【作者】 希尼尼根；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2004， 博士

【摘要】 本研究在对从四川省分离的猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus，PRRSV)SC1和SC2株进行细胞培养特性、电镜观察和RT—PCR鉴定的基础上，对ORF5基因进行克隆、鉴定和序列分析，构建了ORF5基因和猪白细胞介素2(IL-2)真核表达载体(核酸疫苗)，利用Marc-145细胞、小鼠和猪作为模型研究ORF5核酸疫苗在Marc-145细胞中表达、在仔猪和小鼠体内的动态分布及安全性、诱导仔猪体液免疫和细胞免疫的能力、IL-2的免疫增强等作用进行了系统研究，获得如下结果： 1．猪繁殖与呼吸综合征病毒四川分离株ORF5基因的序列及分析：本研究对SC1和SC2分离株的ORF5基因分别进行pGEM-T载体克隆与测序，并进行了同源性比较和进化树分析，结果，SC-1、SC-2分离株ORF5基因序列与VR-2332株的十分相似，其核苷酸同源性达到99％，氨基酸同源性为98-99％，而与LV株相差甚远，其核苷酸及氨基酸同源性仅为57％和54％，在进化程度上四川分离株与美洲型PRRSV有很近的亲缘关系，表明，这两株PRRSV分离毒可能属于美洲型。同时用BioEdit、TMHMM-2.0、TreeView等软件对该基因所推测的GP5进行了分析，结果表明SC1、SC2分离株的GP5各有6个潜在的疏水区、2个跨膜区和一个潜在的信号肽。 2．PRRSV-ORF5基因真核表达质粒的构建及在Marc-145细胞中表达：采用BamHI和EcoRI酶切，通过定向粘末段连接法，分别构建SC1和SC2分离株ORF5基因真核表达质粒pcDNA-SC1-ORF5(核酸疫苗pcDNA-PRRSV-SC1-ORF5)和pcDNA-SC2-ORF5(核酸疫苗pcDNA-PRRSV-SC2-ORF5)，并用脂质体转染法和电穿孔转染法将pcDNA-PRRSV-SC2-ORF5在Marc-145细胞中进行了表达。结果表明，两种转染法都能高效转染pcDNA-PRRSV-SC2-ORF5质粒，但后者比前者转染的表达时间提前3小时；从特异性荧光结果看，两者转染表达的效果差异不大，分别在转染后26h和23h可用荧光抗体技术检测到特异荧光，并在转染后56h达到高峰。 3．PRRSV-ORF5基因真核表达质粒DNA在小鼠和仔猪体内的动态分布及安 四川农业大学博士学位论文全性研究:以pCDNA一PRRSv一sCZ一ORFS免疫小鼠和仔猪，分不同时间段采集组织样品，通过PcR检测pCDNA一PRRSv一ScZ一oRFS质粒在小鼠和仔猪体内的动态分布情况。结果表明，肌注小鼠后，pCDNA一PRRSV一SCZ一ORFS迅速分布到小鼠其他组织器官中，在脑组织中存留时间最短，只有6周;在注射部位存留的时间最长，可达到14周以上;在血液和心肌中能存留10周;肝脏、肾脏、脾脏和肺组织中能存留8周。肌注仔猪后，同样发现pCDNA一PRRSV一SCZ一ORFS分布到仔猪其他组织器官中。同时未发现pcDNA一PRRSv一sCZ一oRFS与宿主细胞染色体整合现象。 4.猪白细胞介素2(IL一2)真核表达质粒的构建及其应用:构建猪白细胞介素2(IL一2)真核表达载体pCDNA一IL一2，并与pCDNA一PRRSV一SCZ一ORFS共同免疫仔猪，观察工L一2对pcDNA一PRRSv一SCZ一ORFS的免疫增强效果。试验结果表明，儿一2对PcDNA一PRRSV一SCZ一ORFS诱导的体液免疫和细胞免疫应答有明显的增强作用。 5.检测猪繁殖与呼吸综合征抗体间接ELISA方法的建立:本实验以Marc一145细胞增殖、梯度离心法获得的PRRSV一SCZ病毒作为包被抗原建立了检测PRRS抗体间接ELISA方法，通过实验证明该方法具有特异性强、敏感度高、重复性好等特点，适宜作为PRRSV ORFS核酸疫苗免疫猪后抗体的检测。 6.PRRSv一ORFS核酸疫苗诱导仔猪体液免疫的研究:pCDNA一PRRSv一SCZ一ORFS分3种剂量和2种免疫途径对仔猪进行免疫后，用间接ELISA法检测仔猪的体液免疫应答情况。结果表明，pCDNA~PRRSV一SCZ一ORFS在3种剂量和2种免疫途径的免疫中，均能诱导仔猪产生体液免疫应答。大剂量组与中剂量组及小剂量组相比，它诱导体液免疫应答更强，差异显著(P<0 .05)，其中以基因枪免疫组的诱导能力最强，其结果差异显著(P<0.05)或极显著(P<0.01)。 7.PRRSV--ORFS核酸疫苗诱导仔猪细胞免疫的研究:采取MTT比色法和流式细胞仪检测仔猪的细胞免疫应答情况。结果表明，pCDNA一PRRsv一ScZ一oRFS在3种剂量和2种免疫途径的免疫中，均能诱导仔猪产生细胞免疫的应答。大剂量组表现出在体液免疫中表现相似的效果，即比中剂量组和小剂量组有较强的细胞免疫应答，差异显著(P<0 .05)，但在两种免疫途径即基因枪免疫组与肌肉注射组中，其诱导的细胞免疫应答差异不显著(P>0.05)。更多还原

【Abstract】 In this research porcine reproductive and respiratory syndrome virus (PRRSV) Sichuan isolates of PRRSV-SCl and PRRSV-SC2 were identified by the method of cell culture, electron microscopy and RT-PCR respectively. ORF5 genes of these two isolates were amplified, cloned and sequenced. Eukaryotic expression plasmid (nucleic acid vaccine) of PRRSV-SCl, PRRSV-SC2-ORF5 and IL-2 were constructed and expression, tissue distribution, security, immunogenicity of SC2-ORF5 and ajduvant effect of IL-2 were studied systematically. Results were as follows:1. Sequencing of ORF5 gene of porcine reproductive and respiratory syndrome virus Sichuan isolates: In this research ORF5 of PRRSV-SCl and PRRSV-SC2 were sequenced after amplified by RT-PCR and cloned into pGEM-T vector. ORF5 of PRRSV-SCl and PRRSV-SC2 showed 98-99% nucleotide identities and 99% amino acid identities with strain ATCC VR-2332, 57% nucleotide identities and 54% amino acid identities with strain LV. Computer analysis by software BioEdit and THMM-2.0 indicates that these two isolates have six potential hydrophobic regions and two potential transmembrane helices and one signal peptide respectively. Phylogenetic tree analysis by Tree View software showed that both PRRSV-SCl and PRRSV-SC2 are closely related to VR-2332 strain, but not closely related to LV strain.2. Construction of eukaryotic expression plasmid of PRRSV-ORF5 gene and its expression in Marc-145 cell line: ORF5 of PRRSV-SCl and PRRSV-SC2 were inserted into the eukaryotic expression plasmid pcDNA3.1 (+) to construct the recombinant plasmid pcDNA-PRRSV-SCl-ORF5 and pcDNA-PRRSV-SC2-ORF5, then pcDNA-PRRSV-SC2-ORF5 was expressed in the Marc-145 cell line by cytofectene?transfection reagent kit processing and electroporation. ORF5 expression was detected by means of immunofluoescence. An intense fluorescence could be observed 23h post transfection in the Marc-145 cells transfected by electroporation, which is 3h earlier thanthat processed by cytofectene?transfection reagent. The highest expression product was detected 56h post transfection in the cells transfected with cytofectene?transfection reagent and (or) electroporation. Results showed that pcDNA-PRRSV-SC2-ORF5 was delivered efficiently by the methods of cytofectene?transfection reagent and electroporation respectively into the cell and expressed.3. Studies on distribution and security of PRRSV-ORF5 gene eukaryotic expression plasmid in pigs and mice: Recombinant plasmids of pcDNA-PRRSV-SC2-ORF5 were transferred into the Balb/C mice and piglets by route of intramuscular injection. Then ORF5 gene was detected by PCR from blood, brain, heart, liver, lung, spleen, kidney and muscle of mice and piglets different time post immunization (PI). Results showed that recombinant plasmid pcDNA-PRRSV-SC2-ORF5 distributed in other tissues of mice 3h post inoculation. pcDNA-PRRSV-SC2-ORF5 was detected in the brain 3h-43d PI, in the liver, lung, spleen, kidney from 3h to 70d PI, in the blood and heart 3h to 84d PI, and was detected from the muscle till 115d PI. ORF5 gene was also detected from the same tissues of piglets. Results also confirmed that no pcDNA-PRRSV-SC2-ORF5 was integrated into the chromosome of these two kinds of animals.4. Constructionn of pig IL-2 gene eukaryotic expression plasmid and its application: Eucaryotic plasmid of pcDNA-IL-2 containing pig interleukin 2 gene was constructed and immune responses of pigs immunized with pcDNA-PRRSV-SC2-ORF5 or pcDNA-PRRSV-SC2-ORF5 and pcDNA-IL-2 were observed. Results showed that pcDNA-IL-2 could enhance both humoral and cellular immune responses induced by pcDNA-PRRSV-SC2-ORF5.5. Establishment of indirect ELISA for detection of antibodies against PRRSV: An indirect ELISA for detection of antibodies against porcine reproductive and respiratory syndrome virus was established employing PRRSV-SC2 isolate as coating antigen and some serum samples were assayed. Results showed that the method is characterized by its specificity, sensitivity and stability.更多还原