【作者】 孔娜；

【导师】 赵宏坤；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 博士

【摘要】 白细胞介素18（Interleukine-18, IL-18）是一种多效细胞因子,具有强烈的IFN-γ诱生能力,对机体起着重要的免疫调节和保护作用,在抗微生物感染、抗肿瘤免疫中具有应用潜力,因此IL-18可作为免疫佐剂与一些病原微生物的保护性基因共表达,从而加强疫苗的免疫效果或制备基因工程疫苗;由于鸡IL-18（ChIL-18）基因发现得比较晚,而对人和哺乳动物IL-18的研究已有许多报道,且其具有与人和哺乳动物相似的生物学功能,因此ChIL-18在比较免疫学研究和禽病治疗中具有重要意义。pFastBac^TM Dual载体含有PH启动子和p10启动子,可以同时表达两个具有独立活性的外源蛋白。His标签的引入为重组蛋白的纯化提供可能,为获得大量有生物活性的蛋白开辟了新途径。传染性法氏囊病（Infectious bursal disease, IBD）是由IBD病毒（IBDV）感染雏鸡引起的以淋巴组织,特别是中枢免疫器官-法氏囊为主要特征的高度接触性传染病。该病主要导致机体免疫抑制,使机体的免疫能力降低和疫苗免疫接种失败。虽然灭活疫苗和减毒活疫苗是相当成熟的,但由于IBDV强毒株和抗原变异的出现使其免疫效价降低。VP2是IBDV的主要保护性抗原,已经鉴定的IBDV中和表位主要在VP2上,并且多为构象依赖性,这意味着VP2的立体结构对其中和表位的形成至关重要,与病毒中和抗体的诱导、抗原和毒力的变异以及细胞凋亡的诱导等有关。禽流感（Avian influenza, AI）是由AI病毒（AIV）感染引起的一种高度接触性人畜共患传染病。HPAI的暴发给养禽业带来了一定的经济损失,H5N1亚型AI的出现不仅产生经济损失,还带来了恐慌。AIV的主要抗原决定簇都位于HA1区域。体外表达HA1涵盖了所有的HA空间中和表位,故其完全可以替代HA作为抗原。所以HA1蛋白是研制基因工程亚单位苗的理想候选抗原。本试验分别以pMD18-T-VP2、pMD18-T-HA1和pMD18-T-mChIL18质粒为模板,以杆状病毒pFastBac^TM Dual为载体,将mChIL18基因与VP2基因或HA1基因分别插入到载体的PH启动子和P10启动子的下游,构建重组转移载体质粒pF-IL18、pF-VP2、pF-IL18-VP2、pF-HA1、pF-IL18-HA1。将其转化DH10Bac感受态细胞,经三重抗性（含卡那霉素、庆大霉素和四环素）与蓝白斑筛选,用小量法提取重组杆状病毒质粒rBac-IL18、rBac-VP2、rBac-IL18-VP2、rBac-HA1、rBac-IL18-HA1,通过一对通用引物M13（Bacmid含有该引物Forward和Reverse两个引物位点,能够从两侧扩增LacZα互补区域内的mini-attTn7位点,有利于PCR分析）进行PCR扩增鉴定。在转染试剂CellfectinII的作用下,通过脂质体介导法将纯化的rBac-IL18、rBac-VP2、rBac-IL18-VP2、rBac-HA1、rBac-IL18-HA1转染至草地贪夜蛾细胞（Spodoptera frugiperda 9,sf9）获得P1代重组杆状病毒,用P1代病毒感染sf9来扩增病毒滴度,将达到一定滴度（10⁷¹0⁸pfu/mL）的P3代重组杆状病毒感染sf9,感染72h后收获sf9细胞及培养上清,进行聚丙烯酰胺凝胶电泳（SDS-PAGE）检测和间接免疫荧光试验（IFA）检测。SDS-PAGE检测显示,mChIL-18、VP2、HA1基因均在sf9中的裂解上清中得到了高效表达,即表达的蛋白是可溶性的,表达的目的条带分子量分别约为19.5 KD, 48.4 KD, 37.4 KD。IFA检测显示mChIL18基因与VP2基因或HA1基因分别同时在同一细胞中独立表达。采用鸡脾淋巴细胞增殖试验（MTT法）、IFN-γ诱导实验和水疱性口炎病毒（VSV）抑制试验对表达的蛋白进行生物学活性测定。鸡脾淋巴细胞增殖试验表明,不同浓度的pIL18、pVP2-IL18和pHA1-IL18均能够明显促进淋巴细胞的增殖,随着蛋白浓度的增加刺激转化作用逐渐增强,均当浓度为200ng/mL时,刺激转化效果最佳,增殖指数可达4.13、4.35、4.09,但随着蛋白浓度的增大淋巴细胞的增殖指数逐渐降低。VSV病毒活性抑制试验表明,当蛋白浓度大于100ng/mL时能刺激脾淋巴细胞产生IFN-γ,并且随着pIL18、pVP2-IL18和pHA1-IL18浓度的增加诱导产生IFN-γ的量随之增加;将不同稀释度的IFN-γ分别作用于VSV,在IFN-γ≥1×10²U/mL时,具有较强的抑制效果,当IFN-γ为10 U/mL时,只能达到约50%的保护。该结果说明pIL18、pVP2-IL18和pHA1-IL18的抗病毒活性是通过IFN-γ实现的,且在一定浓度范围内具有抑制VSV病毒产生细胞病变的作用。将含有pIL18、pVP2、pVP2-IL18的油乳剂疫苗分组免疫动物,14d时一免,28d时二免。I组为传统疫苗组;II组为pIL18与B78减毒疫苗联合疫苗组;III为pIL18与pVP2联合疫苗组;IV组为pVP2-IL18单独免疫组;V组一免注射B78减毒疫苗,二免注射pVP2-IL18;VI组为pVP2单独免疫组;VII组为对照组。从体液免疫和细胞免疫水平上分析试验结果。细胞免疫水平通过流式细胞术检测,从统计学意义上分析处理数据。结果表明,与免疫前和阴性对照组相比,各试验组都促进CD⁴⁺和CD⁸⁺ T细胞增殖。一免后各试验组CD⁴⁺ T细胞都呈上升趋势,但一免后7d后又呈下降趋势;二免后I、III、V呈上升趋势且持续时间比较长,其中IV组CD⁴⁺ T增殖水平最高,在一免后21d、28d、35d都与其它组差异显著（P<0.05）。一免后各试验组CD⁸⁺ T细胞都呈上升趋势,但一免后7d后又呈下降趋势;二免后各实验组仍呈不同下降趋势,其中I组CD⁸⁺ T增殖水平最高,在一免后14d、21d、28d都与其它组差异显著（P<0.05）。体液免疫水平通过使用传染性法氏囊病病毒抗体检测试剂盒检测,从统计学意义上分析处理数据。结果表明,与免疫前相比,各试验组都有较好的促进IBD抗体生成作用,抗体水平都持续增高且维持时间也较长,都在在一免后28d抗体水平达到最高;与阴性对照组相比,IV组效果最好,抗体效价最高,V组次之,然后是II组、I组、III组、VI组。攻毒试验结果表明,IV组保护率可达90%,V组次之可达80%,然后是I组和II组75%、III组为70%、VI组50%,VII组无一幸免。将含有pIL18、pHA1、pHA1-IL18的油乳剂疫苗分组免疫动物,7d时一免,21d时二免。I组为传统疫苗组;II组为pIL18与传统疫苗联合疫苗组;III为pHA1单独免疫组;IV组为pHA1-IL18单独免疫组;V组pIL18与pHA1联合疫苗组;VI组为pHA1单独免疫组;VII组为对照组。从体液免疫和细胞免疫水平上分析试验结果。细胞免疫水平通过流式细胞术检测。结果表明,与免疫前和阴性对照组相比,各试验组都能够明显促进CD⁴⁺和CD⁸⁺ T细胞的增殖反应。一免后各试验组CD⁴⁺和CD⁸⁺T细胞都呈上升趋势,但一免后7d后又呈下降趋势;二免后虽呈上升趋势且持续时间比较长,但增殖幅度不大,以IV组CD⁴⁺ T增殖水平最高。体液免疫水平通过使用血凝抑制试验检测。结果表明,各试验组都有较好的促进ND、H5型AI和H9型AI抗体生成作用,都是在一免后28d抗体水平达到最高;与阴性对照组相比,IV组效果最好,抗体效价最高与其它组差异显著（P<0.05）。更多还原

【Abstract】 Interleukin-18（IL-18） is a novel pleiotropic cytokine, which has the potent inducor of IFN-γproduction.It has been demonstrated that IL-18 plays a major role in immunoregulation and protection, and has large potential in host defense against various micro- organic infection and tumor. So IL-18 which was as a kind of efficient immune adjuvant for vaccine, can coexpress with some protective gene of pathogenic microorganism, thereby enhance and improve the immune effective of vaccine. However, the study on chicken interleukin-18 （ChIL-18） lagged off the mammal interleukin-18. Previously, the gene of ChIL-18 was cloned, but further study is not performed.The discovery of ChIL-18 gene is very meaningful for both the study on comparative immuneology and the therapeutics for poultry diseases. pFastBac^TM Dual has the able to express simultaneously two active foreign proteins, because it has PH promoterand p10 promoter. The purification of recombination protein is possible because of inlet of His tag.Infectious bursal disease （IBD） causes considerable economic losses to the poultry industry worldwide by causing a high rate of morbidity and mortality in an acute form or as a consequence of severe immunosupression provoked by the destruction of immature B-lymphocytes within the bursa of Fabricius. Vaccination against the disease with inactivated or attenuated live vaccines is widely practiced. However, such traditional vaccines have become less effective due to the emergence of very virulent or antigenic variant strains of IBDV in recent years.VP2 and VP3 are major capsid proteins, whereas VP2 is the major antigen of IBDV and contains major epitopes responsible for protection against IBDV. VP2 can stimulate neutralizing antibodies and accordingly induce humoral immuniny in vivo. Therefore, the VP2 protein has been used quite effectively in developing subunit/DNA vaccines with other expression system against IBDV. Protein immunization opens a new approach to the development of gene vaccines for chickens against infectious diseases.Some of the most highly contagious viruses of human and veterinary concern are airborne pathogens, e.g. human and avian influenza virus （AIV）. The highly pathogenic avian influenza viruses （HPAI） have been a threat to the poultry industry for many years. However, when the Asian H5N1 subtype emerged, a trans-boundary spread became evident and a new dimension of zoonotic danger and fear for evolution of a pandemic virus was raised. However, traditional vaccines have become less effective due to the emergence of very virulent or antigenic variant strains of AIV years. So a newly vaccine and its adjuvant are urgently demand to research. And major antigen determinants of AIV lie in HA1 gene, so HA1 protein is the ideal electing antigen of studying gene engineering subunit vaccine.In the experiment, the mature chicken interleukin-18（mChIL-18） gene and VP2 gene/HA1 gene were amplified on the basis of pMD18-T-VP2 plasmid、pMD18-T-HA1 plasmid、pMD18-T-mChIL18 plasmid. Then mChIL-18 gene and VP2 gene/ HA1 gene were inserted into the downstreams of PH promoter and P10 promoter of baculovirus expression plasmid pFastBacTM Dual, respectively. Finally, the constructing eukaryotic expression plasmid pF-IL18、pF-VP2、pF-IL18-VP2、pF-HA1、pF-IL18-HA1 were transformed into DH10Bac competent cells, and screened by three antibiotics （kanamycin, gentamycin and tetracycline） and blue-white patch. The recombinant bacmid DNA is extracted, on the basis of M13 universal primers（The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα-complementation region to facilitate PCR analysis）, we analyze rBac-IL18、rBac-VP2、rBac-IL18-VP2、rBac-HA1、rBac-IL18-HA1 DNA by PCR.In order to harvest the P1 recombinant aculovirus, we transfected the purified bacmid into Spodoptera frugiperda 9（sf9） by using a cationic lipid such as Cellfectin Reagent. Raised the virus titre by infecting sf9 many times and infected the sf9 with the higher baculoviral stock （10⁷¹0⁸ pfu/mL） for expressing protein and harvested the supernatant and cells in different times. The expressed recombinant protein was analyzed by SDS– PAGE and IFA. The results demonstrated that IL18 protein and VP2 protein/HA1 protein were simultaneously expressed in sf9 cells.The biological activities of pIL18、pVP2-IL18 and pHA1-IL18 were detected by the proliferation of lymphocyte, inducing IFN-γproduction and inhibiting the Vesicular stomatitis virus （VSV）. The experiment of MTT showed that every concentration of pIL18、pVP2-IL18 and pHA1-IL18 could enhance proliferation of lymphocyte, and the effect was best when the concentration was 200ng/mL and the proliferation exponent was 4.13、4.35、4.09, respectively. The experiment of VSV inhibition indicated that pIL18、pVP2-IL18 and pHA1-IL18 could induce spleenocytes to produce IFN-γwith high activity when it was more than 100ng/mL, and the amount of recombinant proteins and their inducing effect on IFN-γhad a direct proportion. In the experiment of VSV inhibition, different dilution of IFN-γwas used and they could protect the cells when the concentration was more than 10U/mL. The result showed that pIL18、pVP2-IL18、pHA1-IL18 could inhibit the VSV activity by inducing IFN-γproduction in spleen lymphocyte. So it can be concluded that pIL18、pVP2-IL18 and pHA1-IL18 emerge functional activities by IFN-γ.Two-week-old SPF chickens were randomly divided into 7 groups with 20 chickens per group. Group I received B78. Group II received pIL18 and B78. Group III received pIL18 and pVP2. Group IV received of pVP2-IL18. Group V received B78 in primary immunization and pVP2-IL18 in booster immunization. Group VI received pVP2. Group VII served as PBS control and the normal control. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement. The level of cellular immunity was detected by FACS. The number of CD⁴⁺ and CD⁸⁺ T lympholeukocytes in peripheral blood of chickens immunized with genetically engineered vaccine were obviously higher than the control group and only B78 vaccine immunity class, which demonstrated satisfactory cell-mediated immunity （CMI） induced by genetically engineering vaccine. Group IV was always higher than group III and group I, and then presented significant difference. The number of CD⁴⁺ T lympholeukocytes in pVP2-IL immunized group kept increasing tendency and longer persistence comparing with the B78 group after the secondary immunization. The level of humoral immunity was detected by Infectious Bursal Disease Virus Antibody Test Kit. The anti-IBDV antibody titers of all experiment groups were presented upgrade tendency after secondary immunity and up to maximum in 4 weeks post inoculation. The antibody against IBDV detected in six chickens of group IV was always higher than other groups at days 7, 14, 21, 28 and 35 post-immunization. It presented significant difference （P<0.05） between group IV and group I at days 14, 21, 28 and 35 post-immunization. In addition, it also presented significant difference （P<0.05） between group IV and group III at days 7, 14, 21, 28 and 35 post-immunization.One-week-old SPF chickens were randomly immuned with pIL18、pHA1 and pHA1-IL18. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement. The results showed that compared with pre-immunization, all experiment groups promoted the proliferation of CD⁴⁺ and CD⁸⁺ T lympholeukocytes. The results showed that compared with pre-immunization, all experiment groups had higher titers of antibodies and maintained high titer levels for longer times.更多还原