經(jīng)典和高致病性PRRSV疫苗株嵌合病毒的構(gòu)建及免疫原性分析
發(fā)布時(shí)間：2018-10-12 16:47
【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)是一種由PRRS病毒(PRRS virus,PRRSV)引起的豬病毒性傳染病,主要臨床特征為妊娠母豬繁殖障礙和各年齡階段豬呼吸系統(tǒng)疾病。國內(nèi)于1996年首次分離到PRRSV,2006年由PRRSV變異株引起的高致病性豬繁殖與呼吸綜合癥(HP-PRRS)給我國養(yǎng)豬業(yè)造成了巨大的經(jīng)濟(jì)損失,已成為嚴(yán)重危害我國養(yǎng)豬業(yè)的傳染病之一。目前,疫苗免疫仍是防控PRRS的有效方法,但現(xiàn)有的經(jīng)典和高致病性PRRSV疫苗在誘導(dǎo)機(jī)體免疫反應(yīng)和交叉保護(hù)等方面存在差異,而關(guān)于這種差異產(chǎn)生的分子基礎(chǔ)尚無報(bào)道。因此,本實(shí)驗(yàn)選擇經(jīng)典和高致病PRRSV疫苗株為研究對(duì)象,以期通過構(gòu)建嵌合病毒揭示PRRSV疫苗株產(chǎn)生這種免疫學(xué)反應(yīng)差異的原因或基因區(qū)域,同時(shí)為PRRSV基因組結(jié)構(gòu)與功能的研究提供新方法和思路。本實(shí)驗(yàn)在HP-PRRSV HuN4-F112疫苗株的全長cDNA分子感染性克隆pHuN4-F112基礎(chǔ)上,利用反向遺傳操作技術(shù),將pHuN4-F112基因序列分別替換為經(jīng)典PRRSV CH-1R疫苗株的相應(yīng)基因,最終構(gòu)建CH-1R株的全長cDNA分子感染性克隆,并將CH-1R基因組第454位的C沉默突變?yōu)門產(chǎn)生1個(gè)SpeI酶切位點(diǎn)作為鑒定拯救病毒的分子標(biāo)記。將含CH-1R株基因組全長cDNA質(zhì)粒線性化后體外轉(zhuǎn)錄合成病毒RNA,轉(zhuǎn)染BHK-21細(xì)胞包裝病毒粒子,24 h后將細(xì)胞液上清接種MARC-145細(xì)胞,拯救出病毒。通過細(xì)胞病變(CPE)觀察、間接免疫熒光(IFA)和檢測(cè)分子標(biāo)記證明病毒拯救成功。進(jìn)一步比較拯救病毒與親本病毒的病毒滴度、增殖動(dòng)力學(xué)曲線,發(fā)現(xiàn)兩者的病毒學(xué)及生物學(xué)特性沒有顯著差異,以上結(jié)果表明PRRSV CH-1R疫苗株感染性克隆構(gòu)建成功。本實(shí)驗(yàn)以經(jīng)典PRRSV疫苗株CH-1R的全長感染性克隆pCH-1R為骨架,利用反向遺傳操作技術(shù)分別將其ORF1a、ORF1b和ORF2-7替換為HP-PRRSV HuN4-F112的相應(yīng)片段,構(gòu)建3個(gè)全長cDNA嵌合質(zhì)粒。將構(gòu)建的3個(gè)重組cDNA質(zhì)粒經(jīng)體外轉(zhuǎn)錄后轉(zhuǎn)染BHK-21細(xì)胞,并于MARC-145細(xì)胞中傳代,拯救出嵌合病毒,分別命名為rCH-1R-T1a、rCH-1R-T1b和rCH-1R-T27。病毒一步生長曲線結(jié)果表明,3種嵌合病毒在MARC-145細(xì)胞中的生長特性與親本病毒rCH-1R基本一致;嵌合病毒接種PRRSV陰性仔豬結(jié)果顯示,嵌合病毒試驗(yàn)組血清中PRRSV N蛋白抗體陽轉(zhuǎn)率分別為3/3(rCH-1R-T1a)、2/3(rCH-1R-T1b)和1/3(rCH-1R-T27),而rCH-1R免疫組血清N蛋白抗體檢測(cè)始終為陰性。以上結(jié)果表明,PRRSV ORF1a和ORF1b區(qū)域在病毒誘導(dǎo)N蛋白抗體產(chǎn)生中起關(guān)鍵作用。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is a kind of porcine viral infectious disease caused by PRRS virus (PRRS virus,PRRSV). The main clinical features are reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. The highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) caused by the PRRSV variant strain was first isolated in China in 1996. It has caused huge economic losses to the pig industry in China and has become one of the infectious diseases that seriously harm the pig industry in China. At present, vaccine immunization is still an effective method to prevent and control PRRS, but the existing classical and highly pathogenic PRRSV vaccines are different in inducing immune response and cross-protection, but the molecular basis of this difference has not been reported. Therefore, this study selected classical and highly pathogenic PRRSV vaccine strains as the research object, in order to reveal the cause or gene region of the difference in the immunological response of PRRSV vaccine strain by constructing chimeric virus. At the same time, it provides a new method and train of thought for the study of PRRSV genome structure and function. On the basis of the full-length cDNA molecular infectious clone pHuN4-F112 of HP-PRRSV HuN4-F112 vaccine strain, the pHuN4-F112 gene sequence was replaced with the corresponding gene of classical PRRSV CH-1R vaccine strain by reverse genetic manipulation. Finally, the full-length cDNA molecular infectious clone of CH-1R strain was constructed, and the C-silencing site at position 454th of the CH-1R genome was mutated to T producing a SpeI restriction site as a molecular marker to identify and rescue the virus. The full-length cDNA plasmid containing CH-1R strain was linearized and transcriptional synthesis virus (RNA,) was transfected into the packaging virus particles of BHK-21 cells in vitro. After 24 hours, the supernatant of the cell fluid was inoculated with MARC-145 cells to save the virus. By observing cytopathic (CPE), indirect immunofluorescence (IFA) and detection of molecular markers proved that the virus was successfully saved. Further comparing the virus titer and proliferation kinetics curves of saving virus and parent virus, it was found that there was no significant difference in virology and biological characteristics between them. The above results indicated that the infectious clone of PRRSV CH-1R vaccine strain was successfully constructed. In this experiment, three full-length cDNA chimeric plasmids were constructed by replacing ORF1a,ORF1b and ORF2-7 with the corresponding fragments of HP-PRRSV HuN4-F112 by reverse genetic manipulation with the full-length infectious clone pCH-1R of classical PRRSV vaccine strain CH-1R as the skeleton. Three recombinant cDNA plasmids were transfected into BHK-21 cells after transcriptional in vitro, and then subcultured in MARC-145 cells to save the chimeric virus, named rCH-1R-T1a,rCH-1R-T1b and rCH-1R-T27., respectively. The results of one-step growth curve showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were basically the same as those of parental virus rCH-1R, and the results of inoculation of chimeric viruses with PRRSV negative piglets showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were similar to those of parental viruses. The positive rates of antibody to PRRSV N protein in chimeric virus group were 3 / 3 (rCH-1R-T1a), 2 / 3 (rCH-1R-T1b) and 1 / 3 (rCH-1R-T27), respectively. These results suggest that the PRRSV ORF1a and ORF1b regions play a key role in virus-induced production of N-protein antibodies.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3
本文編號(hào)：2266868
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is a kind of porcine viral infectious disease caused by PRRS virus (PRRS virus,PRRSV). The main clinical features are reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. The highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) caused by the PRRSV variant strain was first isolated in China in 1996. It has caused huge economic losses to the pig industry in China and has become one of the infectious diseases that seriously harm the pig industry in China. At present, vaccine immunization is still an effective method to prevent and control PRRS, but the existing classical and highly pathogenic PRRSV vaccines are different in inducing immune response and cross-protection, but the molecular basis of this difference has not been reported. Therefore, this study selected classical and highly pathogenic PRRSV vaccine strains as the research object, in order to reveal the cause or gene region of the difference in the immunological response of PRRSV vaccine strain by constructing chimeric virus. At the same time, it provides a new method and train of thought for the study of PRRSV genome structure and function. On the basis of the full-length cDNA molecular infectious clone pHuN4-F112 of HP-PRRSV HuN4-F112 vaccine strain, the pHuN4-F112 gene sequence was replaced with the corresponding gene of classical PRRSV CH-1R vaccine strain by reverse genetic manipulation. Finally, the full-length cDNA molecular infectious clone of CH-1R strain was constructed, and the C-silencing site at position 454th of the CH-1R genome was mutated to T producing a SpeI restriction site as a molecular marker to identify and rescue the virus. The full-length cDNA plasmid containing CH-1R strain was linearized and transcriptional synthesis virus (RNA,) was transfected into the packaging virus particles of BHK-21 cells in vitro. After 24 hours, the supernatant of the cell fluid was inoculated with MARC-145 cells to save the virus. By observing cytopathic (CPE), indirect immunofluorescence (IFA) and detection of molecular markers proved that the virus was successfully saved. Further comparing the virus titer and proliferation kinetics curves of saving virus and parent virus, it was found that there was no significant difference in virology and biological characteristics between them. The above results indicated that the infectious clone of PRRSV CH-1R vaccine strain was successfully constructed. In this experiment, three full-length cDNA chimeric plasmids were constructed by replacing ORF1a,ORF1b and ORF2-7 with the corresponding fragments of HP-PRRSV HuN4-F112 by reverse genetic manipulation with the full-length infectious clone pCH-1R of classical PRRSV vaccine strain CH-1R as the skeleton. Three recombinant cDNA plasmids were transfected into BHK-21 cells after transcriptional in vitro, and then subcultured in MARC-145 cells to save the chimeric virus, named rCH-1R-T1a,rCH-1R-T1b and rCH-1R-T27., respectively. The results of one-step growth curve showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were basically the same as those of parental virus rCH-1R, and the results of inoculation of chimeric viruses with PRRSV negative piglets showed that the growth characteristics of the three chimeric viruses in MARC-145 cells were similar to those of parental viruses. The positive rates of antibody to PRRSV N protein in chimeric virus group were 3 / 3 (rCH-1R-T1a), 2 / 3 (rCH-1R-T1b) and 1 / 3 (rCH-1R-T27), respectively. These results suggest that the PRRSV ORF1a and ORF1b regions play a key role in virus-induced production of N-protein antibodies.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 郭寶清,陳章水,劉文興,崔益洙;從疑似PRRS流產(chǎn)胎兒分離PRRSV的研究[J];中國畜禽傳染病;1996年02期
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