表达LX4型IBV流行毒株S1蛋白的重组新城疫病毒LaSota株的构建及免疫效力评价

发布时间：2018-09-10 10:05

【摘要】：传染性支气管炎(infectious bronchitis,IB)是由传染性支气管炎病毒(infectious bronchitis virus,IBV)引起的一种急性、高度接触性禽类传染病,给养鸡业带来了重大的经济损失。自20世纪30年代,Massachusetts型IBV被分离鉴定以来,世界各地陆续分离得到了不同血清型的IBV,且各个血清型间交叉反应小,甚至没有明显的交叉反应,给IB的防制带来了巨大的困难。目前,LX4型IBV毒株已在世界多个国家广泛流行,对养禽业造成了巨大的损失。我国目前广泛应用的Mass型疫苗不能对LX4型IBV提供有效的保护,免疫鸡群体感染LX4型病毒后,体内仍能分离到LX4型IBV。因此,开发出更为安全高效的新型IBV疫苗已成为当务之急。为了研发针对LX4型IBV的新型疫苗,本研究以新城疫病毒(Newcastle disease virus,NDV)LaSota疫苗株为载体,通过反向遗传技术,构建获得了表达LX4型IBV流行株ck/CH/LDL/091022 S1蛋白的重组病毒,命名为rLaSota-S1。生长动力学结果显示,r LaSota-S1与亲本病毒LaSota在鸡胚中生长模式相似,表明IBV S1的插入不明显影响载体病毒的复制。经测定,r LaSota-S1的最小致死量致死鸡胚的平均时间(Mean death time,MDT)为165.3h,1日龄雏鸡脑内接种致病指数(Intracerebral pathogenicity index,ICPI)为0,而LaSota的MDT为100h,ICPI为0.19,这表明IBV S1的插入进一步致弱了LaSota的毒力。间接免疫荧光(Indirect immunofluorescence assay,IFA)以及Western Blot证实S1蛋白能够在r LaSota-S1感染的鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)中稳定表达并包装至病毒粒子中。为了评价rLaSota-S1的免疫保护效力,用rLaSota-S1和LaSota免疫SPF鸡,每周采血分离血清。HI试验结果表明rLaSota-S1与LaSota均能诱导机体产生针对NDV的HI抗体。ELISA试验结果证明rLaSota-S1能够诱导机体产生针对IBV的特异性抗体。免疫后21天分别用ck/CH/LHLJ/1/06型NDV和ck/CH/LDL/091022型IBV强毒株进行攻击。结果显示rLaSota-S1和LaSota免疫组用NDV攻击后没有出现任何临床症状,存活率为100%,对照组鸡在攻毒后第四天全部死亡。攻毒后第4天rLaSota-S1和LaSota免疫组呼吸道排毒率均为10%,攻毒后第8天后没有检测到排毒。rLaSota-S1免疫组及对照组用IBV强毒攻击后,定期采集喉头拭子,通过real-time RT-PCR检测拭子中的IBV RNA,r LaSota-S1免疫组的呼吸道排毒率分别为80%、50%、33.3%和16.7%。而对照组的呼吸道排毒率分别为100%、66.7%、66.7%和33.3%。结果表明,和对照相比,免疫组呼吸道排毒率显著降低。rLaSota-S1免疫组死亡率为40%,而对照组的死亡率高达70%。由于一次免疫效果并不理想,所以本试验对免疫程序进行了调整,在首次免疫2周后以相同剂量加强免疫,并每周对鸡进行采血,通过流式细胞仪对外周血T淋巴细胞亚群比例进行分析,结果表明,免疫鸡只的CD3+CD4+T淋巴细胞、CD3+CD8+T淋巴细胞比例显著高于对照组,并且在加强免疫后显著升高,说明r LaSota-S1能够诱导机体产生细胞免疫反应。ELISA试验结果显示,加强免疫后IBV抗体水平显著升高。加强免疫一周后用IBV强毒进行攻击,攻毒后定期采集攻毒鸡的喉头拭子,通过real-time RT-PCR检测拭子中的病毒载量,结果显示,加强免疫后,鸡只呼吸道排毒率分别为66.7%、35.7%、14.3%和7.1%,远低于对照组。攻毒后第5天对鸡进行捕杀,采集组织样品,通过real-time RT-PCR检测组织中的病毒载量,结果表明,加强免疫后,组织中病毒载量明显低于对照组。上述结果表明,rLa Sota-S1加强免疫后,对IBV强毒株的攻击产生的保护效果相比一次免疫组显著提高,临床保护率可达到93.3%。
[Abstract]:Infectious bronchitis (IB) is an acute, highly contagious avian infectious disease caused by infectious bronchitis virus (IBV), which has caused great economic losses to the chicken industry. Since the 1930s, Massachusetts IBV has been isolated and identified worldwide. At present, LX4 IBV strain has been widely prevalent in many countries in the world, causing great losses to poultry industry. Mass vaccine widely used in China can not provide LX4 IBV. In order to develop a new type of vaccine against LX4 IBV, the LaSota vaccine strain of Newcastle Disease Virus (NDV) was used as the carrier in this study, and the vaccine strain was retrograde. The recombinant virus expressing the protein of ck/CH/LDL/091022 S1 of LX4 IBV epidemic strain was constructed and named rLaSota-S1. The results of growth kinetics showed that the growth pattern of rLaSota-S1 was similar to that of its parent virus LaSota in chicken embryos, suggesting that the insertion of IBV S1 did not significantly affect the replication of the vector virus. Mean death time (MDT) was 165.3 hours, the intracerebral pathogenicity index (ICPI) was 0 in 1-day-old chickens, while the MDT and ICPI of LaSota were 100 hours and 0.19 respectively, indicating that the insertion of IBV S1 further weakened the virulence of LaSota. Tern Blot confirmed that S1 protein could be stably expressed in Chicken embryo fibroblast (CEF) infected with R LaSota-S1 and packaged into virus particles. To evaluate the immunoprotective effect of rLaSota-S1, SPF chickens were immunized with rLaSota-S1 and LaSota, and serum samples were collected weekly. HI test results showed that both rLaSota-S1 and LaSota could induce serum isolation. The results of ELISA showed that rLaSota-S1 could induce the production of specific antibodies against IBV. On the 21st day after immunization, the mice were attacked with strong IBV strains of ck/CH/LHLJ/1/06 and ck/CH/LDL/091022 respectively. The survival rate was 100%. All the chickens in the control group died on the fourth day. The respiratory tract detoxification rate was 10% in the rLaSota-S1 and LaSota immune groups on the fourth day after the attack, and no detoxification was detected on the eighth day after the attack. Respiratory detoxification rates were 80%, 50%, 33.3% and 16.7% in the NA, R LaSota-S1 immunized group, respectively. Respiratory detoxification rates were 100%, 66.7%, 66.7% and 33.3% in the control group, respectively. Respiratory detoxification rates were significantly lower in the immunized group than those in the control group. The mortality rates in the rLaSota-S1 immunized group were 40% and 70% in the control group, respectively. The results were not satisfactory, so the immunization program was adjusted. After the first immunization for two weeks, the immunization was strengthened with the same dose, and the blood samples were collected weekly. The proportion of peripheral blood T lymphocyte subsets was analyzed by flow cytometry. The results showed that the proportion of CD3 + CD4 + T lymphocyte and CD3 + CD8 + T lymphocyte in immunized chickens was significant. The results of ELISA showed that the level of IBV antibody increased significantly after immunization. After one week of immunization, the throat swabs of the chickens were collected regularly and tested by real-time RT-PCR. The virus load in the swabs was measured. The results showed that the virus load of the chickens was significantly lower than that of the control group. The virus load in the tissues was detected by real-time RT-PCR after 5 days of inoculation. The results showed that the protective effect of rLa Sota-S1 on IBV virulent strains was significantly higher than that of the primary immunization group, and the clinical protective rate was 93.3%.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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