【作者】 王永志；

【导师】 文心田；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 猪繁殖与呼吸综合征病毒（PRRSV）是引起猪繁殖障碍和呼吸道疫病的病原之一，病毒的M、N蛋白上具有保守性抗原位点，抗原性好。本研究进行了PRRSV C14-2分离株M、N全基因的克隆分析，构建了N基因的pET-32a-N原核表达载体，建立了以重组N蛋白为抗原检测PRRS的间接ELISA方法。 实验参考PRRSV美洲型毒株VR2332株的基因序列，设计了M、N基因的特异性引物P1/P2及P3/P4，通过RT-PCR扩增出PRRSV C14-2分离株的M、N全基因。以pMD18-T为载体对M、N基因进行了克隆。序列测定表明，M基因长522bp，编码174个氨基酸；N基因长369bp，编码128个核苷酸。序列分析表明，M基因与VR2332株和LV株核苷酸同源性分别为99.0％及67.8％，编码氨基酸同源性分别为98.3％及77.5％；N基因与VR2332株和LV株的核苷酸同源性分别为99.7％及65.1％；编码氨基酸同源性分别为99.2％及57.7％。进化分析表明，C14-2株与美洲型毒株遗传距离较近，从分子水平上证实了C14-2分离株属于美洲型毒株。 将pMD18T-N中的N基因亚质克隆进pET-32a（+），构建了N基因的pET-32a-N原核表达载体，并将其转化进表达宿主菌Ecoli BL21（DE3）中进行诱导表达。表达产物经SDS-PAGE电泳分析，获得了一条约35kDa大小的浓缩蛋白带。优化实验确定了重组蛋白的最佳诱导表达条件：IPTG终浓度0.8～1.0mM，诱导时间3～4h。定位分析表明融合蛋白以可溶性蛋白与不溶性蛋白两种蛋白形式（包涵体）存在于细胞质中。在变性及自然条件下分别以组氨酸纯化试剂盒对重组表达蛋白进行亲合纯化。蛋白质免疫印迹试验结果显示，重组表达蛋白与抗PRRSV的猪血清发生特异性反应，表明其具有抗原活性。 以重组表达融合蛋白作为包被抗原，建立了诊断PRRS的间接ELISA方法。实验确定了最佳抗原包被浓度为3.99μg/ml，血清稀释度为1：40。阳性标准初步确立为：OD₄₅₀＞0.293。特异性实验结果显示，间接rN-ELISA与猪布氏杆菌病、猪瘟、猪大肠杆菌病、猪口蹄疫、猪细小病毒病、猪伪狂犬病阳性血清不发生交叉反应。重复性实验结果显示，间接rN-ELISA批内与批间检测样品结果的平均变异系数分别为3.9493％及6.161％，表明重复性好。间接rN-ELISA与IDEXX HerdCheck ELISA比较结果表明，间接rN-ELISA具有高敏感性及强特异性。 本论文研究进行的PRRSV M、N基因克隆与分析，N基因表达和间接rN-ELISA方法的建立，对PRRSV C14-2株的病原分子生物学特点进行了深入研究，并建立了相应的检测方法。该研究对于PRRS的流行病学调查、病原学研究及诊断防治均具有重要意义。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus (PRRSV) belonging to the Arteriviridae family,which causes reproductive disorders in gilts and sows and respiratory distress in pigs,is an economically important etiological agent of modern porcine industry. Previous studies have demonstrated that M and N proteins contain the conseved epitopes among PRRSV isolates.Especially, N protein is the most abundant protein with high antigenic activity of PRRSV, therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease caused by PRRSV. In this study, complementary DNA corresponding to the entire sequence of M and N gene of the C14-2 strain of PRRSV were cloned into vector pMD18-T after being amplified by reverse transcription polymerase chain reaction (RT-PCR) method. Then N gene was subcloned into prokaryotic vector pET-32a(+) to express recombinant N protein for the purpose of developing an indirect rN-ELISA method to detect antiserum against PRRSV.According to the nucleotide sequence of PRRSV VR-2332. two pairs of primes P1\P2 and P3/P4 for M and N genes were designed ,respectively, to amplify the corresponding target genes byRT-PCR. After purified, the gene fragments were both cloned into a vector pMD18-T. Positive clones were screened and identified by PCR and enzyme-digestion. Except termination codon, sequencing results showed M and N genes of isolate C14-2 contained 522bp and 369bp nucleotides, correspondingly encoding 174 and 123 amino acids, respectively. The M and N genes and the deduced amino acids sequence were compared with corresponding sequence of VR2332 and LV. The results indicated that the homology of M gene and the deduced amino acids were 99.0%, 67.8%, 99.7% and 65.1%, respectively. The homology of N gene and the deduced amino acids were 98.3%, 77.5%, 99.2% and 57.7%, respectively. The phylogenetic relationships which confirmed that the isolate C14-2 blonged to American genotype strains, were established using neighbour-joining and parsimony methods based on M and N gene nucleotides.The N gene contained in pMD18T-N were subloned into prokaryotic vector pET-32a(+) to construct a recombinant plasmid pET-32a-N. After recombinant plasmid更多还原