【摘要】 利用反转录-聚合酶链式反应（RT-PCR）技术扩增出猪繁殖与呼吸综合症病毒（PRRSV）美洲株ATCC VR-2332 ORF6及部分ORF7 586 bp的cDNA。将PCR产物连接进线状克隆载体 pGEM-T easy vector后获阳性重组质粒。用EcoRI及SmaI双酶切阳性重组质粒DNA，回收463 bp的小片段并以此制备出地高辛标记的核酸探针。应用该探针对4头鼻内人工感染ATCC VR-2332的45日龄SPF猪感染后不同时间体内的PRRSV分布进行了研究。结果显示，在感染后30 h，即可在扁桃体、肺脏、脾脏、肝脏、肾脏、胸腺、心脏、脑、淋巴结、鼻粘膜、胰腺、睾丸、附睾等组织中检出病毒，且一直持续至实验结束（人工感染后28 d）。应用探针对人工感染PRRSV北京分离株BJ-4的4头SPF猪及4例死胎的组织进行了检测，均可在肺门淋巴结、胸腺、脑、扁桃体等组织中发现阳性杂交信号。这些结果表明，制备的核酸探针可用于猪繁殖与呼吸综合症的临床诊断。更多还原

【Abstract】 A 586 bp cDNA fragment for ORF6 and part of ORF7 of PRRSV AT CC VR-2332 was amplified by RT-PCR. The PCR product was cloned into p GEM-T easy vector. A 463 bp fragment was obtained by EcoRI/SmaI digest ion of recombinant plasmid and was labeled with digoxigenin-dUTP.Studi es on the distribution of PRRSV in four 45-day-old SPF pigs infected a rtificially with PRRSV ATCC VR-2332 was performed by using the probe. It was shown that tonsil,lung,spleen,liver,kidney,thymus gland, heart , brain, lymph nodes,nasal mucosa,testis, epididymis, pancreas were po sitive at 30 h post- inoculation and the positive signals in these tis sues existed until the termination of the experiment(day28).The contro l tissues were negative.Four 45-day-old SPF pigs infected with PRRSV BJ-4 isolate and four stillborn fetuses were deteced with the probe.Po sitive hybridization signals could be detected in lung,tonsil,lymph no des,thymus gland,brain,etc.All the results indicated that the probe co uld be used for the diagnosing PRRS clinically.更多还原