【作者】 肖运才；

【导师】 毕丁仁； 熊远著；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 禽流感(AI)是由A型流感病毒引起的一种禽类的烈性传染病。自1878年意大利首先发现至今,世界各地都有特定毒株引起的禽流感爆发和流行,导致禽类的大量死亡和生产性能的急剧下降,造成了巨大的经济损失。目前,高致病性禽流感(HPAI)是国际兽疫局规定的A类动物传染病。近年来,禽流感的发生与流行,在我国已造成了巨大的经济损失和严重的社会影响,尤其是香港1997年和东南亚2003-2004年出现的禽流感病毒(AIV)直接感染并致死人的事件,已引了世人的震惊和关注。其防制已直接关系到我国养禽业的持续稳定发展和人民的身体健康。本研究主要是建立一种快速的检测禽流感病毒抗原的ELISA方法,弥补其它检测方法的不足,为该病的早期诊断提供可靠依据和技术保证;另外,由于禽流感病毒亚型之多,不同亚型之间无交叉保护作用,给该病的防制带来了极大的困难,鉴于此,本研究试图利用霍乱毒素B亚基(Cholera toxin B subunit, CTB)强有力的粘膜免疫佐剂作用,将禽流感病毒型特异性NP基因的表达产物与CTB混合或与CTB融合表达后,制成疫苗通过鼻腔粘膜免疫鸡,以期提高鸡体鼻腔、气管粘膜表面sIgA的含量,从而达到预防和控制禽流感的目的。主要研究工作和结果如下: 1.鸡抗AIV、兔抗AIV和山羊抗兔IgG高免血清的制备及山羊抗兔IgG的HRP标记 将H9N2亚型的AIV经9-11日龄鸡胚传代后,收获鸡胚尿囊液,经30,000r/m离心1h后,AIV沉淀用原尿囊液1/30倍体积的0.01mol/L,pH7.2的PBS重悬,所得悬液与福氏佐剂乳化后免疫鸡和兔,得到鸡抗AIV和兔抗AIV的高免血清,其琼扩效价均达到1:64;同时,以纯化的兔IgG免疫山羊,将获得的山羊抗兔IgG高免血清(1:64)纯化后,经辣根过氧化物酶(HRP)标记山羊抗兔IgG,制备出特异性强、免疫学活性和催化活性高的山羊抗兔IgG-HKP,且工作浓度高达1:4000,为禽流感的夹心ELISA诊断方法的建立奠定了坚实的物质基础。 2.检测禽流感病毒的酶联免疫吸附试验(ELISA)方法的建立 以纯化的鸡抗AIV IgG为包被抗体,兔抗AIV IgG为第二抗体,通过ELISA反应条件的优化选择,建立了检测AIV抗原的夹心ELISA法。结果表明,鸡抗AIV IgG的最佳包被浓度为1μg/mL,兔抗AIV IgG的最适工作浓度为5μg/mL;对已知的阳性样品,用夹心ELISA法测得的病毒滴度比血球凝集滴度高16倍以上,且能检出其它亚型的禽流感病毒;与新城疫病毒、传染性支气管炎病毒、减蛋综合征病毒、传染性喉气管炎病毒、传染性法氏囊病病毒、鸡痘病毒、马立克氏病病毒等无交叉反应,说明该方法有很高的特异性和敏感性。对14个鸡场送检的患有呼吸道疾病或有腹膜炎、眼炎、产蛋下降、怀疑为禽流感感染的病鸡进行了检测,结果有7个鸡场为阳性。更多还原

【Abstract】 Avian Influenza (AT) is one of fatal infectious disease caused by influenza A virus in avian. It first appeared in Italy more than 100 years ago(around 1878), and, so far, different strains were found and caused important economic losses in the avian industry throughout the world. At present, highly pathogenic avian influenza (HPAI) is listed in A infectious diseases by World Organization for Animal Health(OIE). Recently, AI has produced important economic losses and bad effect to society in our country, especially, events that AI infected human being happened in Hongkong(1997) and south-eastern Asian (2003-2004), which shocked mankind and caused our attention to commonality sanitation of AI. A common viewpoint is that prevention of AI is closely linked to persistent and steady development of avian industry and humankind’s health. This research is aimed to develop ELISA for detection of AIV in order to provide technique to forepart diagnoses of AI. In addition, Because of negligible cross-protection against different AIV subtypes, it is very difficuly to prevent avian influenza from outbreaking. Therefore, we attempted to mix or fused expression product of Cholera toxin B subunit(CTB), a strong mucosal immunization adjuvant, with NP, a expression product of nucleoprotein(NP) of AIV, then chicken were immunized with mixture(CTB+NP) and fused product(CTB-NP) by nasal cavity in order to enhance secretory IgA(sIgA) titer of nasal cavity and trachea surface, which maybe control and prevent AI from happening. The results of research are summarized as following:1. Preparation of sera of chicken anti-AIV, rabbit anti-AIV, goat anti-rabbit IgG and goat anti-rabbit IgG-HRPAIV (H9N2) was inoculated into 9 to 10-day embryonating chicken eggs by the allantoic route, then allantoic fluid was harvested and centrifuged with 30,000r/m, and AIV deposit was redissolved by 0.01mol/L PBS (pH7.2)which is 1/30 volume of allantoic fluid. Chicken and rabbits were immunized with redissolved fluid of AIV emulsified with Frund’s adjuvant, and sera of chicken anti-AIV, rabbit anti-AIV were acquired after three inoculations, and the titers of AGED reach 1:64. At the same time, purified serum of goat anti-rabbit IgG(AGID: 1:64) from goat immunized with purified rabbit IgG was labeled with horseradish peroxidase(HRP). The conjugate is characteristic of high activity of immunity and its work concentration reached 1:4000. This laid a foundation for the development of sandwich enzyme-linked immunosorbent assay (ELISA) for AIV.2. Development of enzyme-linked immunosorbent assay (ELISA) for AIV.A sandwich ELISA for detection of AIV antigen was developed by using purified chicken anti-ATV IgG as the first antibody coated on the ELISA plate and rabbit anti-AIV IgG regarded as the second antibody. The results showed that the optimum working concentration of chicken anti-AIV IgG and rabbits anti-AIV IgG were lug/mL and 5ug/mL, respectively. The dilution titers of positive AIV sample by ELISA was 16 times higher than that by hemagglutination test, and other AIV subtypes can also be detected by this method. No cross reaction was observed with Newcastle disease virus (NDV), infectious bronchitis virus (IBV), eggs drop syndrome virus (EDS^V), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV), avian pox virus (APV) and Marek’s disease virus(MDV). 7 of 14 suspicious chicken groups showing respiratory tract syndrome or peritonitis, opthalmitis, egg drop were positive detected by this method.3. Development and Application of Sandwich Enzyme-linked Immunosorbent AssayTest Kit for Detecting Avian Influenza Virus AntigenA Sandwich Enzyme-linked immunosobent assay test kit based on previous work was developed for detection of avian influenza virus (ATV) antigen. The kit consists of eleven reagents and a piece of 16-well microtiter plate. There were highly specificity, sensitivity and repeatability in detecting AIV antigen with the kit. It was stable well when storing at below -lO’C for six months. It is very easy to manipulate and the result can be reported within 3 hours. So far, different subtype ATVs including H5 and H9 subtypes from chicken and H5 subtype from Landes Goose were isolated from some provinces such as Hubei, Anhui and Henan and so on detected by the kit.4. Gene fusion and expression of nucleoprotein (NP) of AIV and the Cholera toxin B subunit (CTB)Vector pGEX-KG-CTB expressing CTB gene was developed after CTB gene was cloned into expression vector pGEX-KG containing GST gene. Then NP gene was cloned into the downstream of CTB gene in pGEX-KG-CTB, and the expression vector pGEX-KGCN was constructed. After correct cloning sites and sequences were indentified, CTB and CTB-NP were expressed in Escherichia coli BL21 (codon plus) strain, The results of SDS-PAGE showed that molecular weight of GST-CTB and GST-CTB-NP were about 38.0kD and 94.0kD, respectively; which matches the expected molecular weight. Western blotting showed NP protein in GST-CTB-NP takes on strong biological activity.5. Mucosal immunization study on recombinant nucleoprotein of AIV45 chickens, 15 days old, were divided randomly into 5 groups at the start of experiments. Groups of 9 animals were immunized with NP, or NP+CTB, or CTB-NP, or CTB, at fhe same time, a group was control. All immunizations were given in nasal cavity for three times at 10-day intervals. Before every immunization and 10 days after the last immunization, sera, nasal wash, trachea wash and gut wash were collected for ELISA detection for corresponding immunoglobulin(IgG-NP, IgA-NP, slgA-NP). The results showed that high level IgG-NP and low level IgA-NP in sera happened. SlgA-NP antibody responses(day20 and day30) in nasal wash, trachea wash and gut wash were enhanced significantly by CTB(p<0.05). At day 30, ODs of slgA-NP in nasal wash and gut wash was significantly enhanced by CTB(CTB-NP group > CTB+NP group > NP group), and the difference were significant with each other(p<0.05). In all trachea wash, there was no difference(p>0.05) between CTB-NP group and CTB+NP group, but there was significantly difference(p>0.05)between the two groups and NP group; hi conclusion, when mixed with or fused expression product of recombinant NP, CTB was a candidate adjuvant for enhancing mucosal immunization of recombinant nucleoprotein of AIV.更多还原