本文關(guān)鍵詞： 納米氫氧化鋁佐劑 高致病性禽流感 生產(chǎn)工藝 吸附 疫苗質(zhì)量　出處：《西南大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 禽流感(Avian Influenza,AI)是A型一種禽類的急性、烈性傳染病。國(guó)家預(yù)案規(guī)定,一旦爆發(fā)高致病性AI,疫點(diǎn)周圍3km內(nèi)的所有家禽要全部撲殺,5km內(nèi)的家禽應(yīng)緊急接種疫苗,以建立禽類免疫隔離帶。目前,我國(guó)防控HPAI的常規(guī)途徑是注射油乳劑滅活疫苗。但現(xiàn)行油疫苗免疫后機(jī)體形成有效抗體滴度的間隙期較長(zhǎng),不能滿足緊急情況下快速建立免疫保護(hù)的要求。并且油佐劑刺激性大、難吸收,在肉禽宰殺銷售之時(shí)仍有部分油佐劑殘留于機(jī)體,嚴(yán)重影響產(chǎn)品質(zhì)量。美國(guó)聯(lián)邦政府規(guī)定,畜禽上市前42d內(nèi)不得使用任何礦物油乳劑疫苗。因此,尋找一種高效安全,便于緊急接種的新型佐劑勢(shì)在必行。本試驗(yàn)自制納米氫氧化鋁佐劑,并在此基礎(chǔ)上探討了納米氫氧化鋁與禽流感病毒尿囊液的吸附條件和最佳濃度及該疫苗的質(zhì)量。 本課題內(nèi)容包括: 1納米氫氧化鋁佐劑的制備與檢測(cè) 采用苯扎溴銨-丙三醇-環(huán)己烷-AlCl_3溶液的W/O型微乳液配方制備納米氫氧化鋁佐劑粒子,獲得經(jīng)過(guò)激光粒度儀測(cè)定平均粒徑為197.2nm,滴定法檢測(cè)納米氫氧化的含量為2mg/mL。從而為進(jìn)一步驗(yàn)證納米鋁佐劑的免疫學(xué)效應(yīng)提供了物質(zhì)保證。 2納米氫氧化鋁吸附禽流感病毒H5N1滅活疫苗的研制 本試驗(yàn)選用了HA試驗(yàn)、熒光RT-PCR、單向放射免疫擴(kuò)散測(cè)定尿囊液中的抗原。測(cè)得尿囊液HA=9Log_2,熒光RT-PCR結(jié)果為陽(yáng)性,血凝素含量為3.133μg/ml。這三種方法可以有效地對(duì)疫苗的吸附情況進(jìn)行判斷。在選出的最佳吸附環(huán)境下,制備具有高吸附率的納米氫氧化鋁佐劑吸附禽流感疫苗。分別用血凝試驗(yàn)HA、絡(luò)合滴定法,測(cè)定抗原和佐劑下沉的轉(zhuǎn)速。以尿囊液與佐劑1:2的體積之比混合,選用不同的溫度、吸附時(shí)間和佐劑濃度進(jìn)行吸附,離心取上清液,用HA試驗(yàn)、熒光RT-PCR和絡(luò)合滴定法測(cè)定。結(jié)果選出7000rpm為檢測(cè)疫苗吸附率的離心轉(zhuǎn)速;當(dāng)疫苗中氫氧化鋁終濃度為0.700mg/mL時(shí),25℃下吸附2h,納米佐劑與抗原吸附完全,而常規(guī)佐劑與抗原吸附不完全。 3疫苗質(zhì)量檢測(cè) 鑒于產(chǎn)品開(kāi)發(fā)要求,對(duì)上述工藝生產(chǎn)的禽流感滅活疫苗進(jìn)行相關(guān)檢測(cè),其結(jié)果均符合國(guó)家要求,易儲(chǔ)存在4℃低溫環(huán)境,且納米氫氧化鋁佐劑疫苗比常規(guī)氫氧化鋁佐劑疫苗更穩(wěn)定。 結(jié)論:本研究證實(shí)了納米氫氧化鋁是一種良好的免疫佐劑,能夠有效地吸附禽流感病毒。并且首次報(bào)道了納米氫氧化鋁吸附禽流感H5N1病毒的最佳吸附條件及佐劑濃度,且其吸附率比常規(guī)氫氧化鋁組高。制備的納米氫氧化鋁疫苗質(zhì)量可控,為佐劑的改良和研發(fā)提供了堅(jiān)實(shí)的理論基礎(chǔ),為佐劑的機(jī)理研究提供了理論基礎(chǔ),為進(jìn)一步的臨床試驗(yàn)奠定了牢固的基礎(chǔ)。
[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，

本文編號(hào)：1547078