【作者】 邱璜；

【导师】 夏平安； 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒引起猪的烈性传染病,在分类地位上属于套式病毒目,动脉炎病毒科,动脉炎病毒属,不分节段,有囊膜的单股正链RNA病毒。是能够引起母猪繁殖障碍和新生仔猪呼吸症状的严重危害养猪业的一种重要病原。假型病毒是指一种反转录病毒的囊膜糖蛋白被另外一种病毒的囊膜蛋白所代替,而基因组仍保持反转录病毒本身的特性。带有异源性病毒囊膜糖蛋白的假型病毒作为研究病毒的侵入模型是非常安全的,因为其仅能引起单循环感染,能够获得异源性病毒的宿主范围,因此便于研究病毒的侵入机制、组织嗜性、中和抗体分析以及受体的鉴定等。目前,PRRSV分子生物学方面已进行的大多数研究都针对其主要结构蛋白,特别是针对囊膜蛋白GP5的研究,本实验室也利用病毒假型技术成功构建了PRRSV GP5的MuLV假型病毒体系。但对PRRSV次要结构蛋白的研究相对较少,本试验利用反转录PCR技术扩增了PRRSV次要结构蛋白GP2、GP3、GP4基因,并将GP2、GP3、GP4基因分别克隆至真核表达载体pcDNA3.0中,构建含次要结构蛋白基因的真核表达质粒pcDNA-GP2、pcDNA-GP3、pcDNA-GP4,分别运用脂质体转染方法共转染293T细胞,以空载体pcDNA3.0转染293T细胞作为阴性对照,以pcDNA-GP5(由本实验构建并保存)转染293T细胞作为阳性对照。48h后收获转染细胞,分别与PRRSV特异性多抗及FITC标记的羊抗猪二抗反应,经流式细胞仪检测蛋白表达,结果表明,与阴性对照值0.7%相比,阳性对照pcDNA-GP5的表达量是最高的,达到30.4%,GP2、GP3和GP4重组质粒仅得到少量表达,分别为4.9%、5.4%、7.6%。用pcDNA-GP2、pcDNA-GP3、pcDNA-GP4分别与鼠白血病病毒(MuLV)病毒假型构建体系的两种质粒pHIT60(包括MuLV的结构蛋白基因,即gag和pol)和pHIT111(为MuLV的基因组及报告基因LacZ)瞬时共转染人胚肾细胞293T,48小时后收获转染细胞上清液,超速离心浓缩上清液,利用PRRSV特异性多抗和辣根过氧化物酶标记的二抗通过Western-blot检测,结果未能检测到GP2、GP3、GP4在此浓缩液中表达,说明PRRSV次要结构蛋白不能整合到病毒粒子表面。通过转染上清液感染Marc-145细胞和猪肺巨噬细胞(PAM)等不同的靶细胞,用缺乏囊膜蛋白和已经证实可以形成MuLV假型病毒的水泡性口炎病毒糖蛋白G(VSV-G)作阳性对照,结果未能检测到报告基因LacZ在此转染上清液中的表达,说明这些上清液不具有感染宿主细胞的能力。所以PRRSV次要结构蛋白各自单独不能成功包装成MuLV假型病毒粒子。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome is caused by PRRSV, single strand , normal chain,envelope and no-segmented RNA viruses that belongs to the virales Nidovirales, Arteriviridae,Arterivirus. PRRSV is an important pathogen of pigs which has severely hazarded swine the industry. It primarily not only causes respiratory failure in susceptible sows , but also causes respiratory problems in piglets.The retroviral envelope protein can be exchanged for envelope proteins from non-related viruses,a process called pseudotyping.Pseudotyped viruses with heterogenic glycoprotein incorporated into retroviral particles were proved to be a safe viral entry model,which only go through a single cycle infection and acquired the host range of the parent viruses where the glycoprotein were derived,thus they could facilitate the research on viral entry mechanism,viral tropism,neutralization antibody analysis,and receptor identification.Nowdays,mostly studies about molecular biology of PRRSV is its major structural protein, especially the GP5 of PRRSV, In our laboratory,we had constructed the MuLV-GP5 by pseudotype system,but the study on the secondary structural protein is more less. In this study,the secondary glycoprotein GP2,GP3and GP4 gene of PRRSV were amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.0 to gengerate the expressing plasmid pcDNA-GP2,pcDNA-GP3 and pcDNA-GP4, Respectively transfered 293T cell with liposome method,and the plasmid pcDNA3.0 transfered 293T cell as negative control,the plasmid pcDNA-GP5(constructed by our lab) transferred 293T cell as positive control. collecting cells after 48h, the cells were incubated with specificness polyclonal anti-PRRSV serum and fluorescein-labelled goat anti-swine IgG, respectively. Using a flow cytometry to detect the protein expression quantity.The results showed that the expression quantity of negative control achieved 0.7%, the expression quantity of positive control pcDNA-GP5 was the highest ,achieved 30.4%,the recombinant plasmid pcDNA-GP2,pcDNA-GP3 had a small quantity expression,each expression were achieved 4.9%,5.4% and 7.6%.293T cells were co-transfected with the recombinant plasmid,pHIT60(has the structural genes of MuLV) and pHIT111(has the retroviral genome,containing LacZ as a reporter) plasmids to generate pseudotying virus. The retroviral supernatants were harvested 48 hours post-transfection and centrifuged to remove cellular debris,and used in western-blot ,the results indicated that the GP2,GP3 and GP4 were not expressed in the concentrated supematant fluid, the secondary structural protein could not integrate into the surface of virus particles. Through the transfected supernatants infected Marc-145,PAM and the other different target cells, and parallel transfections were carried out with supernatants produced in absence of a viral envelope and with the vesicular stomatitis virus(VSV)G proteins,which is known to efficiently pseudotype MuLV. The results of infection assays indicated that the transfected supernatants could not detect the expression of reporter gene. The results also indicated that those supernatants had no infection. So the alone secondary structural protein of PRRSV unable construct the infectious MuLV pseudotype virus.更多还原