【作者】 周丹；

【导师】 郭万柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2011， 博士

【摘要】 猪繁殖与呼吸综合征(PRRS)俗称猪蓝耳病,主要以母猪繁殖障碍及各年龄段猪出现肺炎等呼吸道症状为临床特征,是目前导致我国养猪业三大疾病的主要元凶之一。我国1996年首次报道该病,2006年我国爆发了以高发病率和死亡率为特征的高致病性蓝耳病俗称“猪高热病”,使我国养猪业蒙受了巨大的经济损失。研制更安全、高效、廉价的新型疫苗已成为当今PRRS研究领域的热点和重要方向。本研究将PRRSV (SCS株)的6个主要结构蛋白基因分别载入PCI-neo表达载体中,并转染到VERO细胞中进行表达；同时,将重组质粒通过肌肉注射免疫小鼠和猪,检测小鼠和猪不同时期IL-4、IL-2、IFN-γ等相关细胞免疫因子的变化；还通过荧光定量方法对小鼠体内引发的细胞免疫因子BCL10、INF-β、TLR3与TLR4进行表达监控；还检测免疫猪不同时期T淋巴细胞亚群CD3+、CD4+CD8+动态变化,确定各主要结构蛋白核酸疫苗在动物机体内引发的细胞免疫的效果,研究结论如下：1、用RT-PCR方法从PRRSV SCS株病毒培养液中扩增出核酸片段ORF2、ORF3、ORF4、ORF5、ORF6、ORF7,并将此六个经测序鉴定无突变的片段与pCI-neo表达载体连接,构建六个DNA重组表达质粒,并分别转染vero细胞,通过RT-PCR、间接免疫荧光、Western blot等方法检测转染细胞培养物的生物学活性,结果表明pCl重组质粒转染细胞的培养物对PRRSV抗体有反应原性,pCI-ORF2、pCI-ORF3、pCI-ORF4、pCI-ORF5、pCI-ORF6、pCI-ORF7在Vero细胞中得到了高效表达。2、将构建的PRRSV (SCS株)六个结构蛋白的核酸疫苗经肌肉注射免疫小鼠,通过荧光定量PCR,以小鼠β- actin基因作为内参基因,检测胸腺、肺脏和脾脏等三个器官中TLR3、TLR4、INF-β及BCL10基因转录水平,说明核酸疫苗能够激活小鼠产生TLR3、TLR4、INF-β及BCL10蛋白,加强机体免疫信号传递。其中pCI-ORF5的作用最强。3、构建的PRRSV六个结构蛋白的核酸疫苗经肌肉注射免疫小鼠,能刺激小鼠外周血IL-2、IL-4及INF-γ等细胞因子水平从免疫后开始出现升高,提高动物机体抵抗力。其中pCI-ORF5的作用最强,pCI-ORF2作用最弱。4、将小鼠体内免疫效果较好的pCI-ORF3、pCI-ORF4、pCI-ORF5、pCI-ORF6重组质粒经肌肉注射免疫仔猪,检测外周血淋巴细胞动态变化,发现免疫后外周血CD3+、CD4+、CD8+T淋巴细胞数量增加,诱发动物机体的细胞免疫。5、将小鼠体内免疫效果较好的pCI-ORF3、pCI-ORF4、pCI-ORF5、pCI-ORF6经肌肉注射免疫仔猪,能引起仔猪外周血IL-2、IL-4及INF-γ等细胞因子水平自免疫后开始升高,提高动物机体抗该病能力。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome(PRRS) (commonly called blue ear disease) is characterized with either reproductive failure in pregnant sows, or respiratory tract distress particularly in sucking pigs, and is recognized as a serious swine disease. This viral disease was first discovered in China in 1996. During the summer of 2006, the large-scale devastating outbreak of PRRS in China, known locally as "high fever", resulted the remarkably high morbidity and mortality. To date, PRRS has caused enormous economic losses each year. There is an urgent and important for novel control strategies such as a "safe, efficient, and cheap" vaccines against the PRRS infection. Here we amplified six main structural protein gene, then cloned into the mammalian expression vector PCI-neo, expressed these genes in Vero cell, evaluated the humoral and cellular immune responses in BALB/c mice and swine respectively immunized with six DNA recombinant plasmids by intramuscular injection. The results of research are summarized as following:1. The gene segments of ORF2, ORF3, ORF4, ORF5, ORF6, and ORF7 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA of PRRSV SCS strain culture solution. Then, these verified correct genes were respectively cloned into the mammalian expression vector PCI-neo, and these plasmids were transfected into Vero cells. The expression of six genes were determined by RT-PCR, indirect immunofluorescence (IFA), and western blot etc. These results showed that the recombinant plasmids of pCI-ORF2, pCI-ORF3, pCI-ORF4, pCI-ORF5, pCI-ORF6, and pCI-ORF7 were normally transcribed and expressed in Vero cells.2. BALB/c mice were immunized with the DNA vaccines pCI-ORF2, pCI-ORF3, pCI-ORF4, pCI-ORF5, pCI-ORF6, and pCI-ORF7 of PRRSV (SCS strain) by intramuscular injection. The TLR3、TLR4、INF-B、BCL10 transcriptional level of thoracic gland, lung, and spleen were detected by fluorescent quantitation-PCR, using miceβ-actin gene as reference genes. These results demonstrated that DNA immunization against PRRSV activate the function of proteinum TLR3, TLR4, INF-β, and BCL10, and strengthen immunization signal transmission. The plasmid pCI-ORF5 elicited the most strongest specific cellular immune response, compared with other plasmids.3. BALB/c mice immunized DNA vaccine showed higher levels of IL-2, IL-4, and IFN-y than mice immunized with pCI-neo, then enhancing resistance to disease. Those mice receiving injection of pCI-ORF5 showed the most strongest specific cellular immune response, on the contrary, the plasmid pCI-ORF2 elicited the most weakest specific cellular immune response.4. We have previously found that vaccination of BALB/c mice with the combination plasmids pCI-ORF3, pCI-ORF4, pCI-ORF5, and pCI-ORF6 showed good immune effect. Then, swine were inoculated intramuscularly with these plasmids, the change of CD3+, CD4+, and CD8+on lymphocyte of peripheral blood were investigated. The number of antigen-specific CD3+, CD4+, and CD8+ T-lymphocytes cells were up. The swine injected with combination plasmids enhances cellular immune response.5. Swine which immunized with the plasmids of pCI-ORF3, pCI-ORF4, pCI-ORF5, and pCI-ORF6 showed the increasing production of IL-2, IL-4, and IFN-γ. and improve swine’s immune response and resistance to disease.更多还原