【作者】 密金玲；

【导师】 聂奎；

【作者基本信息】 西南大学 ， 预防兽医学， 2007， 硕士

【摘要】 新城疫（Newcastle Disease，ND）是由新城疫病毒（Newcastle disease virus，NDV）所致的一种高度接触性的传染病，能引起鸡、火鸡、鸭、鸽子和鹦鹉等多种禽类的感染和死亡，给世界养禽业造成巨大损失，因此国际兽疫局将其定为Ⅰ类传染病。目前，对该病的防制仍以疫苗免疫为主。核酸疫苗以其无致病性、研制简单、不受母源抗体干扰等诸多优点成为现代疫苗的研究热点，但研究也发现核酸疫苗存在着免疫方式有限、免疫原性不强，常需高剂量多次免疫等问题。因而，人们开始转向对核酸疫苗佐剂和载体的研究，希望能够通过各种佐剂、载体的使用来扬长补短，使其免疫效果得到进一步提高。菌蜕是通过生物分子裂解制备而成，由于G^-菌具有完整的生物亲和性的外膜结构，如菌毛能够进行特异的细胞和组织定位，其表面的脂多糖成分可以作为一种天然佐剂，而且菌蜕的胞周间隙和细胞空腔可接收大量外源物质，菌蜕因而成为一种新型无病原性的生物载体和靶向工具。本课题以此为着眼点，以分子裂解方法制备的菌蜕作为载体，与构建表达NDV F基因的核酸疫苗制备成新城疫核酸菌蜕疫苗，并进行实验动物（鸡）的免疫试验。主要研究内容如下：1．表达新城疫F基因核酸疫苗的构建根据GeneBank中公布的Lasota株F基因的核苷酸序列（AF077761）设计引物，并分别在上下游引入BamHⅠ和KpnⅠ限制性内切酶位点，以利于插入真核表达载体pcDNA3.1+。以新城疫Lasota弱毒株为研究材料，提取出基因组RNA并以其为模板，通过RT-PCR技术扩增新城疫病毒F基因完整片断，扩增产物经纯化回收后与pMD18-T载体相连，转化DH5a感受态细胞，经PCR和酶切电泳鉴定，均得到了大小约1700bp左右的条带，证实扩增产物已插入T载体。获得的重组质粒pT-F经双酶切将F基因插入到pcDNA3.1+载体中，PCR和酶切鉴定显示表达新城疫F基因的核酸疫苗pcDNA3-F构建成功。2．大肠杆菌DH5a菌蜕的制备菌蜕是由裂解蛋白E介导的G^-菌的裂解而产生，编码有噬菌体PhiX174基因E的裂解质粒，可在温敏阻遏元件cI857的控制下进行E基因的限制性表达。在温度低于30℃时，阻遏蛋白cI857的表达使基因E能被很好地抑制，温度高于30℃时，因阻遏蛋白cI857的热失活而使基因E开始表达，在42℃时细菌的裂解达到最佳状态。将含有裂解质粒（pHH43）的大肠杆菌DH5a于28℃200rpm摇床培养，为获得最大量的菌蜕，当OD₆₀₀=0.4～0.6时，迅速升高温度至42℃以启动E裂解蛋白的表达并开始计时，4小时后收集菌蜕。菌落计数（CFU）结果表明有99.998％的细菌被有效裂解。扫描电镜观察可见裂解孔道位于细菌一端或中间，而胞内的物质已经排出，菌蜕保留了较完整的外膜，细菌形态没有发生太大改变。3．新城疫核酸菌蜕疫苗的制备将菌蜕重悬于含有质粒pcDNA3-F的PBS（pH7.4）体系中，加入终浓度25mM CaCl₂溶液，随后在不同条件下，150rpm摇床孵育。为确定质粒DNA的最佳构建浓度，将菌蜕与不同浓度的质粒DNA（5～14mg／ml）孵育，37℃30min后离心收集沉淀和上清；为确定最佳构建温度，将菌蜕与pcDNA3-F（10mg／ml）在不同的温度下进行孵育（4℃、25℃、37℃），30min后离心收集沉淀和上清；为确定孵育时间，将菌蜕与pcDNA3-F（10mg／ml），于25℃进行孵育，分别在2min、4min、10min、30min、60min、120min、180min时，离心收集沉淀和上清。所有离心均为8000rpm 10min，收集的上清通过测定OD_260／280以确定DNA的量。通过改变DNA的浓度、孵育温度和孵育时间发现，装载前DNA的浓度与菌蜕中DNA的量成正相关，孵育温度与装载效果没有明显关联，孵育时间2min足够。4．新城疫核酸菌蜕疫苗免疫效果的评定将7日龄健康雏鸡随机分为7组，每组30只，分别为PBS对照组（Ⅰ组），pcDNA3.1+对照组（Ⅱ组），裸DNA疫苗（pcDNA3-F）组（Ⅲ组），Lasota弱毒疫苗组（Ⅳ组），新城疫核酸菌蜕苗滴鼻组（Ⅴ组），新城疫核酸菌蜕苗口服组（Ⅵ组），新城疫核酸菌蜕苗肌注组（Ⅶ组）。两周后每组各以相同的剂量和途径进行加强免疫一次，二免后第2周用新城疫强毒分离株进行攻毒。通过间接ELISA、血凝抑制抗体检测及攻毒保护试验，对新城疫核酸疫苗的免疫效价进行初步的评定。间接ELISA试验结果表明：首免后一周，与PBS和pcDNA3.1+对照组相比，pcDNA3-F组、Lasota弱毒疫苗组和新城疫核酸菌蜕疫苗组OD值已有所升高，说明所构建的核酸疫苗已在雏鸡体中进行了表达，表达产物作为抗原刺激机体产生了免疫应答反应；首免后两周，除对照组外的所有免疫组的OD值均有显著的升高，并且与对照组相比，差异极显著（P＜0.01）；二免后一周，核酸菌蜕疫苗滴鼻组OD值显著（P＜0.05）高于裸DNA疫苗组，二免后两周达到极显著（P＜0.01），尽管最终没有达到Lasota疫苗组所产生的抗体水平。在同一时期的所有试验组血清抗体OD值中，Lasota疫苗组始终保持最高水平，而空白组和空载体组的OD值在整个试验过程中一直下降。血凝抑制试验结果表明：表达新城疫F蛋白的核酸疫苗诱导产生的抗体与HI抗体无相关性。攻毒试验结果表明：核酸菌蜕疫苗滴鼻组对强毒株的攻击保护率为70％，高于裸DNA疫苗（40％），Lasota弱毒疫苗组的保护率可达90％；而核酸菌蜕疫苗饮水组和肌注组的保护率虽与pcDNA3-F肌注组相同，但推迟了新城疫的发病时间。说明以菌蜕作为新城疫DNA疫苗的运输载体可以刺激机体产生更为有效的免疫反应。更多还原

【Abstract】 Newcastle disease （ND）, caused by Newcastle disease virus （NDV）, is a kind of highly contagious disease with worldwide distribution. NDV can infect many kinds of poultry such as chicken, turkeys, ducks, pigeons, parrots and cause much economic losses to the poultry industry, and the World Organization for Animal Health （namely OIE） determined ND as class I of animal infectious disease. Vaccination is still one of the main measures to control ND . DNA vaccine as a novel vaccine has many advantages, including non-pathogenity, easy to manufacture, non-interference from maternal antibody and becomes the focus of .the vaccine field. However, mass implementation in the field is hampered by the limit of administer routes, less immunogenic and high doses and/or multiple immunizations. Therefore much effort is now turned to increase the efficiency of DNA vaccine through: （i） using of adjuvants to enhance the immune response elicited by DNA vaccines, （ii） development of new delivery systems for specific targeting and/or better DNA uptake. The bacterial ghost system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. The fact that bacterial ghosts preserve a native cell wall, including bioadhesive structures like fimbriae of their living counterparts, makes them suitable for the attachment to specific target tissues, moreover, its periplasmic and cytoplasmic space can accept many foreign matters, so it can be used as a effective tropic carrier and targeting tool. Based on all above mentioned, the main purpose of this study was to examine the immunogenicity of the DNA vaccine against NDV using bacterial ghost as a carrier for delivery of NDV F gene. The main contents are as follows:1. Construction of the eukaryotic plasmid expressing F gene of NDVAccording to the F gene sequence of NDV Lasota strain registered in GenBank （AF077761）, the upstream and downstream primers were designed containing terminal BamH I and Kpn I restriction sites. The F gene was generated by RT-PCR amplification using the viral genome RNA as template. The PCR fragment about 1700bp was obtained, cloned into the vector pMD18-T to construct plasmid pT-F, introduced into DH5a competent cells by calcium shock method and plated on Luria-Bertani （LB） supplement with Ampicillin （50mg/ml）, then the plasmid DNA was prepared for PCR and restriction enzyme digestion. The results of electrophoretic analysis suggested that the F gene was successfully cloned into vector. Subsequently, the fragment of F gene was inserted into the eukaryotic vector pcDNA3.1+ and identified with PCR and restriction enzyme digestion.2. Generation of DH5a ghosts by expression of lysis gene EBacterial ghosts are produced by expression of cloned gene E from bacteriophage PMX174 resulting cell lysis in Gram-negative bacteria, the lysis plasmid carries gene E under transcriptional control of the thermosensitiveλpL/pR-cI857 system. At 28℃or lower, gene E expression is stably repressed by repressor cI857, while was induced at temperatures higher than 30℃for inactivation of cI857, with optimal induction at 42℃Thus, for the production of bacterial ghosts, E. coli DH5a harboring the lysis plasmid pHH43 was grown at 28°C with agitation （200rpm）. When the growing culture reached an optical density at 600 nm （OD600） of 0.4-0.6, the incubation temperature was shifted up to 42°C to induce the gene E-mediated lysis process. After another 4 h of incubation at 42°C with monitoring of the OD600, the culture was harvested. The efficiency of E-mediated killing of E. coli was estimated by plating samples of appropriate dilutions of freshly harvested ghosts on LB agar supplement with chloramphenicol, and the plates were incubated under culture conditions. Colonies were counted to determine the number of CFU, and the result indicated a 99.998% killing efficiency. Electron microscopic studies showed that the cytoplasmic content of the bacteria was expelled leaving an empty internal space and that the protein E-specific transmembrane tunnel structure, which permeabilized the bacterium was not randomly distributed over the cell envelope but was restricted to areas of potential division sites, predominantly in the middle of the cell or at polar sites. Except for the lysis hole, the morphology of the bacteria, including all cell surface structures and appendices, was not affected by the lysis event.3. Preparation of bacterial ghost loading DNA vaccine pcDNA3-FBacterial ghosts were resuspended in PBS buffer saline （pH7.4） containing pcDNA3-F, and after CaCl₂ supplemention （final concentration 25 mM）, they were incubated with agitation （150rpm）. For optimization of DNA concentration, bacterial ghosts were incubated with DNA concentrations ranging from 5 to 14mg/ml in PBS （pH7.4, 25 mM CaCl₂） at 37°C for 30 min. For the optimization of the incubation temperature, bacterial ghosts were incubated with the DNA （10 mg/ml in PBS, pH7.4, 25 mM CaCl₂） at 4, 25, and 37°C for 30 min until pelleted. For incubation time, bacterial ghosts were incubated with the DNA （10mg/ml in PBS, pH 7.4, 25 mM CaCl₂） at 24°C for 2, 4, 10, 30, 60, 120, and 180 min until pelleted. Incubation temperature, DNA concentration, and incubation time were varied systematically to optimize the loading procedure. The results demonstrated that the DNA concentration used for the loading was positively correlated with the amount of pcDNA3-F recovered from the bacterial ghosts. The DNA loads of bacterial ghosts were not significantly altered by incubation at temperatures of 4, 25, or 37°C. A 2-min incubation time was sufficient for the loading of the bacterial ghosts; longer incubation times （up to 180 min） did not increase the pcDNA3-F loading, as the DNA load stayed constant4. The evaluation of bacterial ghost loading the eukaryotic plasmid expressing NDV F geneOne hundred and eighty chickens with similar health condition were regarded as the experimental animals, which were randomly divided into 7 groups, with 30 chickens in each one at 7 day-old. I -II groups as the control were injected with PBS and pcDNA3.1 respectively, III group were injected with the naked pcDNA3-F, IV group were immunized with attenuated Lasota vaccine by eye-dropping, V-VII groups were administered with the bacterial ghost loading pcDNA3-F by eye-dropping, oral application or muscle injection respectively. Blood samples were collected weekly for indirect ELISA and HI assay before challenge. 2^nd week post booster immunization, chickens were challenged with the lethal dose of the isolated virulent NDV. 1^st week after primary immunization, the levels of antibody had risen slightly in pcDNA3-F group, attenuated Lasota vaccine group and bacterial ghost loading pcDNA3-F groups, compared with PBS group and pcDNA3.1+ group, which indicated that the DNA vaccine pcDNA3-F was successfully transformed and expressed into the chicken’s cell, thereby stimulated efficient immune response. 2^nd week after primary immunization, compared with control groups, the levels of all groups except control groups had risen obviously （P<0.01）. What’s more, at 1^st week post booster immunization, the pcDNA3-F-loaded ghost by eye-dropping group was higher than the naked pcDNA3-F group （P<0.05）, and significantly higher at 2^nd week post booster immunization （P<0.01）, although it was lower than the attenuated Lasota vaccine group. In the same period, the antibody level of the attenuated Lasota vaccine group kept the highest in all vaccination groups, and the antibody level of the control groups descent continuously. The result of HI suggested that the antibody induced by DNA vaccine pcDNA3-F had not relation with the antibody of HI. The protective immunization score was 70% for the group III chickens that were inoculated with the bacterial ghost loading pcDNA3-F vaccine, which was higher than the naked pcDNA3-F group, while the group that received the attenuated Lasota vaccine had 90% protection. 100% fatality was recorded for the control groups （PBS and pcDNA3.1+）. The remaining two groups receiving bacterial ghost loading pcDNA3-F either by orally or i.m, whose mortality was same as the naked pcDNA3-F group, but delayed the time of mortility. These results suggested that the DNA vaccine pcDNA3-F could induce more effective immune response against NDV, when used bacterial ghosts as a delivery system更多还原