【作者】 赵会连；

【导师】 贺秀媛； 曹志高；

【作者基本信息】 河南农业大学 ， 兽医， 2010， 硕士

【摘要】 本试验采用生产用毒种重组禽流感病毒(H5N1亚型,Re-5株)毒株一定比例稀释后,接种于11日龄非免疫的发育良好的健康鸡胚尿囊腔,每胚0.2mL,在万级生产区域内,温度36℃,湿度70%条件下继续孵育,培养72h后,取其活鸡胚置0～4℃冷库冷却,12h后,收获鸡胚尿囊液,作为未处理抗原。将未处理抗原经过离心机离心,作为离心抗原。将离心后的抗原采用过滤膜包2倍浓缩,作为浓缩抗原。通过对制备的未处理、离心纯化、浓缩纯化的抗原抽样分别进行灭活前的澄清度、病毒半数感染量(EID50)和对鸡红细胞凝集价(HA)的检测试验,确定抗原通过离心、浓缩纯化处理后质量发生的变化。然后,分别采用灭活剂甲醛溶液1.5‰、2.0‰、2.5‰灭活,恒温37℃灭活24h,通过对灭活后的抗原抽样作灭活后无菌检验、灭活检验、HA检验的灭活检测试验,确定未处理抗原、离心纯化的抗原和浓缩纯化的抗原灭活彻底的最低甲醛溶液浓度。最后,将每组中使用最低甲醛溶液浓度的合格的灭活抗原分装、密封包扎好,进行0~4℃保存试验,通过分别保存1个月、2个月、3个月、4个月、5个月后的HA检测并进行油乳剂苗的配制,对油乳剂苗进行外观、性状、粘度、稳定性的物理性状检测和20日龄非免疫鸡的安全性检测和5周龄非免疫鸡的免疫抗体HI检测,确定未处理抗原、离心纯化的抗原和浓缩纯化的抗原的质量效价的稳定性。通过对制备的未处理、离心纯化、浓缩纯化处理的抗原的澄清度、病毒半数感染量(EID50)和对鸡红细胞凝集价(HA)的检测试验结果对比得出:抗原经过离心、浓缩纯化处理后,不但外观质量得到明显改善,而且效价得到较大提高。浓缩处理的抗原的澄清度高于未处理抗原,低于离心抗原;HA比未处理和离心处理的抗原提高一个滴度;EID50比离心抗原的EID50提高0.2个滴度,比未处理抗原提高0.7个滴度。通过对未处理、离心纯化、浓缩纯化处理抗原的1.5‰,2.0‰,2.5‰甲醛溶液的灭活和灭活后的无菌检验、灭活检验和HA检测试验结果对比得出:未处理抗原灭活彻底的最低甲醛溶液浓度为2.5‰,离心抗原灭活彻底的最低甲醛溶液浓度为1.5‰,浓缩抗原灭活彻底的最低甲醛溶液浓度为2.0‰。通过对2.5‰甲醛溶液灭活的未处理抗原、1.5‰甲醛溶液灭活的离心抗原和2.0‰甲醛溶液灭活的浓缩抗原保存一个月、二个月、三个月、四个月、五个月的HA检测和配制油乳剂苗后的物理性状检测、安全检验和免疫抗体HI检测试验结果对比得出:在五个月保存时间内,浓缩抗原的质量稳定,免疫效果良好。浓缩抗原的HA的算数平均数比离心抗原平均高1个滴度,比未处理抗原平均高3.6个滴度;配制的油乳剂苗稳定、安全,对鸡体接种免疫产生的抗体HI的算数平均数比离心抗原平均高1.08个滴度,比未处理抗原平均高3.36个滴度,且免疫抗体整齐。由此得出:采用离心和浓缩技术,可以提高禽流感病毒抗原的质量,同时,抗原浓缩成倍体积,为今后抗原的有计划储备,灭活多价苗和多联苗的生产打下了坚实的基础。更多还原

【Abstract】 This study uses production poisonous kinds of recombinant avian influenza virus (H5N1 subtype, Re-5 strain) strains of a certain percentage of diluted, inoculated in 11-day-old well-developed non-immune health of chick embryo cysts, each embryo 0.2ml, in the 10000 production in the region, the temperature 36℃, humidity 70% of the conditions for the continuation incubated for 72 hours, whichever is home of live chicken0~ 4℃refrigerator cool, 12 hours later, harvested chicken embryo allantoic fluid, as the untreated antigen. Untreated antigen through the centrifuge centrifuge, as the centrifugal antigen. After centrifugation, the antigen will be a filtration membrane pack twice the concentration, as the antigen concentration. Through the preparation of the untreated, centrifugal purified, concentrated sample of purified antigen were carried out to clarify the degree of pre-inactivated, the virus infected half of the amount of (EID50) and the price of chicken red blood cell agglutination (HA) of the detection tests to determine the antigen by centrifugation, concentrated Purification of the quality of treatment changes. Then, the inactivated agents were used formaldehyde solution 1.5‰, 2.0‰, 2.5‰inactivated, temperature 37℃inactivated 24 hours, by inactivated antigen inactivated sterile samples for testing, inactivated test, HA test inactivated detection tests to determine the untreated antigen, centrifugation purified antigens and inactivated concentrated purified antigens complete a minimum of formaldehyde concentration. Finally, each group with the lowest concentration of formaldehyde inactivated antigens of qualified packaging and sealing wrap is good, for 0~ 4℃preservation test, were saved by 1 month, 2 months, 3 months, 4 months, five months later, the HA test and make an oil-emulsion preparation of seedlings, seedlings of the oil emulsion appearance, properties, viscosity, stability, physical properties testing and 20-day-old non-immune chickens safety testing and 5-week-old non-immune HI antibody detection of immune chickens to determine the untreated antigen, centrifugal concentration of purified antigens and the quality of the purified antigen titer stability. Through the preparation of the untreated, centrifugal purification, concentration, purification of the antigen to clarify the degree of virus infection in half the amount of (EID50) and the price of chicken red blood cell agglutination (HA) test comparison of test results obtained: Antigen After centrifugation, concentration, purification , not only the appearance of significant improvement in quality and potency are greatly enhanced. The enrichment process to clarify the degree of antigen than untreated antigen, less than the centrifugal antigen; HA and centrifugal treatment compared with untreated antigen titer increased by one; EID50 than the centrifugal antigen EID50 increase of 0.2 over the unprocessed antigens increased 0.7. Through untreated, centrifugal purification, concentration, purification antigen 1.5‰, 2.0‰, 2.5‰formaldehyde solution inactivated and inactivated sterile after the inspection, inspection and HA test inactivated comparison of test results obtained: untreated antigen completely inactivated the lowest formaldehyde concentration of 2.5‰, the lowest centrifugal completely inactivated antigen concentration of formaldehyde solution, 1.5‰, the lowest concentration completely inactivated antigen concentration of formaldehyde solution 2.0‰, 2.5‰formaldehyde solution through untreated inactivated antigen, 1.5‰formaldehyde solution inactivated centrifugal antigens and 2.0‰enrichment of antigen inactivated formaldehyde solution to save one month, two months, three, four, five HA months of testing and preparation of the physical properties of oil-emulsion postemergence testing, safety testing and detection of immune antibody HI comparison of test results obtained: In the five months to save time, quality and stability of concentrated antigen, the immune good effect. HA antigen concentration than the average height of a centrifugal antigen titer, compared with an average of 3.6 untreated antigen titer; prepared Miao oil emulsion stability, security, body-immunized chickens produced HI antibody antigen than the average height of 1.08 centrifuge titer was higher than the average of 3.36 untreated antigen titer, and the immune antibody and tidy. The resulting: The centrifuge and enrichment technology can improve the quality of the avian influenza virus antigen, while antigen concentration doubling the size of antigen for the future there are plans to reserve, inactivated polyvalent Miao Miao and multi-joint production of laying a solid basis.更多还原