【作者】 马友记；

【导师】 赵兴绪； 柳纪省；

【作者基本信息】 甘肃农业大学 ， 临床兽医学， 2006， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)和猪2型圆环病毒(Porcine circovirus type 2, PCV2)病是当今养猪业严重关切的两种重大传染病之一,两病的广泛流行和蔓延已给世界各国养猪业的发展造成了严重威胁和经济损失。在现代化规模养猪场中,PCV2与PRRSV往往混合感染,发病率呈上升趋势。为了有效控制这两种传染病的流行,减少由此引起的经济损失,本研究以PCV2和PRRSV当地流行毒株为研究材料,进行了基因克隆、分子结构特征分析、基因图谱绘制、分子诊断、流行病学调查及免疫研究,主要研究成果如下:(1)应用RT-PCR技术,分段克隆了PRRSV GS株基因,采用DNAStar软件将各基因依次拼接,并对其特征进行了分析,绘制了PRRSV GS株全基因图谱和系统进化树。结果表明,该毒株全基因组长15427bp,包含8个阅读框(ORF),其中5’端非编码区长190bp,与国内外参考毒株核苷酸序列的同源性分别为56.3%～97.9%;ORF1长11882,其中ORF1a长7512bp,与国内外参考毒株核苷酸及推导氨基酸序列同源性分别为54.3%～98.7%和49%～99.3%,ORF1b长4374bp,与国内外参考毒株核苷酸及推导氨基酸序列同源性分别为60.8%～95.5%和49%～99.3%;结构蛋白基因长约3819bp,分为6个结构蛋白基因区,与BJ-4、CH-1a、HB-2(sh)/2002和VR-2332美洲株等核苷酸的同源性分别为:ORF2 93.1%～96.6%,ORF3 90.7%～96.5%,ORF4 88.1%～95.5%,ORF5 87.9%～95.0%,ORF6 95.1%～97.1%,ORF7 92.2%～95.7%,推导的氨基酸同源性分别为:ORF2 91.1%～95.7%,ORF3 89.4%～92.5%,ORF4 88.3%～96.6%,ORF5 87.1%～95.0%,ORF6 96%～97.1%,ORF7 90.3%～95.2%;3’端非编码区长151bp,与国内外参考毒株核苷酸序列的同源性分别为70.9%～100%;最后有一个长14个碱基的poly(A)尾。从系统进化树分析,PRRSV GS株同以LV株为代表的欧洲型遗传距离较远,而与以VR-2332株为代表的北美洲型和流行于我国的毒株遗传距离较近,进一步证实了流行于我国的毒株在基因型上属于北美洲型。(2)构建了PRRSV GS株E基因的重组腺病毒质粒,转染AD-293细胞,经荧光检测表明获得了AD-E复制缺陷型腺病毒。用重组腺病毒AD-E免疫小鼠,以间接ELISA和MTT法检测小鼠血清抗体水平和T淋巴细胞增殖情况,结果能刺激T淋巴细胞增殖,但未能检测到小鼠血清中的抗体,其原因有待进一步研究。(3)应用PCR技术对从甘肃猪场分离的3株PCV2毒株进行了全序列测定,其中PCV2 Ww株基因组长度为1768bp,PCV2 LZ和TS株长1767bp,将序列登陆到Genbank获得3个登录更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important virus that affect the swine industry nowadays. Epizootic and spread of PRRS and PCV2 have rised serious threat and significant economic impact on the swine industry worldwide. In modern large-scale pig farms, the PRRSV and PCV2 usually co-infect the pigs, and increasing outbreak is observed. In order to control PRRSV and PCV2 effectively and reduce the economy loss due to these infections, genomic mapping and molecular immunity of one PRRSV strain and three PCV2 strains predominantly prevalent in Gansu province were investigated.Genome of PRRSV Gansu strain was obtained by RT-PCR, sequenced and analyzed by using DNAstar software. The genomic mapping and phylogentic tree of PRRSV were constructed. The results indicated that the complete genome of PRRSV Gansu strain is 15427bp in length with eight open reading frames (ORFs). The 5’end of non-coding region is 190bp and shares 56.3% to 97.9% nucleotide sequence homology with the reference sequence of other PRRSV strains published in GenBank. The ORF1 is 11822bp in length and included ORF1a and ORF1b, of which, the ORF1a is 7512bp and shares 54.3% to 98.7% nucleotide sequence homology and 49% to 99.3% deduced amino acid homology with the reference sequence of other PRRSV strains published in GenBank. The ORF1b is 4374bp and shares 60.8% to 95.5% nucleotide sequence homology and 49% to 99.3% deduced amino acid homology with the reference sequence of other PRRSV strains published in GenBank. The structure protein gene is 3819bp in length and has six structural protein regions, of which,ORF2 shares 93.1% to 96.6% sequence homology and 91.1% to 95.7% deduced amino acid homology, ORF3 shares 90.7% to 96.5% sequence homology and 89.4% to 92.5% deduced amino acid homology, ORF4 shares 88.1% to 95.5% sequence homology and 83.3% to 96.6% deduced amino acid homology, ORF5 shares 87.9% to 95% sequence homology and 87.1% to 95% deduced amino acid homology,更多还原