大熊猫轮状病毒CH-1株外衣壳蛋白(VP7)基因的克隆和生物信息学分析

Gene Cloning and Bioinformatics Analysis of Outer Capsid Protein VP7 gene for Giant Panda Rotavirus CH-1 Strain

为研究大熊猫轮状病毒(Giant Panda Rotavirus,GPRV)CH-1株外衣壳蛋白VP7基因的结构及功能,用MA-104细胞(恒河猴胎猴肾细胞)增殖GPRV,提取总RNA,运用RT-PCR扩增VP7基因,并测序,用生物信息学软件预测其功能,并构建系统进化树。结果显示成功获得了长1042bp的GPRV VP7蛋白基因(GenBank登录号GU188284)。经生物信息学分析,此序列包含1个981bp的完整开放阅读框,编码326个氨基酸;预测GPRV VP7蛋白理论相对分子质量为37354.8u,等电点为4.76,半衰期为30h,不稳定系数为26.71,总平均亲水性为-0.015,疏水性介于-2.344～3.567;1—24位氨基酸可能是信号肽序列;跨膜结构分析表明VP7蛋白有2个跨膜区,其N端和C端都位于病毒膜外区;抗原表位预测显示VP7蛋白有12个抗原决定簇;结构预测显示其可能包含2个N-糖基化作用位点,5个蛋白激酶C磷酸化作用位点、4个酪蛋白激酶Ⅱ磷酸化作用位点、1个酪氨酸激酶磷酸化作用位点、4个N-豆蔻酰化位点、1个原核膜脂蛋白脂附着点和1个革兰氏阳性球菌表面蛋白‘锚’六肽。系统进化树分析显示GPRV VP7基因与人A组轮状病毒VP7基因的进化距离最近。本研究成功获得了GPRV VP7基因,为今后研究此基因的生物学功能以及建立该病毒的诊断方法奠定基础。

The VP7 gene coding outer capsid protein of giant panda rotavirus（GPRV）strain CH-1 was cloned by RT-PCR from GPRV infected MA-104 cells,and sequencing and bioinformatic analysis were then conducted to investigate the structure and function of GPRV VP7 protein. The function of this protein was predicted by bioinformatics software and the phylogenetic tree of VP7 gene was constructed by CLUSTALW software. The results indicated that a length of 1 042 bp GPRV VP7 protein encoding gene was obtained（GenBank accession number：GU188284）. Bioinformatics analysis showed that the sequence contains the complete coding sequence of VP7 protein, which is 981 bp, encoding 326 aa. The molecular weight, isoelectric point, estimated half-life, instability index of GPRV VP7 were 37 354.8 Da, 4.76, about 30 hours and 26.71, respectively. Grand average of hydropathicity of VP7 protein was -0.015, while the hydrophobicity was between -2.344 and 3.567. The protein has one signal peptide between 1 and 24 site, two transmembrane regions 4—23 site and 33—53 site and 12 antigenic determinants predicted by online analysis. Motif searching showed that VP7 protein may have 2 N-glycosylation sites, 5 protein kinase C phosphorylation sites, 4 casein kinase Ⅱ phosphorylation sites, 1 tyrosine kinase phosphorylation site, 4 N-myristoylation sites, 1 prokaryotic membrane lipoprotein lipid attachment site and 1 gram-positive cocci surface proteins ＂anchoring＂ hexapeptide. The phylogenetic tree showed that the evolution distance of giant panda rotavirus VP7 gene is homogeneous to human rotavirus A. The research obtained the clone of GPRV VP7 gene, and displayed some new clues for further studying the biological functions of the gene and establishing diagnosis method of the virus.