小反刍兽疫转基因苜蓿植物疫苗研究

For no systemic and effective controlling means to Peste des Petits Ruminants(PPR), an available and effective plant vaccine wil be developed.After optimization of the protective antigen epitope codon of F and H gene of PPR for the fabaceous plant, the epitope gene and the fusion gene of F and H will be acted as aim gene, respectively. The adjuvant gene LTB and the binding site of ribosome were added into the aim gene and the fusion gene eGFP/GUS was the report gene. 1)The expression vector to posite to chloroplast genome was constructed, which had gene-deletor system to banish all the foreign gene expect aim gene from the special organ in the special growth phage to breed the safe transgenic system on maternal inheritance with non-choosing-marker. 2)The vector pBI121 was acted as the basic expression vector, the MAR and KDEL were added to the vector ,and the promoter CaMV35S was substituded by the promoter CsVMV to establish the effective expression vector of nucleolus genome. The vector will be transferred into the competent agrobacterium EHA105 and LBA4404 by freeze-thawing method, respectively, then be transferred into petiole, hypocotyls and Protoplast of alfalfa by leaf infection method. The transforming condition will be groped. The regenerated transgenic plant will be validated by Southern and Western blot. It will be tested by hemagglutination and serum ELISA method after Mice were raised by the transgenic plant. The plant vaccine with a good immunogene will be gained after immunodetection,that will offer a beneficial attempt for the PPR and correlative ailment defending.

针对小反刍兽疫（PPR）仍未获得系统有效的控制方法，本研究拟研制实用高效的PPR植物疫苗，拟分别将PPR保护性F、H基因和经密码子优化的表位基因与佐剂基因LTB融合，添加核糖体结合位点后作为目的基因,以eGFP/GUS融合基因为报告基因：1）构建叶绿体基因组定点表达载体,其含有基因删除系统lox/frt，培育无选择标记母性遗传的安全转基因系统；2.以pBI121为基本表达载体,添加核基质附着区MAR、内质网驻留蛋白信号序列KDEL,木薯叶脉花叶病毒CsVMV启动子替换CaMV35S启动子,建立核基因组的高效表达载体。继而采用冻融法分别导入感受态的根瘤农杆菌EHA105和LBA4404,以叶盘法侵染苜蓿叶柄、下胚轴、原生质体,确定高效转化条件,再生完整植株,经Southern和Western blot检测,饲喂小鼠，进行血凝法和血清ELISA法检验，获得有较高免疫原性的PPR转基因植物疫苗。

小反刍兽疫（PPR），是由小反刍兽疫病毒(PPRV)引起的山羊、绵羊、野生小反刍兽的高度接触传染性疾病，发病急、传播速度快、致死率高，尚未找到有效的防治方法，本试验拟研制经济、实用、高效的PPR转基因植物疫苗。首先构建PPRV高效表达载体，从PPRV质粒中扩增F基因，F基因终止密码子前加入KDEL序列，形成F-KDEL；烟草SRI中扩增出促进外源基因表达的MAR12序列和MAR34序列；pHL005质粒中扩增出报告基因GFP；人工合成启动子CsVMV，酶切连接构建了载体pBI121-CsVMV-GFP-F-MAR12-MAR34，pBI121-CsVMV-GFP-F-KDEL-MAR12-MAR34，pBI121-CsVMV-F-MAR12-MAR34，pBI121-CsVMV-F-KDEL-MAR12-MAR34, pBI121-F。冻融法将载体分别转入农杆菌LBA4404和EHA105中，侵染中苜3号苜蓿的子叶和下胚轴，结果显示15min为最佳侵染时间，EHA105侵染后的分化率显著高于LBA4404，建立了紫花苜蓿高效组培条件，获得了转基因植株。因原生质体是单细胞系统不易形成嵌合体，对原生质体的游离条件做了初步探索。结果显示，以-70℃保存过夜的种子，取第7d的子叶以震荡的方式进行酶解，酶液组合为2%纤维素酶+0.5%果胶酶+0.3%离析酶，游离效果最佳。还利用叶绿体转化法，制备植物疫苗。分别从PPRV质粒中扩增F基因，苜蓿叶绿体基因组中克隆出用于基因重组的同源片段trnA-trnI，烟草叶绿体基因组中克隆出启动子Prrn和终止子Tpsba，菠菜中克隆出BADH基因和人工合成的aadA基因作为筛选标记基因。使用三引物PCR法将Prrn和Tpsba分别添加到标记基因和F基因两端，构建载体pPFT、pPBT、pPaT。通过酶切连接方法将PFT与PBT相连、PFT与PAT相连，构建载体pPFT-PBT、pPFT-PaT。由于叶绿体表达载体具有原核表达特性，将构建的质粒转入到BL21大肠杆菌中进行原核表达验证。SDS-PAGE验证结果显示目的基因及标记基因均得到了表达，将重组大肠杆菌进行抗逆性检测，结果显示转入重组质粒后的大肠杆菌均显示抗逆性。采用基因枪法将重组质粒转化到苜蓿叶绿体基因组中，获得了转基因植株，实验即将对转基因植株扩繁后饲喂小鼠，进一步验证疫苗的效果。