表達綠色熒光蛋白的北美Ⅱ型PRRSV感染性克隆的制備及特性分析
發(fā)布時間：2018-12-26 16:48
【摘要】：【目的】由豬繁殖與呼吸綜合征病毒(PRRSV)感染引起的豬繁殖與呼吸綜合征(PRRS),一直是危害養(yǎng)豬業(yè)最為嚴重的傳染病之一,給國內外養(yǎng)豬業(yè)帶來巨大的經濟損失。為在分子水平上研究PRRSV與宿主的相互作用及其致病機制,本試驗構建含有綠色熒光蛋白(GFP)基因的PRRSV感染性克隆,并分析了重組PRRSV的某些特性�！痉椒ā恳罁泵繧I型PRRSV病毒株(Gen Bank:AY545985.1)的序列,以其感染性克隆重組質粒(p FL12)和含有gfp基因的質粒(pc DNA-EF1-GFP)為模板,采用重疊PCR的方法,擴增出上、下游含有PRRSV非結構蛋白2(Nsp2)基因片段的gfp基因,并通過Spe I和Xho I位點將其插入到p FL12的PRRSV Nsp2基因中,并與其融合,構建成含gfp基因的PRRSV感染性克隆重組質粒p FL12-GFP。重組質粒經限制性內切酶Acl I酶切線性化后,經蛋白酶K/SDS處理,酚/酚氯仿抽提和乙醇沉淀,得到純化的線性的p FL12-GFP。利用m MESSAGE m MACHINE體外轉錄試劑盒,在T7 RNA聚合酶的催化下,合成含有5′帽子結構的GFP-PRRSV m RNA。然后利用酵母Poly(A)聚合酶對5′端帽子結構的GFP-PRRSV m RNA進行加尾,使其變?yōu)槌墒斓腉FP-PRRSV m RNA,即5′端帶有帽子結構和3′端帶有Poly(A)尾的GFP-PRRSV m RNA。用Transmessenger Transfection試劑將GFP-PRRSV m RNA轉染倉鼠腎(BHK-21)細胞進行病毒的包裝,然后用其細胞裂解液上清感染Marc-145細胞擴增重組病毒,感染48h后,用熒光顯微鏡觀察GFP熒光及PCR方法擴增含gfp基因的Nsp2基因片段確定確實拯救出了含gfp基因的重組PRRSV GFP-PRRSV。然后用親本PRRSV和拯救出的GFP-PRRSV感染Marc-145細胞和豬的肺泡巨噬(PAMs)細胞,并對其感染性和復制能力進行初步分析�！窘Y果】采用重疊PCR的方法將gfp基因成功地插入到了Nsp2基因的特定位點上,并與其融合,構建成含gfp基因的PRRSV感染性克隆重組質粒FL12-GFP。然后將PRRSV感染性克隆重組質粒進行體外轉錄生成GFP-PRRSV m RNA,先后轉染BHK-21和感染Marc-145細胞,成功地拯救出表達GFP的重組GFP-PRRSV。經分析表明,制備的重組GFP-PRRSV與親本PRRSV一樣均能感染Marc-145細胞和豬肺泡巨噬細胞(PAMs),在Marc-145細胞中的增殖速度高于在PAMs細胞中;GFP-PRRSV在兩種細胞中的增殖速度與親本PRRSV相比都有所下降,但未達到顯著水平�！窘Y論】利用重疊PCR策略將gfp基因插入PRRSV感染性克隆的Nsp2基因中并與其融合,成功構建出表達GFP的PRRSV感染性克隆。與親本PRRSV相比,本實驗構建的表達GFP的PRRSV感染性克隆的感染性沒有顯著差異,為將來PRRSV的研究創(chuàng)造了有利條件。
[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.
【學位授予單位】：河北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.3
本文編號：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.
【學位授予單位】：河北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S855.3
【參考文獻】
相關博士學位論文 前1條
1 王麗;PRRSV Nsp2與宿主細胞蛋白BAG6和AIF1相互作用的分子機制[D];中國農業(yè)大學;2014年
，本文編號：2392404
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