR. The target gene was amplified with MDRV-YB RNA template by RT-PCR,thus P10 gene was cloned into PGEM-T vector. The positive plasmid was extracted and identified,then was sequenced. The genetic evolutionary tree was carried out by BLAST on net of NCBI,and the predicted protein structure was analyzed by Prot Param software. The results showed that the length of MDRV-YB P10 gene was 288 bp,which encoded 95 amino acids. Compared with the standard strains of MDRV-89026 strain,the homology was up to 95%,while that was only 21. 9% with ARV-S1133 strains. According to Prot Param software analysis,the theoretical molecular weight of MDRV P10 gene was 10. 7 ku without any signal peptide and glycosylation sites but with 4 Ser and 1 Tyr phosphorylation sites. In addition,MDRV P10 was hydrophobic protein which was not transmembrane domain. Protein secondary structure analysis showed that the amino acids composed with alpha helix and random curl was reduced,but that of beta-fold was increased. Compared with ARV-S1133 P10,it is visual that the nonstructural protein P10 of MDRV has a large variation not only in gene levels,but also in protein properties and structure,which suggests that P10 may be related to MDRV virulence.

关 键 词：番鸭呼肠孤病毒 P10 基因克隆 蛋白质预测与分析 

分 类 号：S851.3[农业科学—预防兽医学]