大熊猫源轮状病毒CH-1株VP3基因的克隆与生物信息学分析  

Cloning and bioinformatics analysis of the VP3 gene of giant panda rotavirus strain CH-1

作　　者：侯蓉 郝中香[2] 廖红[2] 郭玲[2] 刘丹[2] 杨绍林[2] 颜其贵[2] 齐敦武 兰景超 

机构地区：[1]成都大熊猫繁育研究基地,成都610081 [2]四川农业大学动物医学院,四川雅安625014

出　　处：《黑龙江畜牧兽医》2015年第6期4-8,283,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金："973"计划前期研究专项(2012CB722207);"十二五"国家科技支撑计划项目(2012BAC01B06)

摘　　要：为了研究大熊猫轮状病毒VP3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基础,试验采用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VP3基因,将其与p MD19-T Simple载体连接并转化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。结果表明:成功获得大熊猫轮状病毒VP3蛋白基因,长为2 591 bp,包含1个2 508 bp的开放阅读框,编码835个氨基酸,测序结果在Gen Bank中的登录号为HQ641295。VP3蛋白基因编码产物分子质量为97 937.91 u,最大疏水指数为2.256,最小疏水指数为-3.000;无信号肽序列;无跨膜结构;在线预测VP3有37个抗原表位;预测其可能包含10个N-糖基化位点,1个c AMP-c GMP独立蛋白激酶磷酸化位点,20个蛋白激酶C磷酸化作用位点,18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,5个N-豆蔻酰化位点。大熊猫轮状病毒的VP3基因与猪轮状病毒VP3基因的进化距离最近。To study the VP3 gene of giant panda rotavirus, and lay a foundation for further studying the biological functions of the gene and estab-lishing the detection method of the virus,and developing new giant panda mtavirus gene vaccine, the total RNA was extracted from the giant panda rotavirus strain CH- 1 which was cultured in the MA- 104 cells. The outer capsid protein VP3 gene was amplified hy RT - PCR and then connected with the pMD !9 -T Simple Vector. The plasmid was transformed into E. coli DH5 α, and then identified by PCR amplification and sequencing analysis. The function of VP3 was predicted by bioinformatics software and the phylogenetic tree of VP3 gene was constructed. The results showed that the giant panda rotavirus VP3 protein gene was successfully obtained. The complete coding sequence of VP3 protein was 2591 bp in length, and contained an open reading frame of 2 508 bp, encoding for a protein of 835 amino acids, and the accession number in GenBank of the sequencing result was HQ641295. Bioinformatics analysis showed that molecular weight of the encoded product of VP3 protein gene was 97 937.91 u, and the maximum and the Tninimum hydropathy indexes were 2. 256 and - 3. 000, respectvely; the protein had no sig- nal pcptide sequence and transmembrane region; while it had 37 antigenic epitopes predicted by online analysis. It was predicted that the VP3 protein gene might have 10 N - glycosylation sites, 1 cAMP - and cGMP - dependent protein kinase phosphorylation site, 20 protein kinase C phosphorylation sites, 18 casein kinase Ⅱ phosphorylation sites, 1 tyrosine -kinase phosphorylation site and 5 N -myristoylation sites. The phylogenetic tree showed that the VP3 gene of giant panda rotavirus has the nearest phylogenetic distance to that of porcine rotavirus.

关 键 词：大熊猫 轮状病毒 VP3基因 克隆 生物信息学分析 

分 类 号：S852.659.4[农业科学—基础兽医学] S858.9[农业科学—兽医学]