禽传染性支气管炎病毒论文禽传染性支气管炎病毒Sczy3.doc

禽传染性支气管炎病毒论文：禽传染性支气管炎病毒Sczy3株全基因组序列测定分析【中文摘要】鸡传染性支气管炎(IB)是由鸡传染性支气管炎病毒(IBV)引起的一种鸡的急性、高度接触性传染病。现在已经在全球流行蔓延,给世界家禽养殖业带来极大的经济损失。由于禽传染性支气管炎病毒(IBV)频繁的基因点突变和重组,目前已经出现了许多不同的血清型和基因型毒株,并且它们之间缺乏交叉保护性或交叉保护性低,所以分析某地区流行的IBV的基因组结构特点,有助于IB的防控。先前研究证实,近年来四川IBV分离株基因型主要是LX4即QX-like型。从S1基因进化树分析推出Sczy3分离株属于QX型,但是Sczy3基因组的其他基因片段还未被测序。本研究应用RT-PCR方法对Sczy3分离株基因组全序列进行测定,结果表明Sczy3分离株具有典型的禽状病毒基因序列特征5Cap-1a-1b-S-3a-3b-(E)-M-5a-5b-N-3Poly A。Sczy3分离株基因组长27695bp,含有10个开放阅读框(ORF)。Gene 1 (复制转录酶)含有两个开放阅读框ORF1a和ORF1b,分别由11856和7959个核苷酸组成,编码3951个和2652个氨基酸；Gene2(纤突蛋白基因,S)含有一个开放阅读框由3498个核苷酸组成,编码1165个氨基酸,其中S1基因编码540个氨基酸。Gene3(辅助蛋白基因)包括三个开放阅读框ORF 3a、ORF 3b和ORF3c(E),分别编码57、62和108个氨基酸；Gene 4(膜蛋白基因,M)一个开放阅读框,由678个核苷酸组成,编码225个氨基酸；Gene 5(辅助蛋白基因)含三个开放阅读框ORF 5a和ORF 5b,分别编码65和82个氨基酸；Gene 6(核衣壳蛋白基因,N)由1230个核苷酸组成,编码409个氨基酸。部分相邻基因间有重叠现象。Gene 1和Gene 2重叠110 nt, Gene 2和Gene 3重叠32 nt, Gene 3和Gene 4重叠114 nt, Gene 5和Gene 6重叠159 nt。核酸序列同源性比较表明Sczy3与参考毒株基因组序列同源性在86.1%-94.1%之间,与QX-like IBV有核苷酸同源性最高,与Mass型IBV核苷酸同源性最低。S蛋白裂解位点分析表明H-R-R-R-R不是中国分离毒株特有的裂解位点。系统进化树分析显示,与LX4型IBV亲缘关系较近,与Mass型Conn型IBV毒株亲缘关系很远。重组分析推测,Sczy3是一株嵌合IBV毒株,假定亲本毒株是LX4和H120。综上所述,本实验课题为深入研究IBV在中国的演变特征,以及IBV的反向遗传学提供参考依据,同时也丰富了冠状病毒IBV的生物信息数据库。【英文摘要】Infectious bronchitis (IB) is a highly contagious and acute disease of chickens. Now it has been spreaded to the whole world, and has posed a substantial economic losses to the world poultry industry. Because the genome of IBV strains are continuously evolving by the frequent point mutations and genomes recombination, currently, dozens of serotypes and genotypes of IBV have been detected, and the cross-protection between different strains is nonexistent and weak. Therefore, features of the genome structure of IBV that is popular in an area is analysed, which will help to prevent and control IB. Our previous study revealed that isolates in Sichuan province recently were mainly LX4 genotype or QX-like strains. The Sczy3 is a QX-like IBV strain on the basis of the sequence comparison of S1 gene. However, other parts of the genomes of the Sczy3 strain has never been sequenced.The complete genome of Sczy3 was found to consist of 27,695 nucleotides with a genomic structure similar to other the IBV strains,5Cap-1a-1b-S-3a-3b-3c(E)-M-5 a-5b-N-3Poly A.It comprises ten open reading frames (ORFs). The gene 1 (polymerase protein) consists of two open reading frames (ORFs), ORF 1a and 1b, which are 11,856 and 7,959 nt long respectively. Encoding 3,952 and 2,652 aa respectively. Gene 2 (Spike Protein gene, S) contains a single ORF,3498 nt long, encoding 1165 aa, among which, S1 encodes 540 aa. Gene 3(Accessory Proteins gene), which contains three ORFs:3a,3b, and 3c (Envelope Protein gene, E). The ORFs encodes 57,62, and 108 aa, respectively. Gene 4 (Membrane Protein gene, M) encodes 225 aa with a single ORF of 678 nt. Gene 5(Accessory Proteins gene) contains two ORFs:5a and 5b encoding 65 and 82 aa respectively. Gene 6 (Nuclecapsid Protein gene, N) encodes 409 aa, with a single ORF of 1230 nt. In addition, adjacent genes of the Sczy3 genome overlap. Gene 1 and Gene 2, Gene 2 and Gene 3, Gene 3 and Gene 4, Gene 5 and Gene 6 overlap 110,32,114 and 159 nt respectively.Nucleotide sequence identity revealed that Sczy3 had homologies of 86.1%-94.1%of the whole genome nucleotide sequence, and the highest nucleotide sequence identity to QX-like IBVs, while had a lowest nucleotide sequence identity to massachusettes type IBVs. S protein cleavage site analysis showed that HRRRR is not unique to the cleavage site of the strains from China.Phylogenetic tree analysis showed that, strains Sczy3 has closer genetic relationship with IBV LX4-type IBV strains and father genetic relationship with the Mass-type and Conn-type IBV strains. The recombinant analysis found that Sczy3 was a chimera strain, which of putative Parental sequence are LX4 and H120 strains.In summary, this study may help to decipher the evolution character of IBV in China and provide a reference for reverse genetics of IBV. Futhermore, enriched the database of IBV biological information.【关键词】禽传染性支气管炎病毒 基因组 进化树分析 重组【英文关键词】Infectious bronchitis virus Genome Phylogenetic tree analysis Recombination【目录】禽传染性支气管炎病毒Sczy3株全基因组序列测定分析摘要4-5Abstract5-6英汉缩略词对照表7-111 前言11-221.1 传染性支气管炎病毒分子生物学研究进展11-191.1.1 IBV生物学分类地位与病毒粒子特征11-121.1.2 IBV基因组结构12-131.1.3 鸡传染性支气管炎病毒复制机制13-141.1.4 IBV的结构蛋白与生物学功能14-171.1.5 IBV的非结构蛋白与生物学功能17-191.1.6 IBV的非编码区与生物学功能191.2 传染性支气管炎病毒分子遗传变异研究进展19-211.2.1 IBV分子遗传变异的机制191.2.2 基因突变的引起遗传变异19-201.2.3 基因重组的引起遗传变异20-211.3 研究目的和意义21-222 试验材料22-242.1 毒株222.2 菌种、载体222.3 鸡胚222.4 主要试剂222.5 常用试剂配制22-232.6 主要仪器23-243 试验方法24-363.1 病毒培养243.2 病毒RNA提取243.3 Sczy3全基因组内部片段分段扩增24-273.3.1 引物设计243.3.2 RT-PCR扩增24-273.3.3 PCR反应273.4 RACE获取基因组末端片段27-323.4.1 RACE引物设计27-283.4.2 3末端cDNA的扩增(3RACE)28-293.4.3 5末端cDNA的扩增(5RACE)29-323.5 PCR产物回收和纯化323.6 PCR产物克隆32-343.6.1 E.coli DH5感受态细胞的制备32-333.6.2 PCR回收产物与pMD19-T载体连接333.6.3 感受态细胞DNA转化333.6.4 重组质粒的筛选和鉴定33-343.6.5 阳性克隆质粒序列测定343.7 序列拼接和基因组全序列的生物信息学分析34-364 结果36-534.1 基因组内部分段RT-PCR扩增结果36-374.2 基因组末端序列的扩增结果374.3 序列测定和拼接37-384.4 Sczy3全基因组结构分析384.5 转录调控序列分析38-404.6 全基因组及各基因同源性分析40-454.6.1 5UTR和3UTR同源性比对结果404.6.2 Gene 1基因组同源性比对结果40-444.6.3 Gene 2基因组同源性比对结果44-454.6.4 Gene 3基因组同源性比对结果454.6.5 Gene 4基因组同源性比对结果454.6.6 Gene 5基因组同源性比对结果454.6.7 Gene 6基因组同源性比对结果454.7 系统进化树分析4