表達(dá)IBV主要結(jié)構(gòu)蛋白重組DEV的構(gòu)建及其免疫保護(hù)性評(píng)價(jià)
發(fā)布時(shí)間：2018-07-28 20:32
【摘要】：雞傳染性支氣管炎(Infectious bronchitis,IB)是由雞傳染性支氣管炎病毒(Infectious bronchitis virus,IBV)引起的雞的高度接觸性上呼吸道及泌尿生殖道疾病。該病在很多國(guó)家和地區(qū)流行,主要感染呼吸道和腎臟,各種年齡雞都易感,常引起產(chǎn)蛋雞產(chǎn)蛋量和蛋品質(zhì)下降,給養(yǎng)禽業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。鴨腸炎病毒(Duck enteritis virus,DEV),又稱鴨瘟病毒(Duck plague virus,DPV),主要引起鴨、鵝及多種雁形目禽類產(chǎn)生急性、熱性、敗血性傳染病,曾在多個(gè)國(guó)家和地區(qū)發(fā)生流行,給養(yǎng)鴨業(yè)造成巨大的經(jīng)濟(jì)損失。DEV是皰疹病毒科成員,其基因組中部分基因是病毒復(fù)制非必需基因,可作為非復(fù)制型疫苗載體構(gòu)建表達(dá)外源基因的活載體疫苗。本研究在本研究室建立的鴨腸炎病毒重組技術(shù)平臺(tái)基礎(chǔ)上,以US10基因缺失表達(dá)EGFP的重組鴨腸炎病毒為親本病毒,應(yīng)用同源重組技術(shù)分別構(gòu)建表達(dá)雞傳染性支氣管炎病毒主要結(jié)構(gòu)蛋白N、S和S1的3株重組鴨腸炎病毒。鑒別PCR、交叉PCR檢測(cè)及測(cè)序結(jié)果表明,IBV N、S和S1基因分別正確插入鴨腸炎病毒基因組內(nèi),替換病毒US10基因。Western blot和間接免疫熒光結(jié)果表明,IBV N、S和S1蛋白均能夠在重組病毒rDEV-N、rDEV-S和rDEV-S1中檢測(cè)到表達(dá),且隨病毒的復(fù)制時(shí)間增加而表達(dá)量增加。對(duì)3株重組病毒的基本生物學(xué)特性進(jìn)行研究,結(jié)果表明3株重組病毒的蝕斑形態(tài)和蝕斑大小與親本病毒rDEV US10-EGFP和野生型病毒DEV Clone-03相似。與野生型病毒DEV Clone-03相比,重組病毒rDEV-N、rDEV-S和rDEV-S1的病毒滴度略有下降,與其親本病毒rDEV-EGFP病毒滴度相近。復(fù)制動(dòng)力學(xué)研究表明,3株重組病毒的復(fù)制趨勢(shì)與親本病毒和野生型病毒一致,在感染細(xì)胞后72h病毒滴度達(dá)到高峰,在感染后96h病毒滴度開(kāi)始下降,說(shuō)明重組病毒的復(fù)制能力與病毒基因組缺失的位點(diǎn)有關(guān),而與插入的外源基因無(wú)關(guān)。分別將3株重組病毒在雞胚成纖維細(xì)胞上連續(xù)傳代至20代,重組病毒表現(xiàn)出良好的遺傳穩(wěn)定性,外源基因隨病毒復(fù)制而穩(wěn)定遺傳并穩(wěn)定表達(dá)。將獲得的3株雞傳染性支氣管炎重組鴨腸炎病毒免疫1月齡SPF雞,于免疫后21d用IBV強(qiáng)毒株ck/CH/LDL/091022進(jìn)行攻毒,同時(shí)設(shè)對(duì)照組。分別于免疫后1w、2w、3w、4w、5w檢測(cè)免疫雞抗體水平。在免疫后1w,rDEV-N免疫組有84%雞只抗體陽(yáng)轉(zhuǎn),rDEV-S和rDEV-S1免疫組有92%雞只抗體陽(yáng)轉(zhuǎn)。免疫后第2w,各免疫組血清抗體有不同程度下降,rDEV-N免疫組76%抗體陽(yáng)性率,rDEV-S免疫組抗體陽(yáng)性率下降至52%,而rDEV-S1免疫組下降至28%。至免疫后第3w,各免疫組抗體水平均顯著下降,rDEV-N免疫組40%抗體陽(yáng)性率,rDEV-S免疫組抗體陽(yáng)性率下降至16%,而rDEV-S1免疫組下降至8%。攻毒后各組雞只具有不同程度發(fā)病,對(duì)照組發(fā)病率為80%,rDEV-N免疫組發(fā)病率30%,rDEV-S免疫組發(fā)病率20%,rDEV-S1免疫組發(fā)病率40%。攻毒后7d對(duì)照組雞只開(kāi)始死亡,直至攻毒后12天不再有雞只死亡。對(duì)照組死亡率為40%,rDEV-N免疫組發(fā)病率30%,rDEV-S免疫組發(fā)病率10%,rDEV-S1免疫組發(fā)病率30%。于攻毒后5d,采集各組雞只咽拭子,應(yīng)用熒光定量RT-PCR檢測(cè)各組雞只呼吸道的排毒情況。對(duì)照組在攻毒后咽拭子排毒率為90%,rDEV-N免疫組攻毒后經(jīng)呼吸道排毒率為20%,rDEV-S免疫組攻毒后呼吸道排毒率30%,rDEV-S1免疫組攻毒后呼吸道排毒率40%。分別于攻毒后5、10、15、20d進(jìn)行抗體水平檢測(cè),從攻毒后抗體變化水平來(lái)看,各組雞只抗體均有不同程度升高,說(shuō)明攻毒成功,且攻毒后3個(gè)免疫組雞只抗體水平均高于對(duì)照組。結(jié)果提示,重組病毒免疫后的雞只具有抵抗病毒攻擊的能力,與對(duì)照組相比,抗體水平上升較快,說(shuō)明雞只存在免疫記憶。從發(fā)病率、死亡率和攻毒后排毒情況來(lái)看,3株重組病毒均能夠?qū)﹄u只提供免疫保護(hù),但不能夠提供完全保護(hù),其中重組病毒rDEV-S免疫保護(hù)效果好于重組病毒rDEV-N和rDEV-S1。
[Abstract]:Infectious bronchitis (IB) is a highly contact upper respiratory tract and genitourinary tract disease caused by avian infectious bronchitis virus (IBV) virus (Infectious bronchitis virus, IBV). It is prevalent in many countries and regions and is mainly infected with respiratory tract and kidney. All age chickens are susceptible and often produce laying hens. The decline of egg production and egg quality caused serious economic loss to poultry industry. Duck enteritis virus (DEV) and duck plague virus (Duck plague virus, DPV) caused acute, hot, septic infectious diseases of duck, goose and many kinds of wild goose birds, which had been prevalent in many countries and regions, and caused huge duck breeding. The economic loss.DEV is a member of the herpes simplex family. Some of the genes in the genome are non essential genes of the virus, and they can be used as a non replicating vaccine vector to construct a live vector vaccine for expressing foreign genes. On the basis of the recombinant technology platform of duck enteritis virus established in this study room, the recombinant duck gut of EGFP is expressed with the deletion of the US10 gene. 3 strains of recombinant duck enteritis virus, the main structural protein N, S and S1, were constructed by homologous recombination technology to identify PCR. The cross PCR detection and sequencing results showed that IBV N, S and S1 genes were correctly inserted into the duck enteritis virus genome, and the.Western blot was replaced by the virus US10 gene. The results of immunofluorescence and indirect immunofluorescence showed that IBV N, S and S1 proteins were detected in the recombinant virus rDEV-N, rDEV-S and rDEV-S1, and the expression increased with the increase of the replication time of the virus. The basic biological characteristics of the 3 recombinant viruses were studied. The results showed that the plaque morphology and plaque size of the 3 strains of the virus and the parent virus were the size of the virus. RDEV US10-EGFP was similar to the wild virus DEV Clone-03. Compared with the wild virus DEV Clone-03, the virus titer of the recombinant virus rDEV-N, rDEV-S and rDEV-S1 decreased slightly, and the titer of the virus rDEV-EGFP virus was similar to that of its parent virus. The replication dynamics study showed that the reproduction trend of the 3 recombinant viruses was consistent with the parent virus and the wild type virus. After infected cells, the titer of 72h virus reached a peak, and the titer of 96h virus began to decline after infection, indicating that the replication ability of the recombinant virus was related to the site of the missing genome of the virus, but it had nothing to do with the inserted foreign gene. 3 recombinant viruses were continuously transmitted to 20 generations on the chicken embryo fibroblasts, and the recombinant virus showed good inheritance. Stability, the exogenous gene was stable and stable with the replication of the virus. 3 chicken infectious bronchitis recombinant duck enteritis virus was immune to 1 month old SPF chickens. After immunization, 21d was attacked by IBV strong strain ck/CH/LDL/091022, and the control group was set at the same time. After immunization, 1W, 2W, 3W, 4W, and 5W were used to detect the antibody level of the immune chicken. 1W, rDEV-N immunization group had 84% chickens with only antibody positive rotation, and 92% chickens in rDEV-S and rDEV-S1 immunization groups were only antibody positive. After immunization 2W, the serum antibody of each immune group decreased in varying degrees, the positive rate of 76% antibody in rDEV-N immunization group, the positive rate of rDEV-S immunization group decreased to 52%, and rDEV-S1 immune group decreased to 28%. to 3W after immunization, and antibody of every immune group. The positive rate of the 40% antibody in the rDEV-N immunization group, the positive rate of the antibody in the rDEV-S immune group decreased to 16%, while the chickens in the rDEV-S1 immunization group decreased to 8%. after the attack, the incidence of the control group was 80%, the incidence of the rDEV-N immunization group was 30%, the incidence of the rDEV-S immune group was 20%, and the incidence of 40%. in the rDEV-S1 immunization group was 7d after 40%. attack 7d. The chickens in the control group died only until 12 days after the attack. The mortality of the control group was 40%, the incidence of the rDEV-N immunization group was 30%, the incidence of the rDEV-S immunization group was 10%, the incidence of rDEV-S1 in the immune group was 30%. after the attack of 5D, and the only pharynx swab of each group was collected, and the control group was used to detect the detoxification of the respiratory tract in each group by fluorescence quantitative RT-PCR. The rate of detoxification of the pharynx swab after attack was 90%, the rate of detoxification of the respiratory tract after the rDEV-N immunization group was 20%, and the rate of detoxification of the respiratory tract after the attack of the rDEV-S immune group was 30%. The rate of detoxification of the respiratory tract after the attack of the rDEV-S1 immunization group was 40%. after the attack of 5,10,15,20d, respectively. The antibody level of the 3 immunized groups was higher than that of the control group. The results suggested that the chickens after the recombinant virus were only able to resist the attack of the virus. Compared with the control group, the level of antibody increased rapidly, indicating that the chicken had no epidemic memory. 3 recombinant viruses can provide immune protection to chickens, but they can not provide complete protection. The recombinant virus rDEV-S has better immune protection than recombinant virus rDEV-N and rDEV-S1..
【學(xué)位授予單位】：甘肅農(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)】：S852.65
本文編號(hào)：2151490
[Abstract]:Infectious bronchitis (IB) is a highly contact upper respiratory tract and genitourinary tract disease caused by avian infectious bronchitis virus (IBV) virus (Infectious bronchitis virus, IBV). It is prevalent in many countries and regions and is mainly infected with respiratory tract and kidney. All age chickens are susceptible and often produce laying hens. The decline of egg production and egg quality caused serious economic loss to poultry industry. Duck enteritis virus (DEV) and duck plague virus (Duck plague virus, DPV) caused acute, hot, septic infectious diseases of duck, goose and many kinds of wild goose birds, which had been prevalent in many countries and regions, and caused huge duck breeding. The economic loss.DEV is a member of the herpes simplex family. Some of the genes in the genome are non essential genes of the virus, and they can be used as a non replicating vaccine vector to construct a live vector vaccine for expressing foreign genes. On the basis of the recombinant technology platform of duck enteritis virus established in this study room, the recombinant duck gut of EGFP is expressed with the deletion of the US10 gene. 3 strains of recombinant duck enteritis virus, the main structural protein N, S and S1, were constructed by homologous recombination technology to identify PCR. The cross PCR detection and sequencing results showed that IBV N, S and S1 genes were correctly inserted into the duck enteritis virus genome, and the.Western blot was replaced by the virus US10 gene. The results of immunofluorescence and indirect immunofluorescence showed that IBV N, S and S1 proteins were detected in the recombinant virus rDEV-N, rDEV-S and rDEV-S1, and the expression increased with the increase of the replication time of the virus. The basic biological characteristics of the 3 recombinant viruses were studied. The results showed that the plaque morphology and plaque size of the 3 strains of the virus and the parent virus were the size of the virus. RDEV US10-EGFP was similar to the wild virus DEV Clone-03. Compared with the wild virus DEV Clone-03, the virus titer of the recombinant virus rDEV-N, rDEV-S and rDEV-S1 decreased slightly, and the titer of the virus rDEV-EGFP virus was similar to that of its parent virus. The replication dynamics study showed that the reproduction trend of the 3 recombinant viruses was consistent with the parent virus and the wild type virus. After infected cells, the titer of 72h virus reached a peak, and the titer of 96h virus began to decline after infection, indicating that the replication ability of the recombinant virus was related to the site of the missing genome of the virus, but it had nothing to do with the inserted foreign gene. 3 recombinant viruses were continuously transmitted to 20 generations on the chicken embryo fibroblasts, and the recombinant virus showed good inheritance. Stability, the exogenous gene was stable and stable with the replication of the virus. 3 chicken infectious bronchitis recombinant duck enteritis virus was immune to 1 month old SPF chickens. After immunization, 21d was attacked by IBV strong strain ck/CH/LDL/091022, and the control group was set at the same time. After immunization, 1W, 2W, 3W, 4W, and 5W were used to detect the antibody level of the immune chicken. 1W, rDEV-N immunization group had 84% chickens with only antibody positive rotation, and 92% chickens in rDEV-S and rDEV-S1 immunization groups were only antibody positive. After immunization 2W, the serum antibody of each immune group decreased in varying degrees, the positive rate of 76% antibody in rDEV-N immunization group, the positive rate of rDEV-S immunization group decreased to 52%, and rDEV-S1 immune group decreased to 28%. to 3W after immunization, and antibody of every immune group. The positive rate of the 40% antibody in the rDEV-N immunization group, the positive rate of the antibody in the rDEV-S immune group decreased to 16%, while the chickens in the rDEV-S1 immunization group decreased to 8%. after the attack, the incidence of the control group was 80%, the incidence of the rDEV-N immunization group was 30%, the incidence of the rDEV-S immune group was 20%, and the incidence of 40%. in the rDEV-S1 immunization group was 7d after 40%. attack 7d. The chickens in the control group died only until 12 days after the attack. The mortality of the control group was 40%, the incidence of the rDEV-N immunization group was 30%, the incidence of the rDEV-S immunization group was 10%, the incidence of rDEV-S1 in the immune group was 30%. after the attack of 5D, and the only pharynx swab of each group was collected, and the control group was used to detect the detoxification of the respiratory tract in each group by fluorescence quantitative RT-PCR. The rate of detoxification of the pharynx swab after attack was 90%, the rate of detoxification of the respiratory tract after the rDEV-N immunization group was 20%, and the rate of detoxification of the respiratory tract after the attack of the rDEV-S immune group was 30%. The rate of detoxification of the respiratory tract after the attack of the rDEV-S1 immunization group was 40%. after the attack of 5,10,15,20d, respectively. The antibody level of the 3 immunized groups was higher than that of the control group. The results suggested that the chickens after the recombinant virus were only able to resist the attack of the virus. Compared with the control group, the level of antibody increased rapidly, indicating that the chicken had no epidemic memory. 3 recombinant viruses can provide immune protection to chickens, but they can not provide complete protection. The recombinant virus rDEV-S has better immune protection than recombinant virus rDEV-N and rDEV-S1..
【學(xué)位授予單位】：甘肅農(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)】：S852.65
【共引文獻(xiàn)】
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1 胡北俠;楊少華;張秀美;張偉;曹三杰;許傳田;黃慶華;張琳;黃艷艷;文心田;;雞傳染性支氣管炎病毒變異株全基因組序列分析[J];病毒學(xué)報(bào);2014年04期
2 霍亞飛;胡北俠;張秀美;張琳;楊少華;黃慶華;黃艷艷;許傳田;;Real-Time PCR方法對(duì)IBV弱毒苗致弱分析[J];山東農(nóng)業(yè)科學(xué);2015年04期
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1 董波;豬血凝性腦脊髓炎病毒S蛋白受體結(jié)合域的初步鑒定[D];吉林大學(xué);2014年
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1 劉隨新;豬流行性腹瀉病毒及其N蛋白核仁定位信號(hào)對(duì)細(xì)胞周期的影響[D];中國(guó)農(nóng)業(yè)科學(xué)院;2013年
2 焦洋;豬流行性腹瀉病毒、豬傳染性胃腸炎病毒和豬博卡病毒多重PCR檢測(cè)方法的建立[D];南京農(nóng)業(yè)大學(xué);2013年
3 武存霞;豬流行性腹瀉病毒S2基因片段重組豬痘病毒的構(gòu)建和免疫原性分析[D];南京農(nóng)業(yè)大學(xué);2013年
4 曹宇航;PRRSV分離、序列分析及Annexin A2與Hsp27蛋白影響PRRSV感染的初步研究[D];吉林大學(xué);2014年
5 陳玲鳳;中國(guó)部分Massachusetts型雞傳染性支氣管炎病毒的生物學(xué)特性[D];中國(guó)農(nóng)業(yè)科學(xué)院;2014年
6 陳雨昕;H9N2亞型禽流感病毒與雞傳染性支氣管炎病毒的協(xié)同致病作用[D];揚(yáng)州大學(xué);2014年
7 陳啟穩(wěn);QX型雞傳染性支氣管炎弱毒疫苗的研制[D];揚(yáng)州大學(xué);2014年
8 魏巍;PRRSV感染骨髓組織實(shí)時(shí)定量PCR內(nèi)參基因的選擇及ASAH1基因表達(dá)影響研究[D];山西農(nóng)業(yè)大學(xué);2014年
，本文編號(hào)：2151490
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