【作者】 卢林静；

【导师】 毕丁仁； 刘梅；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 本研究室刘梅利用苏云金芽胞杆菌的S-层蛋白在苏云金芽胞杆菌细胞表面成功展示了禽流感病毒核衣壳蛋白(NP)、血凝素蛋白(HA1)和鸡毒霉形体粘附素蛋白(pMGA)等禽类病原体抗原,虽然实验证实苏云金芽胞杆菌BMB171对小鼠和鸡都安全无毒,但其是否具有益生作用,还未见相关报道;蜡样芽胞杆菌在畜牧业中常被用作微生态制剂来使用,本文在刘梅等研究的基础上利用细菌细胞表面展示技术,探索在蜡样芽胞杆菌细胞表面展示外源抗原的可行性,为研制能耐高温同时具有益生作用的禽用口服疫苗提供理论基础。1.蜡样芽胞杆菌重组菌株的构建以苏云金芽胞杆菌CH和BCCH菌株为出发菌株,进行质粒pCSHA1P、pCTC-HA1P和pMIL-CSA的抽提和酶切鉴定,将鉴定正确的重组质粒分别电转化至蜡样芽胞杆菌中,其中包含质粒pCSHA1P的重组菌株命名为BcA,同时含有两个质粒pCTC-HA1 P和pMIL-CSA的重组菌株命名为BcC。其中质粒pMIL-CSA携带有csaAB操纵元,在转化不含csaAB操纵元的重组质粒时要同时转化该质粒,以便给受体菌株提供csaAB操纵元。2.检测外源抗原蛋白在重组菌株细胞表面的展示情况用血凝和血凝抑制试验检测BcA和BcC这两个重组菌株的表面展示情况。结果,这两个重组菌株均表现出了预计的生物学活性,说明所构建的S-层融合蛋白基因在受体菌株中得到表达并展示到了细胞表面且具有活性。3.检测重组菌株对雏鸡的免疫原性将展示HA1蛋白的重组菌株BcA和BcC培养至芽胞形成期,用重组菌株的芽胞液通过口服途径免疫艾维因商品代肉雏鸡,用血凝抑制试验测定接种免疫鸡的血清抗体效价。结果,实验鸡未产生针对相应外源抗原的抗体,其机理有待于进一步研究。更多还原

【Abstract】 S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avain influenza virus nucleoprotein (NP), hemagglutinin (HA1) or protein of Mycoplasma gallisepticum agglutinin (pMGA) on the cell surface of Bacillus thuringiensis. Although the experiment demonstrated Bacillus thuringiensis was safe and non-toxic for mice and chickens, but the role of probiotics, not related to reporting; Bacillus cereus, often used as probiotics, This study was used bacterial cell surface display technology to test the possibility of displaying heterologous antigens on the cell surface of bacillus cereus for the development of heat-stable, probiotic oral vaccine using B.cereus as a carrier.1. Construction recombinant strains of Bacillus cereusBacillus thuringiensis recombinant strains CH and BCCH as the original beginning bacterial strains for plasmids extraction and endonuclease, being used for electroporation to Bacillus cereus. The resulting strains include plasmid PCSHAIP named BcA, include two plasmids: CTC- HA1P and pMIL-CSA named BcC. The plasmid pMIL-CSA harboring csaAB operon was co-transferred into B.cereus with the recombinant plasmids which did not harboring csaAB operon.2. Assaying the display of heterologous antigens on the recombinant strainsHaemagglutination assay showed recombinant HAI proteins were displayed on the cell surface of respective recombinant strains. Hemagglutination inhibition assay showed recombinant HA1 proteins displayed on the cell surfaee of recombinants respectively were specific to standard positive serum of avian influenza virus, respectively.3. Immunogenicity of recombinant strains to chiekensThe spores of recombinant strains BcA and BcC were prepared. The AV commercial broiler chickens were immunized by oral route with spores. The serum antibody titers of chiekens immunized with spores of BcA and BcC were assayed by haemagglutination inhibition test. The results showed that the recombinant strains did not elicite humoral response of chickens to respective heterologous antigens displayed on the cell surfaces and didn’t exhibite immunogenieity. the mechanism need to be further studied.更多还原