大熊猫轮状病毒CH-1株外衣壳蛋白VP4基因的克隆与生物信息学分析

Cloning and bioinformatics analysis of outer capsid protein VP4 gene of rotavirus strain CH-1 in giant panda

利用MA-104细胞培养增殖大熊猫轮状病毒CH-1株,提取总RNA,运用RT-PCR扩增外衣壳蛋白VP4基因,将其与pMD19-T simple vector连接并转化DH5α,经PCR扩增和测序分析进行鉴定,用生物信息学软件预测其功能,并构建系统进化树。结果显示,成功获得大熊猫轮状病毒VP4蛋白基因,长2 362bp,包含一个2 331bp的开放阅读框,编码776个氨基酸,测序结果在GenBank中的登录号为HQ641296。生物信息学分析表明,VP4蛋白基因编码产物分子质量为86 768.8u,理论等电点为5.56,半衰期为30h(体外哺乳类网状细胞),不稳定系数为28.64,脂肪指数为82.53,总平均亲水性为-0.265,最大疏水性为2.244,最小疏水性为-2.911;无信号肽序列;跨膜结构分析显示VP4蛋白无跨膜区,位于病毒膜外区;在VP4序列中的抗原决定簇主要位于VP4的N-末端和C-末端。预测其可能包含8个N-糖基化作用位点,15个蛋白激酶C磷酸化作用位点、15个酪蛋白激酶Ⅱ磷酸化作用位点、12个N-豆蔻酰化位点。系统进化分析显示,大熊猫轮状病毒的VP4基因与猪轮状病毒的VP4基因的进化距离最近。本试验成功获得大熊猫轮状病毒VP4基因,为以后研究此基因的生物学功能、建立该病毒的检测方法以及研制新型大熊猫轮状病毒基因疫苗奠定了基础。

The total RNA was extracted from the rotavirus（RV） CH-1 strain in giant panda which was cultured in MA-104 cells.The outer capsid protein VP4 gene was amplified by RT-PCR and directly cloned into pMD19-T simple vector.The plasmid was transformed into Escherichia coli DH5α,and then identified by PCR amplification and sequencing.Then the function of VP4 was predicted by bioinformatics software and the phylogenetic tree of VP4 gene was constructed by CLUSTALW software.In results,the VP4 gene contained the complete coding sequence of VP4 protein,including 2 331 bp in size,encoding 776 aa,and its GenBank accession is HQ641296.Bioinformatics analysis showed that molecular weight of VP4 is86 768.8u,and theoretical pI,estimated halflife and instability index of VP4 protein was 5.56,30 hours（mammalian reticulocytes in vitro）and 28.64,respectively.Grand average of hydropathicity of VP4 protein was-0.265,while the maximum and the minimum hydrophobicity were 2.244 and-2.911.The protein had no signal peptide and transmembrane region. The antigenic determinants of VP4 were located in the N-terminal and C-terminal.Motif searching showed that VP4 protein might have 8 N-glycosylation sites,15 protein kinase C phosphorylation sites,15 casein kinaseⅡ phosphorylation sites,12 N-myristoylation sites. The phylogenetic tree showed that the giant panda RV VP4 gene was the most homogeneous to that of porcine rotavirus. The present study obtained the clone of VP4 gene of RV in giant panda,and displayed some new clues for further studying the biological functions of the gene and establishing diagnosis method of the virus.