1株Ⅶ型新城疫病毒融合蛋白基因的克隆与生物信息学分析

中国畜牧兽医

1株Ⅶ型新城疫病毒融合蛋白基因的克隆与生物信息学分析

吴运谱¹^，2，赵晓彤^1，2，薛树山^1，2，石霖^1，2，何利昆^1，2，张晓华³，曹明慧⁴，顾贵波¹^，2，谷志大^1，2，魏澍^1，2，曹东^1，2

（1.辽宁省动物疫病预防控制中心，辽宁沈阳 110164；2.辽宁省动物医学研究院，辽宁沈阳 110164；3.辽宁省畜牧兽医局，辽宁沈阳 110001；4.中国兽医药品监察所，北京 100081）

收稿日期:2014-01-09 出版日期:2014-07-20 发布日期:2014-08-21
通讯作者: 曹东，研究员。E-mail：crz1965@126.com
作者简介:吴运谱（1980—），男，湖北人，硕士，兽医师，研究方向：动物疫病诊断。
基金资助:
辽宁省自然科学基金（201202099）。

Cloning and Bioinformatics Analysis of F Gene of Genotype Ⅶ Newcastle Disease Virus

WU Yun-pu¹^，2, ZHAO Xiao-tong¹^，2, XUE Shu-shan¹^，2, SHI Lin¹^，2, HE Li-kun¹^，2, ZHANG Xiao-hua³, CAO Ming-hui⁴, GU Gui-bo¹^，2, GU Zhi-da¹^，2, WEI Shu¹^，2, CAO Dong¹^，2

(1.Liaoning Center for Animal Disease Control and Prevention, Shenyang 110164, China；2.Liaoning Veterinary Medicine Academy, Shenyang 110164, China；3. Liaoning Province Bureau of Animal Health and Production, Shenyang 110001, China；4.China Institute of Veterinary Drug Control, Beijing 100081, China)

Received:2014-01-09 Online:2014-07-20 Published:2014-08-21

摘要/Abstract

摘要： 为了研究Ⅶ型新城疫病毒（Newcastle disease virus，NDV）CY1株的进化情况，分析其遗传变异和基因功能，试验应用RT-PCR扩增NDV辽宁分离株CY1株的融合蛋白(fusion，F)基因，进行测序分析，构建系统进化树，并用生物信息软件对蛋白结构功能进行分析。结果显示，NDV F基因全长1662 bp，编码553个氨基酸。生物信息学分析结果表明，F蛋白的分子质量为58991.0 u，理论等电点为8.48，半衰期为30 h（哺乳动物类网状细胞），不稳定系数31.62，脂肪指数为108.12，总平均疏水性0.174，最大疏水性为3.078，最小疏水性为-2.578，信号肽序列为第1-31位氨基酸残基，跨膜区结构分析结果表明该蛋白为跨膜蛋白，预测可能含有6个N-糖基化作用位点、9个酪蛋白激酶Ⅱ磷酸化位点、7个蛋白激酶C磷酸化位点、14个N-豆蔻酰化位点、1个酰胺化位点、1个亮氨酸拉链、1个微体C端靶信号位点、1个双向核定位信号。系统进化分析结果表明，NDV CY1株与zj1株进化距离最近。

关键词: font-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">新城疫病毒; F 基因; 生物信息学分析

Abstract: The assay was aimed to analyze the protein structure and function of fusion(F) gene of genotype Ⅶ Newcastle disease virus(NDV) strain CY1. The total RNA was extracted from NDV CY1 strain, F gene was amplified by RT-PCR and sequenced, then using some related computer software and database, the sequence of F gene of NDV CY1 strain and the deduced amino acid sequence were analyzed, and the secondary structure and hydrophobicity of the protein were determined and predicted. The results showed that F gene of NDV CY1 strain was consisted of 1662 nucleotides encoding a 553-amino acid protein. The protein had two transmembrane domains, and its subcellular localization was in plasma membrane and endoplasmic reticulum. The secondary structure contained several kinds of conformations. Grand average of hydropathicity of F protein of NDV CY1 strain was 0.174. The theoretical pI, estimated half-life, instability index and fatty index of F protein was 8.48, 30 h （mammalian reticulocytes in vitro）,31.62 and 108.12, respectively. The maximum and minimum hydropathicities were 3.078 and -2.578，respectively. The protein included a 31-amino acid signal peptide.Motif search showed that F protein had 6 N-glycosylation site, 9 casein kinase Ⅱ phosphorylation site, 7 protein kinase C phosphorylation site, 14 N-myristoylation site,1 amidation site,1 leucine zipper pattern,1 microbodies C-terminal targeting signal, 1 bipartite nuclear localization signal profile. Analysis of phylogenetic tree indicated that the F gene of NDV CY1 strain had the highest genetic relationship with zj1 strain.

Key words: font-size: 10.5pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-font-size: 12.0pt; mso-font-kerning: 1.0pt; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA" lang="EN-US">Newcastle disease virus; F gene; bioinformatics analysis

吴运谱，赵晓彤，薛树山，石霖，何利昆，张晓华，曹明慧，顾贵波，谷志大，魏澍，曹东. 1株Ⅶ型新城疫病毒融合蛋白基因的克隆与生物信息学分析[J]. 中国畜牧兽医.

WU Yun-pu ZHAO Xiao-tong, XUE Shu-shan SHI Lin, HE Li-kun, ZHANG Xiao-hua, CAO Ming-hui, GU Gui-bo, GU Zhi-da, WEI Shu, CAO Dong. Cloning and Bioinformatics Analysis of F Gene of Genotype Ⅶ Newcastle Disease Virus[J]. .

参考文献

1 仇旭升，孟春春，于洋，等. 新城疫病毒P/V/W基因编码产物功能结构域的分析及定位［J］.生物技术通报，2011，1:191～196.
2 田献礼，宁红梅，银梅，等.新城疫病毒囊膜糖蛋白生物学特性的研究进展［J］.中国畜牧兽医，2011，38(8):189～192.
3 农业部.中华人民共和国兽用生物制品质量标准［S］.北京：中国农业科技出版社，2001.
4 张士强，易悦，徐志文，等. 猪伪狂犬病病毒SL株TK基因的克隆与生物信息学分析［J］.中国畜牧兽医，2012，39(6):26～30.
5 周思旋，徐健，周碧君，等. 高致病性猪繁殖与呼吸综合征病毒JL株Nsp10基因生物信息学分析［J］.中国畜牧兽医，2012，39(1):6～10.
6 秦卓明，马保臣，何叶峰，等. 新城疫病毒HN和F基因遗传变异相关性的研究［J］.微生物学报，2006，46(2):227～232.
7 秦卓明，徐怀英，刘玉山，等. 疫苗免疫压力下新城疫病毒的动态演化［J］.中国兽药杂志，2013，2:5～10.
8 董国英，徐连壁，顾贵波，等. 新城疫F-Ⅶ病毒辽宁强毒株的分离鉴定及防控对策［J］.当代畜禽养殖业，2003，6:56～59.
9 GB/T 16550—2008. 新城疫诊断技术［S］.
10 Bagai S，Lamb R A. Quantitative measurement of paramyxovirus fusion: Differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus［J］. J Virol，1995，69(11):6712～6719.
11 Cao H W，Li D S，Zhang H. Analysis of synonymous codon usage in Newcastle disease virus hemagglutinin-neuraminidase(HN) gene and fusion protein(F) gene［J］.Indian J Virol，2014，25(1):132～136.
12 Czegledi A，Ujvari D，Somogyi E，et al.Third genome size category of avian paramyxovirus serotype Ⅰ(Newcastle disease virus) and evolutionary implications［J］. Virus Res，2006，120(1～2):36～48.
13 Glickman R L，Syddall R J，Iorio R M，et al. Quantitative basic residue requirement in the cleavage activation site of the fusion glycoprotein as a determinant of virulence for NDV［J］. Journal of Virology，1998，62(1):354～356.
14 Liang R，Cao D J，Li J Q，et al.Newcastle disease outbreaks in western China were caused by the genotypes Ⅶa and Ⅷ［J］. Vet Microbiol，2002，87:193～203.
15 Liu X F，Wan H Q，Ni X X，et al. Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985—2001［J］. Arch Virol，2003，148(7):1387～1403.
16 Miller P J，Kim L M，Ip H S，et al. Evolutionary dynamics of Newcastle disease virus［J］.Virology，2009，391(1):64～72.
17 Miller P J，Decanini E L，Afonso C L. Newcastle disease: Evolution of genotypes and the related diagnostic challenges［J］. Infect Genet Evol，2010，10:26～35.
18 Mondal S，Jaishankar S P，Ramakumar S. Role of context of the relationship between form and function: Structural plasticity of some PROSITE patterns［J］. Biochem Biophys Res Commun，2003，305:1078～1084.
19 Munir M，Abbas M，Khan M T，et al.Genomic and biological characterization of a velogenic Newcastle disease virus isolated from a healthy backyard poultry flock in 2010［J］. Virol J，2012，16(9):46～56.
20 Nakai K，Horton P. PSORT: A program for detecting sorting signals in proteins and predicting their subcellular localization［J］. Trends Biochem Sci，1999，24:34～36.
21 Wang M，Liu Y S，Zhou J H，et al. Analysis of codon usage in Newcastle disease virus［J］. Virus Genes，2011，42(2):245～253.

[1]	黄建文，王晓平，王念璐，徐刚毅. 山羊组织蛋白酶L基因克隆、序列分析及组织表达研究[J]. 中国畜牧兽医, 2014, 41(7): 1-9.
[2]	崔保亮，陈利苹，张娈娈，李华芳，刘思国. 结核分枝杆菌rv0199基因在耻垢分枝杆菌中的表达及检测[J]. 中国畜牧兽医, 2014, 41(7): 9-13.
[3]	王欣然，聂虹霞，李岩松，谭彩云，胡盼，冯小丽，任洪林，郭一平，周玉，柳增善，卢士英. 展青霉素人工抗原的制备与鉴定[J]. 中国畜牧兽医, 2014, 41(7): 14-20.
[4]	李兴玉，李金海，林毅，曹冶，罗丹丹，廖党金，魏甬，李江凌，于吉锋，张先惠. 猪细小病毒疫苗株NS1基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2014, 41(7): 21-26.
[5]	张文东，宋建领，赵焕云，刘庆亮，胡媛媛，曾伟，张富强. 云南2株基因Ⅱ型新城疫病毒F基因变异特性分析[J]. 中国畜牧兽医, 2014, 41(7): 54-59.
[6]	张双翔，程振涛，唐宇，冉隆仲，李涛，周碧君，文明，王开功，王慧. 绵羊肺炎支原体热休克蛋白Hsp70的生物信息学分析[J]. 中国畜牧兽医, 2014, 41(7): 74-80.
[7]	李丹丹，徐义刚，王绥家，高慎阳，李一经. 肠出血性大肠埃希菌O157∶H7实时荧光定量PCR快速检测方法的建立[J]. 中国畜牧兽医, 2014, 41(7): 81-84.
[8]	陈国顺，李冲，俞瑞山，冯光彧，陈胜. 日粮中添加腐植酸钠对肉仔鸡成活率及生长性能的影响[J]. 中国畜牧兽医, 2014, 41(7): 122-125.
[9]	孙焕林，张文举，刘艳丰，唐淑珍. 甘草酸的生物学功能、提取工艺及其在单胃动物中的应用[J]. 中国畜牧兽医, 2014, 41(7): 126-130.
[10]	安培培，李春义. 威尔姆氏瘤基因1对肾脏和性腺发育影响的研究进展[J]. 中国畜牧兽医, 2014, 41(7): 136-141.
[11]	王轶敏，代阳，刘新峰，刘中伟，李吉霞，郭宏，丁向彬. 牛骨骼肌卫星细胞的分离鉴定和诱导分化[J]. 中国畜牧兽医, 2014, 41(7): 142-147.
[12]	王昕，王岩，秦顺义，王宏祥，王景申. 不同剂量硒蛋白粉对大鼠血常规指标和血液生化指标的影响[J]. 中国畜牧兽医, 2014, 41(7): 148-152.
[13]	张谊，何自标，陈晨，鲜红，张明. TLR4在妊娠异常终止中的作用机制和研究进展[J]. 中国畜牧兽医, 2014, 41(7): 153-158.
[14]	王海军，赵德良，李树伟，谢飞，铁鑫. 舍饲和田羊与卡拉库尔羊生长发育指标的测定[J]. 中国畜牧兽医, 2014, 41(7): 163-168.
[15]	薛倩，王金玉，张跟喜，王亚男，唐莹，魏岳，施会强. 京海黄鸡甲状腺激素应答蛋白Spot14α基因外显子1多态性与屠体和腹脂性状的相关性分析[J]. 中国畜牧兽医, 2014, 41(7): 169-174.