【作者】 张淼涛；

【导师】 张彦明； 谢庆阁；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 博士

【摘要】 猪瘟是危害养猪业的重要疾病之一。猪瘟病毒(CSFV)C-株是世界范围内公认的免疫性能良好的弱毒疫苗株。为探讨CSFV C-株组织毒在SK-6 细胞上的嗜性及其检测方法,以CSFV C-株兔脾组织毒为对象,以SK-6 细胞作为病毒宿主,借助直接荧光抗体染色、夹心ELISA 和RT-PCR 等方法研究了病毒抗原的表达并检测了病毒核酸序列,结果表明,C-株兔脾组织毒可在SK-6 细胞中低水平增殖和表达。ELISA 法和RT-PCR 法适于检测CSFV 弱毒疫苗株,但直接荧光抗体染色法不宜用于CSFV 弱毒疫苗株的检测。该研究为CSFV 弱毒疫苗株的检测提供了一定指导,为C-株兔脾组织毒在SK-6 细胞中的增殖培养奠定了基础,也为CSFV 的逆向遗传学研究提供了基础条件。经过测序发现本研究室前期构建的CSFV 全长cDNA 分子中存在几个致死性突变位点,致使该cDNA 缺乏感染性,为了恢复其感染性,采用RT-PCR、Nested PCR 和Half-nested PCR 技术从实验感染兔脾组织的总RNA 中得到了CSFV C-株全长cDNA 的3 个待改造片段,分别克隆于pMD18-T 载体后进行测序。用重组技术分别从前期构建的5’半长cDNA或3’半长cDNA 中替换F1、F3 和F51,构建成两个新的半长cDNA,进一步连接成新的全长cDNA,经测序证实全长cDNA中3个致死性突变位点均得到改正。借助脂质体转染技术转染SK-6 细胞,检测了CSFV 的抗原表达和特异性核酸序列,结果表明该全长cDNA 具有感染性。成功地建立了CSFV C-株反向遗传操作系统,为从事CSFV 分子生物学研究提供了重要的技术平台。本研究进一步利用该感染性克隆作为骨架,利用猪繁殖与呼吸综合征病毒GP5 基因作为标记基因,通过基因工程技术插入CSFV Npro基因的起始密码子ATG 后,借助脂质体转染SK-6 细胞,检测了包含PRRS 病毒GP5 基因的一段重组序列及CSF 病毒的抗原表达,结果表明GP5 基因在细胞连续传代5 到10 代后仍然稳定的插入在CSFV的序列中,培养细胞中存在猪瘟抗原,由于构建的重组病毒可通过检测GP5 区段的特异序列区别于猪瘟野毒,另外,标记基因为PRRSV 的主要抗原基因,因而该重组体可望作为CSFV C-株活病毒标记疫苗,用于CSF 和PRRS 的免疫预防。更多还原

【Abstract】 Classical swine fever is the most harmful disease in swine breeding. The C strain of CSFV is one of the best attenuated vaccine in the world. To discuss the tropism of spleen derived virus in SK-6 cell, SK-6 cell was selected as the tropism cell and the antigen express and the sequence of CSFV was detected by direct immuno-fluorescent staining method, ELISA and RT-PCR. The result indicated that the spleen derived C strain of CSFV can growth at a low level in SK-6 cell. The ELISA and RT-PCR method is suitable for the detection of cultured CSFV, while the direct immuno-fluorescent staining method is not suitable. The study can served as a useful guide for the detection of attenuated CSFV. And it also constructed a solid foundation for its culture in SK-6 and its reverse genetics study. By sequencing, several leathal mutation sites were fount in the full-length cDNA clone we constructed before. To resume its infectivity, it was reconstructed. Using RT-PCR, nested PCR and half nested PCR, three fragments were obtained from the total RNA of experimentally infected rabbit spleen. After correspondingly cloned in pMD18-T vector, they were sequenced. The corresponding sequence in 5’half cDNA and 3’half cDNA were substituted by recombinant. The two half cDNA were then ligated into full-length cDNA. After sequencing, it was verified that the 3 lethal mutation sites were corrected. Then it was transfected into SK-6 by lipofectin. The antigens and special sequence of CSFV was detected. The result indicated that the full-length cDNA have infectivity. The study successfully constructed the reverse genetic system of CSFV. The infective cDNA clone was used as a skeleton, the GP5gene of PRRSV was used as a marker gene and was inserted into the genome of CSFV right down the initiation codon ATG in Npro. After transfected the SK-6, a special sequence containing GP5 gene the antigens of CSFV were detected. The results indicated that the GP5gene was stably inserted in the CSFV genome even after 5or 10 passages and the antigen of CSFV can express in SK-6. Due to the field virus can be differentiated from the recombinant by detecting the special sequence containing GP5, and the marker is the major antigen gene of PRRSV, the recombinant maybe used as a marker vaccine to control CSF and PRRS. It is hopefully a live marker vaccine of CSFV C strain.更多还原