【作者】 李雪峰；

【导师】 苏布达； 相文华； 曹贵方；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 马流感(Equine influenza,EI)是由马流感病毒引起的一种急性、高度接触性的马属动物呼吸道传染病。近年来,随着国际马术赛事的蓬勃兴起以及马属动物流动范围扩大等原因,致使EI在全球上频繁暴发、流行并由此对赛马业等构成了严重威胁。因此,从分子水平上阐明EIV H3N8亚型的流行规律,不仅在病毒学研究领域中有重要的学术意义,而且对EI防控也具有实用价值。本研究先将内蒙古株A/equine/Inner Mongolia/8/08(H3N8)在SPF鸡胚中增殖,提取病毒核酸,根据EIV 8个基因片段的3’末端高度保守的核苷酸序列特点,设计一条反转录引物,根据GenBank已发表的H3N8亚型EIV 8个基因的cDNA序列,利用Oligo6.0设计并合成14对特异性引物,通过RT-PCR方法扩增出8个基因的序列片段,将它们连接到pMD18-T载体上。序列测定及分析结果显示,EIV 8个基因的cDNA包含该病毒完整的开放阅读框,与美洲株Kentucky/02的同源率最高,HA,NA,NP,NS1,NS2,M1,M2,PA,PBl,PB2基因核苷酸序列的同源性分别为98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8%,99.2%,两者各基因有着相似的遗传演化过程。本研究将全长700bp的NS1基因亚克隆到原核表达载体pET-32a中,经双酶切鉴定为阳性的重组质粒再转化到感受态细胞BL21(DE3)中,挑选含重组质粒pET-NS1的菌落,经SDS-PAGE分析表明,用IPTG(终浓度为0.8mmol/L)诱导培养5h,pET-NS1融合蛋白表达量达到高峰,以包涵体形式存在,目的蛋白与载体上的标签蛋白融合后大小约为46kDa,与理论值相符。Western-blot分析表明,融合蛋白能与H3N8亚型马流感病毒感染马阳性血清发生特异性反应,具有良好的抗原性。更多还原

【Abstract】 Equine influenza (EI) is an acute contagious respiratory infectious disease which is common for horses.In recent years, with the international horse racing increasing and the scope enlarging,it seriously threats horse racing because of the outbreak and epidemic ofequine influenza.It is very significant not only on virology and veterinary medicine study but also on prevent and control equine infuenza that master the epidemic regularity of EIV subtype H3N8 on molecular level.In this study. Virus of the local isolate A/equine/Inner Mongolia/8/08(H3N8) was propagated in SPF chicken embryo and nudeic acid was extracted. According to the conservation sequence of 3’terminus of eight gene fragment from EIV, a piece of primer was designed to reverse transcript RNA. Accordmg to the cDNA sequence from H3N8 EIV published in GenBank, fourteen pairs of specific primers were designed to amplify all of the eight gene by software Oligo6.0 and the products of RT-PCR was cloned into pMD18-T vector. The sequencing results show that all the cDNA has a complete open reading frame.By comparing with some strains of the same subtype published in GenBank,there is the highest percent homology of nucleotide with American strain Kentucky/02.The homology of nucleotide sequence of H A , N A , N P , N S 1 , N S 2 , M 1 , M 2 , P A , P B l a n d P B 2 i s 98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8% and 99.2% respectively,comparing with the sequence of the strain Kentucky/02.It showed that there is the same evolutional process of all the gene and this strain and the strain Kentucky/02 may derive from the same ancestor.The NS1 gene,700bp, was subcloned into prokaryotic expression vector pET-32a, and the positive recombinant plasmid was transformed into competence cell BL21 (DE3). The colony containing the positive plasmid pET-32a was screened and induced by 0.8mmol/L IPTG and analyzed by SDS-PAGE. The result in gel showed that the targat protein was mostly in inclusions when induced by IPTG,and the product was 46kDa fused with tag protein.The Western-blot result showed that the recombinant protein can specific bind with EIV(H3N8) positive serum.更多还原