表达绿色荧光蛋白的北美Ⅱ型PRRSV感染性克隆的制备及特性分析
发布时间：2018-12-26 16:48
【摘要】：【目的】由猪繁殖与呼吸综合征病毒(PRRSV)感染引起的猪繁殖与呼吸综合征(PRRS),一直是危害养猪业最为严重的传染病之一,给国内外养猪业带来巨大的经济损失。为在分子水平上研究PRRSV与宿主的相互作用及其致病机制,本试验构建含有绿色荧光蛋白(GFP)基因的PRRSV感染性克隆,并分析了重组PRRSV的某些特性。【方法】依据北美II型PRRSV病毒株(Gen Bank:AY545985.1)的序列,以其感染性克隆重组质粒(p FL12)和含有gfp基因的质粒(pc DNA-EF1-GFP)为模板,采用重叠PCR的方法,扩增出上、下游含有PRRSV非结构蛋白2(Nsp2)基因片段的gfp基因,并通过Spe I和Xho I位点将其插入到p FL12的PRRSV Nsp2基因中,并与其融合,构建成含gfp基因的PRRSV感染性克隆重组质粒p FL12-GFP。重组质粒经限制性内切酶Acl I酶切线性化后,经蛋白酶K/SDS处理,酚/酚氯仿抽提和乙醇沉淀,得到纯化的线性的p FL12-GFP。利用m MESSAGE m MACHINE体外转录试剂盒,在T7 RNA聚合酶的催化下,合成含有5′帽子结构的GFP-PRRSV m RNA。然后利用酵母Poly(A)聚合酶对5′端帽子结构的GFP-PRRSV m RNA进行加尾,使其变为成熟的GFP-PRRSV m RNA,即5′端带有帽子结构和3′端带有Poly(A)尾的GFP-PRRSV m RNA。用Transmessenger Transfection试剂将GFP-PRRSV m RNA转染仓鼠肾(BHK-21)细胞进行病毒的包装,然后用其细胞裂解液上清感染Marc-145细胞扩增重组病毒,感染48h后,用荧光显微镜观察GFP荧光及PCR方法扩增含gfp基因的Nsp2基因片段确定确实拯救出了含gfp基因的重组PRRSV GFP-PRRSV。然后用亲本PRRSV和拯救出的GFP-PRRSV感染Marc-145细胞和猪的肺泡巨噬(PAMs)细胞,并对其感染性和复制能力进行初步分析。【结果】采用重叠PCR的方法将gfp基因成功地插入到了Nsp2基因的特定位点上,并与其融合,构建成含gfp基因的PRRSV感染性克隆重组质粒FL12-GFP。然后将PRRSV感染性克隆重组质粒进行体外转录生成GFP-PRRSV m RNA,先后转染BHK-21和感染Marc-145细胞,成功地拯救出表达GFP的重组GFP-PRRSV。经分析表明,制备的重组GFP-PRRSV与亲本PRRSV一样均能感染Marc-145细胞和猪肺泡巨噬细胞(PAMs),在Marc-145细胞中的增殖速度高于在PAMs细胞中;GFP-PRRSV在两种细胞中的增殖速度与亲本PRRSV相比都有所下降,但未达到显著水平。【结论】利用重叠PCR策略将gfp基因插入PRRSV感染性克隆的Nsp2基因中并与其融合,成功构建出表达GFP的PRRSV感染性克隆。与亲本PRRSV相比,本实验构建的表达GFP的PRRSV感染性克隆的感染性没有显著差异,为将来PRRSV的研究创造了有利条件。
[Abstract]:[objective] Porcine reproductive and respiratory syndrome (PRRS),) caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection has been one of the most serious infectious diseases in pig industry, and has brought huge economic losses to pig industry at home and abroad. In order to study the interaction between PRRSV and host and its pathogenic mechanism at molecular level, PRRSV infectious clones containing green fluorescent protein (GFP) gene were constructed in this study. Some characteristics of recombinant PRRSV were analyzed. [methods] based on the sequence of North American II PRRSV virus strain (Gen Bank:AY545985.1), the infectious clone recombinant plasmid (p FL12) and the plasmid containing gfp gene (pc DNA-EF1-GFP) were used as templates. The upstream and downstream gfp genes containing PRRSV nonstructural protein 2 (Nsp2) gene fragments were amplified by overlapping PCR, and inserted into the PRRSV Nsp2 gene of p FL12 by Spe I and Xho I sites, and fused with them. Construction of PRRSV Infectious cloning Recombinant plasmid p FL12-GFP. containing gfp Gene The recombinant plasmid was linearized by restriction endonuclease Acl I, then treated with protease K/SDS, phenol / phenol chloroform extraction and ethanol precipitation, and purified linear p FL12-GFP. was obtained. Synthesis of GFP-PRRSV m RNA. with 5 'hat structure by using m MESSAGE m MACHINE in vitro transcription kit and catalyzed by T7 RNA polymerase Then the yeast Poly (A) polymerase was used to tail the GFP-PRRSV m RNA with 5 'end cap structure to make it become mature GFP-PRRSV m RNA, that is, GFP-PRRSV m RNA. with hat structure at 5' end and GFP-PRRSV m RNA. with Poly (A) tail at 3 'end. GFP-PRRSV m RNA was transfected into hamster kidney (BHK-21) cells with Transmessenger Transfection reagent to package the virus, and then the recombinant virus was amplified from the supernatant of the cell lysate and infected with Marc-145 cells. After 48 h of infection, the recombinant virus was amplified. Fluorescence microscope observation of GFP fluorescence and PCR Amplification of Nsp2 Gene fragment containing gfp Gene confirmed that the Recombinant PRRSV GFP-PRRSV. containing gfp Gene was indeed saved Then they infected Marc-145 cells and porcine alveolar macrophages (PAMs) cells with parental PRRSV and saved GFP-PRRSV. Results the gfp gene was successfully inserted into the specific site of the Nsp2 gene by overlapping PCR and fused with it. Construction of PRRSV Infectious cloning Recombinant plasmid FL12-GFP. containing gfp Gene Then the PRRSV infectious clone recombinant plasmid was transcribed in vitro to produce GFP-PRRSV m RNA, which was then transfected into BHK-21 and infected Marc-145 cells. The recombinant GFP-PRRSV. expressing GFP was successfully saved. The results showed that the recombinant GFP-PRRSV could infect Marc-145 cells and porcine alveolar macrophages (PAMs),) as well as parental PRRSV. The proliferation rate of (PAMs), in Marc-145 cells was higher than that in PAMs cells. The proliferation rate of GFP-PRRSV in both cells was lower than that of parent PRRSV, but it was not significant. [conclusion] gfp gene was inserted into Nsp2 gene cloned by PRRSV infection using overlapping PCR strategy and fused with gfp gene. PRRSV infectious clone expressing GFP was successfully constructed. There was no significant difference in the infectivity of PRRSV clones expressing GFP compared with parental PRRSV, which provided favorable conditions for the study of PRRSV in the future.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2392404
[Abstract]:[objective] Porcine reproductive and respiratory syndrome (PRRS),) caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection has been one of the most serious infectious diseases in pig industry, and has brought huge economic losses to pig industry at home and abroad. In order to study the interaction between PRRSV and host and its pathogenic mechanism at molecular level, PRRSV infectious clones containing green fluorescent protein (GFP) gene were constructed in this study. Some characteristics of recombinant PRRSV were analyzed. [methods] based on the sequence of North American II PRRSV virus strain (Gen Bank:AY545985.1), the infectious clone recombinant plasmid (p FL12) and the plasmid containing gfp gene (pc DNA-EF1-GFP) were used as templates. The upstream and downstream gfp genes containing PRRSV nonstructural protein 2 (Nsp2) gene fragments were amplified by overlapping PCR, and inserted into the PRRSV Nsp2 gene of p FL12 by Spe I and Xho I sites, and fused with them. Construction of PRRSV Infectious cloning Recombinant plasmid p FL12-GFP. containing gfp Gene The recombinant plasmid was linearized by restriction endonuclease Acl I, then treated with protease K/SDS, phenol / phenol chloroform extraction and ethanol precipitation, and purified linear p FL12-GFP. was obtained. Synthesis of GFP-PRRSV m RNA. with 5 'hat structure by using m MESSAGE m MACHINE in vitro transcription kit and catalyzed by T7 RNA polymerase Then the yeast Poly (A) polymerase was used to tail the GFP-PRRSV m RNA with 5 'end cap structure to make it become mature GFP-PRRSV m RNA, that is, GFP-PRRSV m RNA. with hat structure at 5' end and GFP-PRRSV m RNA. with Poly (A) tail at 3 'end. GFP-PRRSV m RNA was transfected into hamster kidney (BHK-21) cells with Transmessenger Transfection reagent to package the virus, and then the recombinant virus was amplified from the supernatant of the cell lysate and infected with Marc-145 cells. After 48 h of infection, the recombinant virus was amplified. Fluorescence microscope observation of GFP fluorescence and PCR Amplification of Nsp2 Gene fragment containing gfp Gene confirmed that the Recombinant PRRSV GFP-PRRSV. containing gfp Gene was indeed saved Then they infected Marc-145 cells and porcine alveolar macrophages (PAMs) cells with parental PRRSV and saved GFP-PRRSV. Results the gfp gene was successfully inserted into the specific site of the Nsp2 gene by overlapping PCR and fused with it. Construction of PRRSV Infectious cloning Recombinant plasmid FL12-GFP. containing gfp Gene Then the PRRSV infectious clone recombinant plasmid was transcribed in vitro to produce GFP-PRRSV m RNA, which was then transfected into BHK-21 and infected Marc-145 cells. The recombinant GFP-PRRSV. expressing GFP was successfully saved. The results showed that the recombinant GFP-PRRSV could infect Marc-145 cells and porcine alveolar macrophages (PAMs),) as well as parental PRRSV. The proliferation rate of (PAMs), in Marc-145 cells was higher than that in PAMs cells. The proliferation rate of GFP-PRRSV in both cells was lower than that of parent PRRSV, but it was not significant. [conclusion] gfp gene was inserted into Nsp2 gene cloned by PRRSV infection using overlapping PCR strategy and fused with gfp gene. PRRSV infectious clone expressing GFP was successfully constructed. There was no significant difference in the infectivity of PRRSV clones expressing GFP compared with parental PRRSV, which provided favorable conditions for the study of PRRSV in the future.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
【参考文献】
相关博士学位论文 前1条
1 王丽;PRRSV Nsp2与宿主细胞蛋白BAG6和AIF1相互作用的分子机制[D];中国农业大学;2014年
，本文编号：2392404
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