纳米氢氧化铝吸附禽流感病毒灭活疫苗的研制

发布时间：2018-02-28 11:19

  本文关键词： 纳米氢氧化铝佐剂 高致病性禽流感 生产工艺 吸附 疫苗质量　出处：《西南大学》2009年硕士论文　论文类型：学位论文

【摘要】： 禽流感(Avian Influenza,AI)是A型一种禽类的急性、烈性传染病。国家预案规定,一旦爆发高致病性AI,疫点周围3km内的所有家禽要全部扑杀,5km内的家禽应紧急接种疫苗,以建立禽类免疫隔离带。目前,我国防控HPAI的常规途径是注射油乳剂灭活疫苗。但现行油疫苗免疫后机体形成有效抗体滴度的间隙期较长,不能满足紧急情况下快速建立免疫保护的要求。并且油佐剂刺激性大、难吸收,在肉禽宰杀销售之时仍有部分油佐剂残留于机体,严重影响产品质量。美国联邦政府规定,畜禽上市前42d内不得使用任何矿物油乳剂疫苗。因此,寻找一种高效安全,便于紧急接种的新型佐剂势在必行。本试验自制纳米氢氧化铝佐剂,并在此基础上探讨了纳米氢氧化铝与禽流感病毒尿囊液的吸附条件和最佳浓度及该疫苗的质量。 本课题内容包括: 1纳米氢氧化铝佐剂的制备与检测 采用苯扎溴铵-丙三醇-环己烷-AlCl_3溶液的W/O型微乳液配方制备纳米氢氧化铝佐剂粒子,获得经过激光粒度仪测定平均粒径为197.2nm,滴定法检测纳米氢氧化的含量为2mg/mL。从而为进一步验证纳米铝佐剂的免疫学效应提供了物质保证。 2纳米氢氧化铝吸附禽流感病毒H5N1灭活疫苗的研制 本试验选用了HA试验、荧光RT-PCR、单向放射免疫扩散测定尿囊液中的抗原。测得尿囊液HA=9Log_2,荧光RT-PCR结果为阳性,血凝素含量为3.133μg/ml。这三种方法可以有效地对疫苗的吸附情况进行判断。在选出的最佳吸附环境下,制备具有高吸附率的纳米氢氧化铝佐剂吸附禽流感疫苗。分别用血凝试验HA、络合滴定法,测定抗原和佐剂下沉的转速。以尿囊液与佐剂1:2的体积之比混合,选用不同的温度、吸附时间和佐剂浓度进行吸附,离心取上清液,用HA试验、荧光RT-PCR和络合滴定法测定。结果选出7000rpm为检测疫苗吸附率的离心转速;当疫苗中氢氧化铝终浓度为0.700mg/mL时,25℃下吸附2h,纳米佐剂与抗原吸附完全,而常规佐剂与抗原吸附不完全。 3疫苗质量检测 鉴于产品开发要求,对上述工艺生产的禽流感灭活疫苗进行相关检测,其结果均符合国家要求,易储存在4℃低温环境,且纳米氢氧化铝佐剂疫苗比常规氢氧化铝佐剂疫苗更稳定。 结论:本研究证实了纳米氢氧化铝是一种良好的免疫佐剂,能够有效地吸附禽流感病毒。并且首次报道了纳米氢氧化铝吸附禽流感H5N1病毒的最佳吸附条件及佐剂浓度,且其吸附率比常规氢氧化铝组高。制备的纳米氢氧化铝疫苗质量可控,为佐剂的改良和研发提供了坚实的理论基础,为佐剂的机理研究提供了理论基础,为进一步的临床试验奠定了牢固的基础。
[Abstract]:Avian Influenza A (AIV) is a type A acute, acute infectious disease of poultry. The National Preparedness Plan stipulates that in the event of an outbreak of highly pathogenic AI1, all poultry within 3 km of the epidemic site should be immediately vaccinated against all poultry within 5 km. At present, the routine way to prevent and control HPAI in China is to inject oil emulsion inactivated vaccine. However, the gap period of effective antibody titer is long after the current oil vaccine is immunized. It cannot meet the requirements of establishing immunity protection quickly in emergency situations. Moreover, oil adjuvants are highly irritating and difficult to absorb. Some oil adjuvants remain in the body when meat and poultry are slaughtered and sold, seriously affecting the quality of products. No mineral oil emulsion vaccine should be used within 42 days before the marketing of livestock and poultry. Therefore, it is imperative to find a new adjuvant with high efficiency, safety and convenience for emergency inoculation. On this basis, the adsorption conditions and optimal concentration of nano-aluminum hydroxide and avian influenza virus allantoic fluid and the quality of the vaccine were discussed. This topic includes:. Preparation and detection of nano-aluminum hydroxide adjuvant. Nanometer aluminum hydroxide adjuvant particles were prepared by using the W- / O microemulsion formula of benzalkonium bromide-propanol-cyclohexane-AlCl3 solution. The average particle size measured by laser particle size analyzer is 197.2nmand the content of nano-hydroxide detected by titration is 2mg / mL, which provides a material guarantee for further verification of immunological effect of nano-aluminum adjuvant. Development of inactivated vaccine against avian influenza virus H5N1 adsorbed by 2 nanometer aluminum hydroxide. In this experiment, HA test, fluorescence RT-PCR and one-way radioimmunodiffusion were used to determine the antigen in allantoic fluid. The hemagglutinin content is 3.133 渭 g / ml. These three methods can effectively judge the adsorption of the vaccine. The avian influenza vaccine adsorbed by nano-aluminum hydroxide adjuvant with high adsorption rate was prepared. The antigenic and adjuvant sinking speed was determined by hemagglutination test (HA) and complexometric titration. The volume ratio of allantoic solution to adjuvant 1: 2 was mixed at different temperatures. Adsorption time and concentration of adjuvant were adsorbed, supernatant was obtained by centrifugation, HA test, fluorescence RT-PCR and complexometric titration were used. Results 7000rpm was selected as centrifugal speed to detect the adsorption rate of vaccine. When the final concentration of aluminum hydroxide in the vaccine was 0.700 mg / mL, the adsorption of nano-adjuvant and antigen was complete at 25 鈩，

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