鵝源呼腸孤病毒油乳劑滅活疫苗的制備
發(fā)布時間：2018-12-23 11:41
【摘要】：鵝呼腸孤病毒病(goosereovirus,GRV)是由鵝呼腸孤病毒引起的一種傳染病,近年來該病在我國多個省份流行發(fā)生,給養(yǎng)殖業(yè)造成了很大的經(jīng)濟損失,極大的危害了我國養(yǎng)鵝業(yè)的健康發(fā)展。2015年下半年江蘇省某鵝場的鵝群發(fā)生了一種以肝脾壞死、運動障礙為主要特征的傳染病,疑似感染呼腸孤病毒。為探明該病病原,本研究將病死鵝的組織勻漿液,卵黃囊接種10日齡鵝胚,從尿囊液中分離到一株病毒,病毒分離株對雞紅細胞沒有血凝活性,其ELD50為10-3.75/0.2 mL。參考GenBank鵝呼腸孤病毒基因序列的保守區(qū)設計合成了特異性引物,用該引物對提取的病毒RNA進行RT-PCR,可擴增到約380bp目的條帶,將目的條帶回收后和pMD18-T載體連接,將重組載體轉(zhuǎn)化到DH5α大腸桿菌并在AMP+/LB培養(yǎng)基中培養(yǎng),對陽性菌落測序分析。結果表明,克隆的基因片段為鵝呼腸孤病毒σC基因片段;用生物學軟件分析該分離株與水禽源呼腸孤病毒存在于同一分支,同源性在94.8%~96.5%,與雞呼腸孤病毒同源性僅39.9%~50.5%;在6日齡的雛鵝上進行回歸實驗可復制該病;上述結果表明該分離株為鵝呼腸孤病毒,分離的病毒由實驗室保存。將保存的鵝呼腸孤病毒分離株經(jīng)鵝胚傳代三次,收獲病毒尿囊液,8000rpm離心15min,取上清,加雙抗。將10號白油,Span-80和硬脂酸鋁按一定比例混合作油相,經(jīng)雙抗處理的病毒液加2%的吐溫-80制作水相,按照油水比2:1混合制備油乳劑滅活疫苗,并進行包括外觀、乳劑類型、粘度、穩(wěn)定性、無菌、安全性和保存期的質(zhì)量檢驗。為評價制備的滅活疫苗的免疫效力,將90只6日齡的雛鵝隨機分為3組,每組30只,1-2組為免疫組,免疫劑量分別為0.2mL和0.5mL,第3組為對照組,免疫后進行抗體水平監(jiān)測和免疫保護試驗,并觀察試驗鵝的發(fā)病和死亡情況。結果表明疫苗外觀為乳白色,呈油包水型,無菌,粘度符合標準,穩(wěn)定性良好,對動物無不良反應,4℃下可保存12個月以上;免疫保護試驗中,免疫組攻毒后出現(xiàn)短暫的精神沉郁,1組在在免疫兩周時仍不能完全保護雛鵝,2組免疫一周后即可完全保護,對照組攻毒后雛鵝均出現(xiàn)典型的病理變化并死亡;1組攻毒鵝泄殖腔拭子于第3天開始檢測到病毒核酸,第12天時仍有部分樣品檢測核酸為陽性,2組檢測病毒核酸為陰性;對免疫后雛鵝血液中的抗體監(jiān)測,免疫后6d抗體開始明顯上升,20d左右達到峰值,此后稍有降低但仍可維持高峰2周以上。上述結果表明制備的鵝呼腸孤病毒滅活疫苗安全、穩(wěn)定、易于儲存運輸,雛鵝免疫后能獲得堅強保護力,抗體水平能維持高峰數(shù)周,可覆蓋易感日齡。本研究從疑似感染鵝呼腸孤病毒的病料中成功分離了一株鵝源呼腸孤病毒,為開展該病毒的進一步研究奠定基礎,用該毒株制備了鵝呼腸孤病毒滅活疫苗,疫苗安全有效,能在雛鵝的易感日齡提供堅強保護,為鵝呼腸孤病毒的疫苗研制提供了依據(jù)。
[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 偽 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，
本文編號：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 偽 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，
本文編號：2389937
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