Molecular Characteristics Analysis of D-strain Vaccine Virus of Muscovy Duck-origin Goose Parvovirus NS Protein Epitopes
-
摘要: 为获得番鸭小鹅瘟病毒弱毒株(MDGPV-D)非结构蛋白NS基因的相关信息,根据已发表的MDGPV-PT株全基因序列,应用DNAStar分子生物学软件设计1对引物,采用高保真PCR技术扩增MDGPV-D株NS全基因序列。对番鸭小鹅瘟病毒疫苗弱毒D株(MDGPV-D)NS基因进行克隆、测序,并利用生物信息学技术分析MDGPV-D株NS蛋白的同源性、遗传衍化、N-糖基化位点、磷酸化位点、B细胞抗原表位、T细胞抗原表位及其二级结构。结果表明,D株NS全基因大小为1 884bp,编码627个氨基酸(GenBank登录号:JF926696),与MDPV NS基因的亲缘性最近核苷酸及其推导氨基酸同源性分别为97.9%~98.6%,97.6%~98.2%;MDGPV-D株NS蛋白具有3个潜在的N-糖基化位点和27个磷酸化位点,可能存在11个B细胞抗原表位,13个CD8+CTL表位,10个CD4+Th抗原表位;二级结构分析显示,α螺旋和无规则卷曲含量高,分别为40.67%、41.15%,而β转角仅占4.63%。与亲本强毒PT株相比,弱毒D株的NS蛋白存在2个定点突变,分别位于核苷三磷酸区域(第338位氨基酸)与CD4+Th抗原表位(第225位氨基酸)。Abstract: To analyze the non-structural protein NS gene of vaccine muscovy duck (Cairina moschata) origin Goose parvovirus (GPV) D strain,apair of primers were designed by DNAStar software based on the published sequences of MDGPV-PT strain from GenBank. We examed the homology,heredity evolution,N-glycosylation sites,phosphorylation sites,B cell epitopes,T cell epitopes and their secondary structure of NS protein with bioinformatics software.The full length NS gene was amplified by PCR and cloned into pMD18-T vector. Sequencing demonstrated that the NS gene of MDGPV-D strain contained 1 884bps,encoding 627amino acids (GenBank number : JF926696) .The MDGPV-Dstrain NS gene was highly similar to that of MDPV (nt:97.9%98.6%,aa : 97.6% -98.2%) .It was also shown that there were 3 potential N-glycosylation sites and 27 phosphorylation sites existed in MDGPV-D strain NS protein,and 11Bcell epitopes,13CD8 + CTL epitopes and 10CD4 + Th epitopes as well.Then the prediction of secondary structure of NS protein showed that alpha helix,random coil and beta angle account for 40.67%,41.15% and 4.63% respectively.Comparison of the parental virulent strain PT with its attenuated vaccine strain D revealed two site-specific mutagenesis:one was in the nucleoside triphosphate (NTP) binding motif at amino acid 338,and the other in a CD4 + Th epitope at amino acid 225.These studies has provided a molecular basis for investigating attenuation mechanisms of MDGPV,and benefitted for further researches on genetically engineered MDGPV vaccines.
-
Key words:
- MDGPV /
- NS protein /
- epitope /
- molecular characteristics
-
[1] 方定一.小鹅瘟的介绍[J].中国兽医杂志,1962,(8):19-20. [2] 陈少莺,胡奇林,程晓霞,等.鹅细小病毒弱毒株选育的研究[J].中国预防兽医学报,2002,24(4):286-288. [3] ZADORI Z,ERDEI J,KISARY J.Characteristics of the genome of goose parvovirus[J].Avian Pathology,1994,23(2):359-364. [4] Z DORI Z,STEFANCSIK R,RAUCH T,et al.Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus2[J].Virology,1995,212(2):562-573. [5] SHACKELTON L A,HOELZER K,PARRISH C R,et al.Comparative analysis reveals frequent recombination in the parvoviruses[J].Journal of General Virology,2007,88(12):3294-3301. [6] COTMORE S F,CHRISTENSEN J,N ESCH J P,et al.The NS1polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif[ACCA]2-3[J].Journal of Virology,1995,69(3):1652-1660. [7] DING C,URABE M,BERGOIN M,et al.Biochemical characterization of Junonia coenia Densovirus Nonstructural Protein NS-1[J].Journal of Virology,2002,76(1):338-345. [8] 孔宪刚,李桂霞,刘胜旺,等.鹅细小病毒分离株HG5/82的分子特征分析[J].中国病毒学,2005,20(1):28-32. [9] KATARZYNA DOMANSKA-BLICHARZ,ANNA JACUKOWICZ,ANNA LISOWSKA,et al.Genetic characterization of parvoviruses circulating in turkey and chicken flocks in Poland[J].Arch Virol,2012,157(12):2425-2430. [10] 潘兹书,张楚瑜,丁建华,等.伪狂犬病毒胸苷激酶基因变异引起的病毒致弱作用[J].科学通报,2001,46(16):1364-1367. [11] 李昌主,韩先杰,邹玲,等.鸭瘟病毒AV1221株TK基因的克隆与序列分析[J].青岛农业大学学报:自然科学版,2007,24(3):162-164. [12] 刘文波,周斌,黄兵,等.传染性喉气管炎病毒烟台株TK基因序列测定及TK蛋白功能初步分析[J].西北农林科技大学学报:自然科学版,2005,33(10):75-79. [13] TATáR-KIS T,MATóT,MARKOS B,et al.Phylogenetic analysis of Hungarian goose parvovirus isolates and vaccine strains[J].Avian Pathol,2004,33(4):438-444. [14] SHIEN J H,WANG Y S,CHEN C H,et al.Identification of sequence changes in live attenuated goose parvovirus vaccine strains developed in Asia and Europe[J].Avian Pathol,2008,37(5):499-505. [15] GL VITS R,ZOLNAI A,SZAB E,et al.Comparative pathological studies on domestic geese(Anser anser domestica)and Muscovy ducks(Cairina moschata)experimentally infected with parvovirus strains of goose and Muscovy duck origin[J].Acta Vet Hung,2005,53(1):73-89. -
点击查看大图
计量
- 文章访问数: 226
- HTML全文浏览量: 50
- PDF下载量: 2
- 被引次数: 0
下载:
