摘要
猪繁殖与呼吸综合征(Porcinereproductiveandrespiratorysyndrome,PRRS),俗称蓝耳病,是由猪繁殖与呼吸综合征病毒(porcinereproductiveandrespiratorysyndromevirus,PRRSV)引起的一种以母猪流产、早产、产木乃伊胎儿和仔猪呼吸道症状及死亡为特征的传染病。该病自上世纪出现以来,给世界养猪业造成了严重的经济损失,被世界动物卫生组织(OIE)列为法定报告的动物疫病,我国将其列为二类动物疫病。对蓝耳病的诊断和预防一直是研究热点。本研究旨在建立一种高致病性蓝耳病的分子诊断方法,探讨研制核酸疫苗的可行性,取得了如下初步结果。
1.用Marc-145细胞分离了高致病性蓝耳病病毒甘肃株(GS株),对其基因全序列进行了测定,利用生物信息学软件对其结构蛋白进行了分析,并对其抗原表位做了预测。
2.通过序列分析设计合成了3条特异性引物,运用一步法RT-PCR,初步建立了高致病性蓝耳病病毒的分子诊断方法,试验结果显示,该法具有快速、特异和敏感等特点,可用于临床样品的快速检测。
3.以ORF3和ORF5抗原基因作为核酸疫苗研究的目的基因,用pBudCE4.1载体构建了真核表达载体pBud-GP3和pBud-GP5。用脂质体法将pBud-GP3和pBud-GP5分别转染Marc-145细胞,通过RT-PCR和荧光抗体检测,目的基因在Marc-145细胞中获得了表达;以BALB/c小鼠为动物模型,通过肌肉注射重组质粒进行免疫接种,用间接ELISA检测抗PRRSV抗体。结果显示,pBud-GP3和pBud-GP5能够有效诱导机体产生体液免疫,说明ORF3和ORF5抗原基因作为PRRSV核酸疫苗的候选基因是可行的。
4.选用IFN-γ基因作分子佐剂,构建了pBud-IFN-GP3和pBud-IFN-GP5重组质粒。将pBud-IFN-GP3和pBud-IFN-GP5质粒转染Marc-145细胞,通过RT-PCR和荧光抗体检测重组质粒在体外的表达情况。然后通过肌肉接种免疫BALB/c小鼠,用ELISA法检测免疫抗体水平;流式细胞术检测T淋巴细胞亚型。结果显示:pBud-IFN-GP3和pBud-IFN-GP5重组质粒可增强小鼠细胞免疫应答,IFN-γ发挥了免疫佐剂的作用,增强了DNA疫苗的免疫原性,是一种很良好的免疫佐剂。同时,为了研究CD58分子作为免疫佐剂的可行性,构建了pBud-CD-GP3和pBud-CD-GP5重组质粒。将pBud-CD-GP3和pBud-CD-GP5重组质粒分别转染Marc-145细胞,RT-PCR和荧光抗体检测结果表明,CD58分子、GP3和GP5在体外获得了表达。为进一步通过动物模型来研究CD58分子的免疫作用奠定了基础。
Porcine reproductive and respiratory syndrome (PRRS) is often called blue ear disease, which is characterized by respiratory disease and reproductive failure in sows and high motality in piglets. The aetiological agent, porcine reproductive and respiratory syndrome virus (PRRSV), belongs to the member of arteriviruses, a group of small, enveloped, positive-strand RNA virus. The disease appeared in the last century and caused enormous economic losses. PRRS is classified as a notifiable disease by OIE and as a list B disease by China. The diagnosis and prevention of PRRS is a very important research field. In this study, the genes of PRRSV GS strain were obtained by RT-PCR and sequencing. A molecular diagnostic method of PRRS was established by designing the specific primers. Eukaryotic expression vectors containing ORF3 and ORF5 were constructed and used to immunize mice. The results were as follows:
1. The PRRSV GS strain was separated by using Marc-145 cells and its whole genome was sequenced. The structural proteins were analyzed with Accelrys and the epitopes were predicted.
2. A molecular diagnostic method of PRRSV was primarily established using one-step RT-PCR. The results revealed that it is able to detect PRRSV quickly and exactly.
3. The ORF3 and ORF5 sequences were inserted into pBudCE4.1 vector. The expression vector pBud-GP3 or pBud-GP5 was transfected into the Marc-145 cells by liposome mediated method. RT-PCR and immunofluorescent test showed transcription and expression of the target genes in the Marc-145 cells. The recombinant plasmids were intramuscularly injected into BALB/c mice and the anti-PRRSV antibody was tested by indirect ELISA. The result indicated that the humoral immune response was effectively induced.
4. A IFN-γgene was inserted into the pBud-GP3 and pBud-GP5, respectively. The pBud-IFN-GP3 or pBud-IFN-GP5 recombinant plasmid was transfected into the Marc-145 cells. RT-PCR and immunofluorescent test showed that the target genes were transcribed and expressed in the Marc-145 cells. The recombinant plasmids were intramuscularly injected into BALB/c mice. The anti-PRRSV antibody was tested by ELISA and the T lymphocyte subset was investigated by flow-cytometry. The results showed that the pBud-IFN-GP3 and pBud-IFN-GP5 recombinant plasmids enhanced cellular immunologic response, in which the IFN-γwas acted as an immunologic adjuvant enhances immunogenicity. At the same time, to evaluate the feasibility of the CD58 as an immunologic adjuvant. The vectors pBud-CD-GP3 and pBud-CD-GP5 were constructed and transfected into the Marc-145 cells, respectively. The result showed that CD58, GP3 and GP5 were successfully expressed in vitro. These results will lay a foundation for further study of immunization of CD58 in animal models.
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