表达IBV主要结构蛋白重组DEV的构建及其免疫保护性评价
发布时间：2018-07-28 20:32
【摘要】：鸡传染性支气管炎(Infectious bronchitis,IB)是由鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)引起的鸡的高度接触性上呼吸道及泌尿生殖道疾病。该病在很多国家和地区流行,主要感染呼吸道和肾脏,各种年龄鸡都易感,常引起产蛋鸡产蛋量和蛋品质下降,给养禽业造成严重的经济损失。鸭肠炎病毒(Duck enteritis virus,DEV),又称鸭瘟病毒(Duck plague virus,DPV),主要引起鸭、鹅及多种雁形目禽类产生急性、热性、败血性传染病,曾在多个国家和地区发生流行,给养鸭业造成巨大的经济损失。DEV是疱疹病毒科成员,其基因组中部分基因是病毒复制非必需基因,可作为非复制型疫苗载体构建表达外源基因的活载体疫苗。本研究在本研究室建立的鸭肠炎病毒重组技术平台基础上,以US10基因缺失表达EGFP的重组鸭肠炎病毒为亲本病毒,应用同源重组技术分别构建表达鸡传染性支气管炎病毒主要结构蛋白N、S和S1的3株重组鸭肠炎病毒。鉴别PCR、交叉PCR检测及测序结果表明,IBV N、S和S1基因分别正确插入鸭肠炎病毒基因组内,替换病毒US10基因。Western blot和间接免疫荧光结果表明,IBV N、S和S1蛋白均能够在重组病毒rDEV-N、rDEV-S和rDEV-S1中检测到表达,且随病毒的复制时间增加而表达量增加。对3株重组病毒的基本生物学特性进行研究,结果表明3株重组病毒的蚀斑形态和蚀斑大小与亲本病毒rDEV US10-EGFP和野生型病毒DEV Clone-03相似。与野生型病毒DEV Clone-03相比,重组病毒rDEV-N、rDEV-S和rDEV-S1的病毒滴度略有下降,与其亲本病毒rDEV-EGFP病毒滴度相近。复制动力学研究表明,3株重组病毒的复制趋势与亲本病毒和野生型病毒一致,在感染细胞后72h病毒滴度达到高峰,在感染后96h病毒滴度开始下降,说明重组病毒的复制能力与病毒基因组缺失的位点有关,而与插入的外源基因无关。分别将3株重组病毒在鸡胚成纤维细胞上连续传代至20代,重组病毒表现出良好的遗传稳定性,外源基因随病毒复制而稳定遗传并稳定表达。将获得的3株鸡传染性支气管炎重组鸭肠炎病毒免疫1月龄SPF鸡,于免疫后21d用IBV强毒株ck/CH/LDL/091022进行攻毒,同时设对照组。分别于免疫后1w、2w、3w、4w、5w检测免疫鸡抗体水平。在免疫后1w,rDEV-N免疫组有84%鸡只抗体阳转,rDEV-S和rDEV-S1免疫组有92%鸡只抗体阳转。免疫后第2w,各免疫组血清抗体有不同程度下降,rDEV-N免疫组76%抗体阳性率,rDEV-S免疫组抗体阳性率下降至52%,而rDEV-S1免疫组下降至28%。至免疫后第3w,各免疫组抗体水平均显著下降,rDEV-N免疫组40%抗体阳性率,rDEV-S免疫组抗体阳性率下降至16%,而rDEV-S1免疫组下降至8%。攻毒后各组鸡只具有不同程度发病,对照组发病率为80%,rDEV-N免疫组发病率30%,rDEV-S免疫组发病率20%,rDEV-S1免疫组发病率40%。攻毒后7d对照组鸡只开始死亡,直至攻毒后12天不再有鸡只死亡。对照组死亡率为40%,rDEV-N免疫组发病率30%,rDEV-S免疫组发病率10%,rDEV-S1免疫组发病率30%。于攻毒后5d,采集各组鸡只咽拭子,应用荧光定量RT-PCR检测各组鸡只呼吸道的排毒情况。对照组在攻毒后咽拭子排毒率为90%,rDEV-N免疫组攻毒后经呼吸道排毒率为20%,rDEV-S免疫组攻毒后呼吸道排毒率30%,rDEV-S1免疫组攻毒后呼吸道排毒率40%。分别于攻毒后5、10、15、20d进行抗体水平检测,从攻毒后抗体变化水平来看,各组鸡只抗体均有不同程度升高,说明攻毒成功,且攻毒后3个免疫组鸡只抗体水平均高于对照组。结果提示,重组病毒免疫后的鸡只具有抵抗病毒攻击的能力,与对照组相比,抗体水平上升较快,说明鸡只存在免疫记忆。从发病率、死亡率和攻毒后排毒情况来看,3株重组病毒均能够对鸡只提供免疫保护,但不能够提供完全保护,其中重组病毒rDEV-S免疫保护效果好于重组病毒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..
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：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..
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
【共引文献】
相关期刊论文 前2条
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2 霍亚飞;胡北侠;张秀美;张琳;杨少华;黄庆华;黄艳艳;许传田;;Real-Time PCR方法对IBV弱毒苗致弱分析[J];山东农业科学;2015年04期
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