【作者】 王旭荣；

【导师】 杨增岐； 宋长绪；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2005， 硕士

【摘要】 本试验通过对PRRSV 5 株广东流行株ORF3, 5, 6 3 个基因的分析,探讨广东省5株流行株之间及其与其他毒株间的遗传变异关系,为探索广东省PRRSV 的来源、分子流行病学特征和抗原变异规律提供依据,同时为研制基因工程疫苗奠定了基础。另外将腺病毒表达系统与PRRSV 的主要抗原多肽的基因有机地结合起来,利用腺病毒对动物体细胞的广泛感染性,改善PRRSV 的GP3、GP5 对动物免疫系统的刺激效率和方式,模拟PRRSV病毒的自然感染过程,以期更好地发挥PRRSV GP3、GP5 的抗原活性,为研制PRRSV高效安全的基因工程疫苗开辟新途径。本试验的主要内容及结果如下: 1.5 株PRRSV GD 株ORF3、ORF5、ORF6 基因的变异分析根据GeneBank 中发表的序列分别设计合成了针对PRRSV ORF3, ORF5 和ORF6 基因的引物,利用RT-PCR 方法从广东省送检的5 份可疑病料中均分别扩增得到大小约787bp、630bp、550bp 的片段,克隆入pMD18-T 载体并测序。应用DNAStar 软件分析所测的序列,与国内外已发表毒株和疫苗株(RespPRRS/Repro、RespPRRS MLV)及LV4.2.1 等毒株的序列进行比较,结果表明:广东省的5 个毒株在遗传关系上接近HB-1(sh)株,本研究中分离于不同猪场的5 个毒株间的核苷酸序列差异不显著,可能有共同的来源。2.PRRSV ORF3-5串联基因重组腺病毒的构建用WP3L和WP3R引物从pT-ORF3阳性质粒上扩增完整的ORF3 片段,利用T-A 连接的原理将片段亚克隆入pMD18-T 载体EcoV 处,按常规方法转化DH5a,筛选出正向重组质粒并测序,命名为pMD-ORF3。用WP5L和WP5R引物从pT-ORF5 阳性质粒上扩增ORF5 基因,用Sph I/Hind III 双酶切后回收目的片段,将重组质粒pMD-ORF3 也经Sph I/Hind III 双酶切并去磷酸化,与目的片段于16℃连接,按常规方法筛选出重组阳性质粒并测序,命名为pMD-ORF3-5。用Not I/HindIII 从重组质粒载体pMD-ORF3-5上切取ORF3-5 基因插入pAdTrack-CMV穿梭载体,挑取卡那霉素(Kan~+)抗性的单菌落,按常规方法获得阳性重组子pAdTrack-CMV-3-5。将构建的携有PRRSV ORF3-5 串联基因的重组穿梭载体Pme I 线性化后与腺病毒骨架载体pAdEasy-1 共转染E.coli BJ5183,使之同源重组,经PCR、酶切鉴定获得重组腺病毒质粒pAdCMV-3-5,然后用Pac I 线性化,用脂质体介导转染入人更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus( PRRSV) was one of the most economically important pathogens of swine diease. ORF3, 5, 6 gene of PPPSV GD strains was amplified by RT-PCR and the sequence was analysised in this study. Both the relationship among PRRSV GD isolates each other, and relationship between GD isolates and other strains were discussed. So that to show the basis of origin searching for PRRSV in GD provicine and manufacturing the gene engineering vaccine. The major antigen gene ORF3, 5, 6 of PRRSV were combined to the recombinant adenovirus express system. Based on the extensive infection of adenovirus to body cell of animal, the ORF3-5 fusion gene were developed and iserted into the genome of the adenovirus expressed system inorde to raise the activation of antigen to the system of animal. The paper include two parts: (1)Genetic variation analysis ORF3, 5, 6 genes of PRRSV GD isolates According to the complete sequence of American PRRSV (the ATCC VR2332), three pairs of primers with special restrict enzyme sites were designed. ORF3, 5, 6 genes of PRRSV GD isolates were obtained from 5 PRRSV infected swine samples by RT-PCR, and sequenced after directly cloning into the pMD18-T vector. The sequences of ORF3, 5, 6 genes of GD isolates were compared with the other PRRSV strains in Genebank. The results showed that the sequences of ORF3, 5, 6 of the GD isolates were related closely with ATCC VR-2332 and farther with PRRSV LV strain. And the phylogenetic trees also revealed the GD isolates was related closely with HB-1(sh). (2)Construction of recombinant adenovirus carrying ORF3-5 fusion gene According to the sequence of ATCC VR2332 gene in Genebank, two pairs of PCR primers were designed. The ORF3 gene, a fragment of 780 bp was amplified from the pT-ORF3 by PCR while using WP3L and WP3R, then cloned into pMD 18-T vector and identified by Sal I , Not I/Hind III digestion, and the recombinant plasmids named pMD-ORF3 were obtained. The ORF5 gene were amplified from pT-ORF5 by PCR. The PCR products were cloned into pMD18-ORF3, the recombinant plasmids obtained. The pAdTrack-CMV vector and the interesting gene(ORF3-5)were all digested by the two restriction enzymes Not I and Hind III , then pAdTrack-CMV vector and ORF3-5 gene were linked together with T4 DNA ligase. The ligation mixture was transformed into competent E. Coli DH5a preparaed with Calcium Chloride method. The plasmid DNAs with kanamycin resistant gene were identified by PCR analysis and restriction analysis. The plasmid DNA pAdTrackCMV-3-5 was linearized with Pme I and subsequently cotransfected into the E.Coli BJ5183 together with the backbone plasmid pAdEasy-1. After their homogeneous recombination, screened the positive recombinant adenovirus genome plasmid DNA, the recombinant adenovirus genome DNA was digested with PacI and transfected into 293 cells with Lipofectamine to package the recombinant adenovirus. At last, The recombinant adenovirus carrying ORF3-5 gene was screened under fluorescent microscopy and identified by PCR analysis. The study above offer the scientific basis for the epidemic spread of PRRSV in GD and preventive measures establishing。更多还原