【作者】 赵双成；

【导师】 姜永萍；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2012， 硕士

【摘要】 高致病性禽流感（HPAI）是由一些H5和H7亚型流感病毒所引起的高致病性和高死亡率的烈性禽类传染病，已对养禽业构成严重威胁，并具有重要的公共卫生意义。DNA疫苗是预防禽流感最具潜力的基因工程疫苗之一，DNA免疫不仅能诱导较好的抗体反应和特异性的细胞免疫反应，还具有安全稳定、不受宿主种属限制、可反复加强免疫、免疫持续期长等突出优点。表达H5亚型禽流感病毒A/Goose/Guangdong/1/96(H5N1)[GS/GD/1/96]HA基因的DNA疫苗质粒pCAGGoptiHA5免疫效果良好，目前已完成临床试验，正在申请新兽药证书注册。鉴于现地免疫影响因素众多以及目前我国H5亚型禽流感不同抗原群并存的复杂状况，了解和评价DNA疫苗免疫后的细胞免疫应答状况，探讨不同抗原群DNA疫苗的联合免疫的可能性，将为DNA疫苗的推广应用提供有用的数据支撑。为获得GS/GD/1/96的HA蛋白，将GS/GD/1/96的HA基因片段克隆于杆状病毒表达系统的转移载体pFastBacHTA中，构建重组质粒pFastBacHTA-HA，转化至DH10Bac感受态细胞，构建重组杆粒，在脂质体介导下转染Sf9昆虫细胞，获得重组杆状病毒（rBacmid-HA5）。采用间接免疫荧光（IFA)、Western blot和血凝试验对所表达的蛋白进行抗原性和生物学活性分析。结果表明，表达了分子量约为64ku的重组蛋白；IFA和western blot试验证明重组HA蛋白能与H5亚型禽流感阳性血清特异结合，具备良好的抗原性；血凝试验和淋巴细胞增殖试验显示重组蛋白具备良好生物学活性。重组蛋白的正确表达为评价DNA疫苗的细胞免疫水平、研究HA基因功能活性及制备禽流感HA基因亚单位疫苗奠定了基础。为了解H5亚型DNA疫苗免疫后的细胞免疫应答反应，将DNA质粒pCAGGoptiHA5以15μg剂量间隔3周两次免疫SPF鸡，第二次免疫后2周用105EID50的GS/GD/1/96进行致死性攻击，免疫以及攻毒后，用流式细胞术检测试验鸡外周血及脾脏CD3+、CD4+、CD8+T淋巴细胞亚类百分含量的动态变化，利用MTT法进行T淋巴细胞增殖实验分析，同时采用ELISA方法检测血清中IFN-γ、IL-2的含量变化。结果表明：外周血及脾脏CD4+、CD8+T淋巴细胞亚类百分含量具有类似的变化趋势，免疫后CD4+、CD8+T淋巴细胞相对百分含量明显高于非免疫组（p<0.05），CD4+T细胞于第二次免疫后一周达到峰值，攻毒后CD4+含量略有下降但变化不明显；CD8+T淋巴细胞含量也于加免后一周达到峰值，攻毒后二周仍然处于上升趋势；首次免疫后CD4+/CD8+比例逐渐升高，并于加免后一周达到峰值。ConA刺激免疫组和对照组淋巴细胞后，细胞大量增殖，但两组间差异不显著（p>0.05）；表达蛋白刺激后，两组间差异显著（p<0.05）。免疫组血清中IFN-γ、IL-2的含量均高于同期对照组（p<0.05），并于第二次免疫后一周达到峰值，攻毒后略有上升。而非免疫组SPF鸡外周血中CD4+、CD8+淋巴细胞的数量、CD4+/CD8+比例以及血清中IFN-γ、IL-2的含量并没有显著变化，并于攻毒后一周内全部死亡。结果表明，DNA疫苗质粒pCAGGoptiHA5免疫后可诱导较强的细胞免疫应答。为探讨H5亚型不同抗原群禽流感DNA疫苗联合免疫的可能性和协同免疫保护效力，将DNA疫苗质粒pCAGGoptiHA5、pCAGGoAHHA和pCAGGSXHA分别等量混合，以15μg、30μg、45μg、60μg和90μg总剂量联合免疫3周龄SPF鸡，首次免疫后3周以相同的剂量和途径进行第二次免疫，加强免疫后2周分别用105EID50的A/Goose/Guangdong/1/96(H5N1)[GS/GD/1/96]、A/Duck/Fujian/31/2007(H5N1)[DK/FJ/31/2007]及A/Chicken/Shanxi/2/2006(H5N1)[CK/SX/2/2006]通过鼻腔途径进行攻击，每天观察发病和死亡情况，并于攻毒后第3、5、7天分别采集喉头和泄殖腔拭子，进行病毒的分离和滴定。同时检测免疫后和攻毒后血清HI抗体水平。结果表明，3种质粒以60μg和90μg剂量两次联合免疫后，免疫鸡均可对不同抗原群病毒的攻击提供100%的完全保护（不发病、不排毒、不致死），15μg剂量联合免疫后可对GS/GD/1/96和DK/FJ/31/2007的攻击提供90%和80%的死亡保护；CK/SX/2/2006攻击后，15μg和30μg剂量组的免疫保护率分别为80%和90%,45μg剂量能提供100%的致死保护。结果显示，3种质粒疫苗联合免疫效果良好，可实现有效的交叉保护，减少免疫次数，DNA疫苗联合免疫有望成为抵御不同抗原群H5亚型禽流感的有效免疫策略。本研究结果表明，DNA疫苗质粒pCAGGoptiHA5可激发SPF鸡产生较强的细胞免疫应答；3种质粒pCAGGoptiHA5、pCAGGoAHHA和pCAGGSXHA联合免疫效果良好，这将为禽流感DNA疫苗的产业化以及现地应用提供必要的科学依据和支持。更多还原

【Abstract】 Avian Influenza is a serious disease, which causes huge economic losses for the domestic poultryin rescent years. H5subtype Highly pathogenic avian influenza virus(HPAIV) not only infect poultryand wild birds, but also threat our human health.Therefore, it is essential to research new vaccineagainst Avian Influenza. DNA vaccine has lots of advantages. It can induce both humoral and cellimmune responses. It is also easier to store, transport and manufacture. Otherwise, it will inducelong-lasting protective immune response.DNA vaccine pCAGGoptiHA5which express HA gene has good immune effect and has completedits clinical tests. DNA vaccine is applying for a new veterinary drug registration certificate. In view ofmany influent facters of immune condition and H5subtypes of AI different antigen coexist, evalution ofcell immune response and discussion of different DNA vaccine joint immunity will provide useful datasupports for the popularization and application of DNA vaccine.For eukaryotic expression of avian influenza virus (AIV) hemagglutinin (HA), the HA gene ofA/Goose/Guangdong/1/96(H5N1)(GS/GD/1/96)was cloned into the baculovirus transfer vectorpFastBacHTa and the resultant recombinant plasmid was transformed into DH10Bac competent cells togenerate recombinant bacmid (rBacmid-HA), which was identified by PCR and sequencing. Therecombinant baculovirus stock was prepared by transfecting rBacmid-HA into the Sf9insect cells. Theexpression of the recombinant HA in sf9cells was identified by indirect immunofluorescence assay andwestern blot, which shown that the recombinant HA were about64ku. Further identificationsdemonstrated that the HA was able to induce high titer of HI antibody and effectively stimulateproliferation of lymph cells in peripheral blood of SPF chicken detected by MTT assay. The preparationof the recombinant protein in sf9cells was facilitated the further study of HA functions, preparation ofthe subunit vaccine against AIV infection and evaluation of DNA vaccines cellular immune responses.To verify cellular immune responses in SPF chickens after immunization, SPF chickens wereimmunized with15μg AIV HA gene DNA vaccine pCAGGoptiHA5.The percent of CD3+,CD4+andCD8+T lymphocytes and proliferation of lymph cells in periphery blood and spleen were detected byflow cytometer and MTT assay, respectively. The dynamic changes of IFN-γ、IL-2in sera werechecked with ELISA assay.The results demonstrated that the percent of CD4+and CD8+T lymphocytesin periphery blood and spleen had the same tendency. The percent of CD4+and CD8+T lymphocytes ofimmunized groups were higher than that of the control groups and had significant difference betweentwo groups(p<0.05).The amount of CD4+T lymphocytes reached the peak one week after boostingimmunization, then decreased slightly until the experiment finished. In the first immunization, thepercent of CD8+T lymphocytes consistently increased and reached the peak one week after boostingand maintained in a steady level two weeks after challenge. The ratios of CD4+/CD8+increasedgradually in the first immunization and reached the peak one week after boosting. The amount of CD4+and CD8+T lymphocytes as well as ratios of CD4+/CD8+from unchickens had increasing trend butlower than that of vaccinated chickens and then all chickens died one week after challenge. The lymphocytes in vaccinated group and control group stimulated by ConA were proliferation, but nosignificant difference between two groups(p>0.05). While the HA stimulation made significantdifference between two groups(p<0.05). The amount of IFN-γ and IL-2in sera of vaccinated groupswere higher than that of the control groups and reached the peak one week after boosting and thenincreased slightly after challenge.While the amount of IFN-γ and IL-2in control chickens hardly had nochanges and chickens died one week after challenge.The two groups had significant difference(p<0.05).These results also showed that DNA vaccine could induce strong cellular immune responses inSPF chickens.For evaluating the protective efficacy of H5subtype avian influenza vaccine pCAGGoptiHA5、pCAGGoAHHA and pCAGGSXHA, groups of3-week-old SPF chickens were intramuscular inoculatedwith different doses of three DNA vaccines in15μg、30μg、45μg、60μg and90μg. Groups of chickenswere injected with200μl PBS as controls. Two weeks after the boost, all chickens were challenged with105EID50of highly pathogenic A/Goose/GuangDong/1/96strain, A/Duck/FuJian/31/07strain andA/Chicken/ShanXi/2/06strain, respectively. Oropharyrigeal and cloacalswab specimens were collectedfrom all chickens3,5and7days after inoculation for titration of virus in eggs and chickens wereobserved daily for disease signs and deaths for two weeks. Sera were collected weekly after vaccinationand challenged for detecting HI antibodies. Results demonstrated that60μg and90μg vaccines boostedchickens were completely protected from virus challenge (no disease sign, no virus shedding and nodeath).The protective rates in15μg groups which challenged by A/Goose/GuangDong/1/96(H5N1) orA/Duck/FuJian/31/07(H5N1) were90.0%(9/10)and80%(8/10), respectively. The protective rates in15and30μg groups which challenged by A/Chicken/ShanXi/2/06(H5N1) were80.0%(8/10)and90.0%(9/10), respectively.45μg doses of the vaccine can provide100%of the lethal protection.Resultsshow that the combination of vaccine immune can protecte chickens from different lethal H5N1viruschallenge to achieve purpose of cross protection. The joint DNA vaccine is a highly efficient geneengineering vaccine and can be used for preventing HPAIV effectively in near future.In summary, the study imply that DNA vaccine pCAGGoptiHA5can stimulate strong cellularimmune response in SPF chickens.The effect of three plasmid joint immune is good and this willprovide necessary scientific basis and support for industrialization and application of AI DNA vaccine.更多还原