【作者】 许丽娜；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 Ⅰ型干扰素是由病毒诱导的外周血单核细胞（PBMC）和成纤维细胞产生的一种糖蛋白，具有显著的抗病毒作用。它能干扰病毒的合成与复制，是机体抗病毒感染的第一道防御系统。在1995年，Schultz等首先克隆出鸭干扰素（DuIFN），并分别在大肠杆菌（E．coli）和COS7细胞中高效表达，发现重组的DuIFN对一些RNA病毒有很强的抗病毒能力，对原代鸭胚肝细胞中DHBV的复制有强烈抑制作用。 本实验参考GenBank发表的DuIFN全序列，应用Oligo 4.1设计一对引物，采用RT-PCR方法扩增得到一段大约600bp的DNA片段。将这个片段克隆至pMD18-T载体，序列分析结果表明，克隆的鸭Ⅰ型干扰素（DuIFN-1）基因的开放阅读框包括576bp，编码蛋白为191个氨基酸，N末端的30个疏水性氨基酸是信号肽，成熟的多肽由161个氨基酸组成。同GenBank上公布的DuIFN基因进行序列比较分析表明，在核苷酸水平上的同源性为99.65％，在氨基酸水平上的同源性为98.95％。但是，DuIFN-Ⅰ与DuIFN-Ⅱ相比，核苷酸水平的同源性仅为31.73％，氨基酸水平的同源性为7.58％。与鸡、犬和人的Ⅰ型干扰素基因进行同源性比较，在核苷酸水平分别为71.82％、42.86％和39.21％，在氨基酸水平分别为46.88％、16.36％和16.50％。 将DuIFN-Ⅰ基因特异性的亚克隆到表达载体pGEX-6p的BamH Ⅰ和Sal Ⅰ酶切位点上，重组质粒转化大肠杆菌TGI和BL21（DE₃）PlysS，经1.0mmol／L IPTG诱导表达，表达产物用SDS-PAGE检测，得到大约47ku的融合蛋白，与预期的相符。 用Antheprot 5.0软件对DuIFN-Ⅰ和DuIFN的二硫键、二级结构进行预测及比较，表明二者的二硫键、二级结构无明显差异。故推断DuIFN-Ⅰ也有生物学活性。 本研究为了解国内外鸭Ⅰ型干扰素核苷酸序列演化上的关系提供了理论依据，同时也为抗鸭病毒性疾病提供了一种新型的治疗制剂。更多还原

【Abstract】 Interferon-I was a giycoprotein produced by PBMC or fibroblasts which is induced by viruses. Interferon-I was notable for its functions of anti-virus. It can interfere virus in synthesis and reproduction. It is the first defending system of antiviral infection.Duck interferon (DuIFN) gene was first cloned by Schultz in 1995, and was expressed in E.coli and COS7 cells. Recombinant DuIFN had a strong antiviral effect toward several RNA viruses and was a potent inhibitor of Duck hepatitis B virus (DHBV) in primary duck hepatocytes.According to the complete nucleotide sequence of DuIFN published in GenBank, a pair of primers were designed with Oligo 4.1. By reverse transcription-polymerase chain reaction (RT-PCR), a DNA fragment of about 600bp was amplified. And then, the fragment was cloned into pMD18-T vector. The result of sequencing shows that the open reading frame (ORF) of Duck interferon-I (DuIFN-I) gene was consist of 576bp, and encodes a protein of 191 amino acids. The 30 hydrophobic amino acids at the N-terminus may serve as signal peptide. Mature polypeptide of DuIFN-I seems to consist of 161 amino acids. Sequence comparison with the published DuIFN gene in GenBank revealed 99.65% identity at the nucleotide level. Identity at the amino acid level was 98.95%. But it only shared 31.73% identity in nucleotide level and 7.58% identity in amino acid level with DuIFN-II gene. By comparing of the homology, DuIFN-I gene share 71.82%, 42.86%, 39.21% identities with Chicken, Canine and human IFN gene in nucleotide respectively, and shares 46.88%, 16.36%, 16.50% identities in amino acid respectively.Then, the DuIFN-I gene was cloned specially into pGEX-6p vector between the BamH I and Sal I cleavage site, the recombinant plasmid was transformed into E.coli TGI and BL21(DE3)PlysS, and was induced with 1.0mmol/L IPTG for DuIFN-I gene expression. The expressed product was identified by SDS-PAGE, a fusion protein about 47ku as we expected was found.The secondary structure and disulfide bonds of DuIFN-I and DuIFN were predicted and compared by Antheprot5.0 software. The result suggested that there was no difference between DuIFN-I and DuIFN. It demonstrated that DuIFN-I had biological ability as soon as DuIFN published in GenBank.This study provided the basis evidence for the further research about DuIFN molecular biological properties and nucleotide sequence evolution relationship between domestic and exterior countries. It also provided a new reagent for resisting duck viral disease.Candidate: Xu LinaMajor: Preventive Veterinary ScienceSupervisor: Prof. Wang Junwei更多还原