鹅源呼肠孤病毒油乳剂灭活疫苗的制备
发布时间：2018-12-23 11:41
【摘要】：鹅呼肠孤病毒病(goosereovirus,GRV)是由鹅呼肠孤病毒引起的一种传染病,近年来该病在我国多个省份流行发生,给养殖业造成了很大的经济损失,极大的危害了我国养鹅业的健康发展。2015年下半年江苏省某鹅场的鹅群发生了一种以肝脾坏死、运动障碍为主要特征的传染病,疑似感染呼肠孤病毒。为探明该病病原,本研究将病死鹅的组织匀浆液,卵黄囊接种10日龄鹅胚,从尿囊液中分离到一株病毒,病毒分离株对鸡红细胞没有血凝活性,其ELD50为10-3.75/0.2 mL。参考GenBank鹅呼肠孤病毒基因序列的保守区设计合成了特异性引物,用该引物对提取的病毒RNA进行RT-PCR,可扩增到约380bp目的条带,将目的条带回收后和pMD18-T载体连接,将重组载体转化到DH5α大肠杆菌并在AMP+/LB培养基中培养,对阳性菌落测序分析。结果表明,克隆的基因片段为鹅呼肠孤病毒σC基因片段;用生物学软件分析该分离株与水禽源呼肠孤病毒存在于同一分支,同源性在94.8%~96.5%,与鸡呼肠孤病毒同源性仅39.9%~50.5%;在6日龄的雏鹅上进行回归实验可复制该病;上述结果表明该分离株为鹅呼肠孤病毒,分离的病毒由实验室保存。将保存的鹅呼肠孤病毒分离株经鹅胚传代三次,收获病毒尿囊液,8000rpm离心15min,取上清,加双抗。将10号白油,Span-80和硬脂酸铝按一定比例混合作油相,经双抗处理的病毒液加2%的吐温-80制作水相,按照油水比2:1混合制备油乳剂灭活疫苗,并进行包括外观、乳剂类型、粘度、稳定性、无菌、安全性和保存期的质量检验。为评价制备的灭活疫苗的免疫效力,将90只6日龄的雏鹅随机分为3组,每组30只,1-2组为免疫组,免疫剂量分别为0.2mL和0.5mL,第3组为对照组,免疫后进行抗体水平监测和免疫保护试验,并观察试验鹅的发病和死亡情况。结果表明疫苗外观为乳白色,呈油包水型,无菌,粘度符合标准,稳定性良好,对动物无不良反应,4℃下可保存12个月以上;免疫保护试验中,免疫组攻毒后出现短暂的精神沉郁,1组在在免疫两周时仍不能完全保护雏鹅,2组免疫一周后即可完全保护,对照组攻毒后雏鹅均出现典型的病理变化并死亡;1组攻毒鹅泄殖腔拭子于第3天开始检测到病毒核酸,第12天时仍有部分样品检测核酸为阳性,2组检测病毒核酸为阴性;对免疫后雏鹅血液中的抗体监测,免疫后6d抗体开始明显上升,20d左右达到峰值,此后稍有降低但仍可维持高峰2周以上。上述结果表明制备的鹅呼肠孤病毒灭活疫苗安全、稳定、易于储存运输,雏鹅免疫后能获得坚强保护力,抗体水平能维持高峰数周,可覆盖易感日龄。本研究从疑似感染鹅呼肠孤病毒的病料中成功分离了一株鹅源呼肠孤病毒,为开展该病毒的进一步研究奠定基础,用该毒株制备了鹅呼肠孤病毒灭活疫苗,疫苗安全有效,能在雏鹅的易感日龄提供坚强保护,为鹅呼肠孤病毒的疫苗研制提供了依据。
[Abstract]:Goose reovirus disease (goosereovirus,GRV) is an infectious disease caused by geese reovirus. In recent years, the disease is prevalent in many provinces of China, which has caused great economic losses to the breeding industry. In the second half of 2015, the goose herd in a goose farm in Jiangsu Province developed an infectious disease characterized by liver and spleen necrosis and dyskinesia, which was suspected to be infected with reovirus. In order to identify the pathogeny of the disease, the yolk sac of the dead goose was inoculated with 10 day-old goose embryo, and a virus was isolated from the allantoic fluid. The virus isolate had no hemagglutination activity on chicken red blood cells, its ELD50 was 10-3.75 / 0.2 mL.. According to the conserved region of GenBank geese reovirus gene sequence, a specific primer was designed and synthesized. Using this primer to amplify the extracted virus RNA by RT-PCR, the target band of about 380bp was amplified, and the target band was recovered and connected with the pMD18-T vector. The recombinant vector was transformed into DH5 伪 Escherichia coli and cultured in AMP / LB medium. The positive colonies were sequenced. The results showed that the cloned gene fragment was a 蟽 C gene fragment of geese reovirus. Biological software was used to analyze that the isolate existed in the same branch as the waterfowl origin reovirus, and the homology was at 94.8 and only 39.9% and 50.5, respectively. The results showed that the isolated strain was geese reovirus and the isolated virus was preserved in laboratory. The preserved isolate of goose reovirus was passed through goose embryo for three times. The virus allantoic fluid was harvested and centrifuged by 8000rpm for 15 min. The supernatant was obtained and the double antibody was added. White oil No. 10, Span-80 and aluminum stearate were mixed as oil phase in a certain proportion, the virus solution treated with double antibody was added 2% Tween-80 to make water phase, and oil emulsion inactivated vaccine was prepared according to 2:1 oil / water ratio, and the appearance was included. Emulsion type, viscosity, stability, asepsis, safety and quality inspection for storage life. In order to evaluate the immunological efficacy of the inactivated vaccine, 90 6-day-old goslings were randomly divided into 3 groups, 30 in each group, and 1 or 2 groups were immunized with 0.2mL and 0.5mL, respectively, and the third group was the control group. Antibody level and immune protection test were carried out after immunization, and the incidence and death of goose were observed. The results showed that the vaccine had milky white appearance, water-in-oil type, aseptic viscosity, good stability, no adverse reaction to animals, and could be preserved for more than 12 months at 4 鈩，
本文编号：2389937
[Abstract]:Goose reovirus disease (goosereovirus,GRV) is an infectious disease caused by geese reovirus. In recent years, the disease is prevalent in many provinces of China, which has caused great economic losses to the breeding industry. In the second half of 2015, the goose herd in a goose farm in Jiangsu Province developed an infectious disease characterized by liver and spleen necrosis and dyskinesia, which was suspected to be infected with reovirus. In order to identify the pathogeny of the disease, the yolk sac of the dead goose was inoculated with 10 day-old goose embryo, and a virus was isolated from the allantoic fluid. The virus isolate had no hemagglutination activity on chicken red blood cells, its ELD50 was 10-3.75 / 0.2 mL.. According to the conserved region of GenBank geese reovirus gene sequence, a specific primer was designed and synthesized. Using this primer to amplify the extracted virus RNA by RT-PCR, the target band of about 380bp was amplified, and the target band was recovered and connected with the pMD18-T vector. The recombinant vector was transformed into DH5 伪 Escherichia coli and cultured in AMP / LB medium. The positive colonies were sequenced. The results showed that the cloned gene fragment was a 蟽 C gene fragment of geese reovirus. Biological software was used to analyze that the isolate existed in the same branch as the waterfowl origin reovirus, and the homology was at 94.8 and only 39.9% and 50.5, respectively. The results showed that the isolated strain was geese reovirus and the isolated virus was preserved in laboratory. The preserved isolate of goose reovirus was passed through goose embryo for three times. The virus allantoic fluid was harvested and centrifuged by 8000rpm for 15 min. The supernatant was obtained and the double antibody was added. White oil No. 10, Span-80 and aluminum stearate were mixed as oil phase in a certain proportion, the virus solution treated with double antibody was added 2% Tween-80 to make water phase, and oil emulsion inactivated vaccine was prepared according to 2:1 oil / water ratio, and the appearance was included. Emulsion type, viscosity, stability, asepsis, safety and quality inspection for storage life. In order to evaluate the immunological efficacy of the inactivated vaccine, 90 6-day-old goslings were randomly divided into 3 groups, 30 in each group, and 1 or 2 groups were immunized with 0.2mL and 0.5mL, respectively, and the third group was the control group. Antibody level and immune protection test were carried out after immunization, and the incidence and death of goose were observed. The results showed that the vaccine had milky white appearance, water-in-oil type, aseptic viscosity, good stability, no adverse reaction to animals, and could be preserved for more than 12 months at 4 鈩，
本文编号：2389937
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