猪细小病毒非结构蛋白NS1基因的克隆、序列分析及蛋白质结构预测

Cloning,Sequence Analysis and Structure Prediction of Non-structural Protein NS1 Gene of Porcine Parvovirus

根据GenBank登录的猪细小病毒NADL-2株和China株序列,利用Oligo 6.0软件设计1对扩增NS1全基因的特异性引物,对从贵州省安顺地区检测到的PPV的NS1基因进行扩增、克隆、测序、序列分析和蛋白质结构预测分析。测序结果表明,扩增的目的基因长度为1986bp,共编码659个氨基酸,其中NS1基因的1287、1288和1298位碱基发生了缺失,导致429、430和433位氨基酸发生缺失。系统发生树结果表明,本试验测序的NS1基因与NADL-2弱毒株处在同一进化分支;氨基酸同源性与NADL-2弱毒株和Kresse毒株最高,均为98.6%。蛋白质结构预测分析结果表明,非结构蛋白NS1的分子质量为75269.76u,理论等电点pI为7.25,不稳定系数为41.57,推测其为不稳定蛋白质;脂肪指数为73.58,总体平均亲水性为-0.565,推测该蛋白质是一种亲水性蛋白质;该蛋白质含有丰富的α-螺旋、β-折叠、β-转角和无规则卷曲,柔性区域较多,呈连续分布;非结构蛋白NS1无跨膜区和信号肽;预测非结构蛋白NS1含有3个N-糖基化位点、3个cAMP和cGMP依赖性蛋白激酶磷酸化位点、5个蛋白激酶C磷酸化位点、22个酪蛋白激酶Ⅱ磷酸化位点、1个酪氨酸激酶磷酸化位点、8个N-肉豆蔻酰化位点、1个酰胺化位点及1个ATP/GTP结合位点基序A(P-环);该蛋白质抗原表位较多,存在10个主要的抗原表位。

A pair of specific primers was designed based on the sequence of NADL-2 strain and China strain of porcine parvovirus published in GenBank.The NS1 gene of PPV detected in Guizhou province was amplified,cloned,sequenced,sequence analyzed and protein structure predicted.The sequencing results showed that NS1 gene was 1986 bp in size,encoding 659 amino acids,the bases deletion of NS1 gene were found at site 1287,1288 and 1298;amino acids deletion were found at site 429,430 and 433.Phylogenetic tree showed that the NS1 gene sequencing in this experiment was at the same evolutionary branch with NADL-2 attenuated strain;the homologies of coding amino acids between this strain and NADL-2 attenuated strain and Kresse strain were both 98.6%.Protein structure prediction showed that molecular weight of non-structural protein NS1 was 75269.76 u,isoelectric pI was 7.25,instability coefficient was 41.57,speculating this protein was an instable protein.Aliphatic index was 73.58,the grand average of hydropathicity（GRAVY） was-0.565,suggesting that the protein was a hydrophilic protein.This protein contained rich α-helix,β-pleated sheet,β-turn and random coil,and there were many flexible regions,showing continuous distribution.No transmembrane region and signal peptide sequence were found in this protein;motif searching showed that non-structural protein NS1 might contain 3 N-glycosylation sites,3 cAMP-and cGMP-dependent protein kinase phosphorylation sites,5 protein kinase C phosphorylation sites,22 casein kinase Ⅱ phosphorylation sites,1 tyrosine kinase phosphorylation site and 8 N-myristoylation sites,1 amidation site and 1 ATP/GTP-binding site motif A（P-loop）;10 major antigenic epitopes distributed in this protein.