【作者】 杨冬梅；

【导师】 陈创夫；

【作者基本信息】 石河子大学 ， 预防兽医学， 2007， 硕士

【摘要】 为了构建表达大肠杆菌菌毛蛋白K88ac和热稳定肠毒素STII融合基因的无抗性减毒鼠伤寒沙门氏菌疫苗株,并对其免疫效果进行研究。根据GenBank中登录号为m29375的K88ac基因序列以及减毒鼠伤寒沙门氏菌原核表达载体pYA3334的多克隆位点设计特异性引物P1、P2,并在上下游引物的5’端分别引入NcoI和BamHI酶切位点,从大肠杆菌DE3(pET- K88ac-STⅡ)中通过聚合酶链式反应(PCR)扩增得到K88ac-STⅡ融合基因。回收纯化后将K88ac-STⅡ融合基因用T4DNA连接酶连于克隆载体pBS-T上,热击转化受体大肠杆菌Top10,蓝白斑筛选阳性菌落,酶切鉴定证明其中含有K88ac-STⅡ融合基因片段,再测序鉴定该融合基因序列的正确性,命名为pBS-K88ac-STⅡ。之后采用缺失腺苷酸环化酶基因(Δcya)、环腺苷酸受体蛋白基因(Δcrp)以及天冬氨酸β-半醛脱氢酶基因(Δasd)的鼠伤寒沙门氏菌(X4550)作为宿主,将编码K88ac-STII的融合基因切下插入Asd+组成型表达载体pYA3334中,鉴定后通过电转化引入宿主菌X4550中,再次酶切鉴定正确后,过夜培养进行SDS-PAGE和Western-Blot试验,检测其特异性蛋白表达情况。最后分别以重组菌株X4550(pYA3334-K88ac-STII)灌服小鼠作为试验组,以转化了空载体的菌株X4550(pYA3334)作为空载体对照组,同时设PBS空白对照组,分别对三个分组的小鼠进行免疫,采集血清检测其抗体效价,之后免疫小鼠用强毒株C83915(STⅡ﹢)攻击,观察结果。本实验成功构建了表达K88ac-STII融合基因平衡致死的减毒鼠伤寒沙门氏重组菌株X4550(pYA3334-K88ac-STII),该重组菌株在33Kda处有特异性表达的蛋白条带,与预期分子量大小一致,而空载体对照菌X4550(pYA3334)在该处未有相应条带出现。Western-blot免疫印迹结果显示重组菌株X4550(pYA3334-K88ac-STII)表达的蛋白能够被K88ac阳性血清特异性结合,在相应位置出现特异性显色条带。这证明重组菌株能够表达K88ac-STII特异性融合蛋白,重组菌的体外稳定实验也证明其有良好的稳定性,从而保证了K88ac-STII抗原能够得以持续表达。而且试验小鼠灌喂重组减毒鼠伤寒沙门氏菌X4550 (pYA3334-K88ac-STII)后,能够产生特异性的抗体,而且对大肠杆菌强毒株具有一定的抵抗力。这为进一步研制相应的抗仔猪大肠杆菌病及相应的沙门氏菌病的双价活疫苗侯选株奠定了重要基础。更多还原

【Abstract】 Object:To construct a live oral Salmonella typhimurium vaccine strain expressing K88ac-STII fusion gene of enterotoxigenic Escherichia coli.Methods: Primers(P1,P2) were designed according to the nucleotide sequence of K88ac gene of enterotoxigenic Escherichia coli(ETEC) reported respectively and the expression vector pYA3334 . With the specific primers, the gene encoding the fusion protein K88ac-STII was amplified by PCR of the plasmid pET- K88ac-STⅡand was inserted into the linearized plasmid vector PBS-T. By enzyme analysis, PCR and sequencing, the recombinant plasmid was proved to be true. The target fragment was obtained by digestion of the plasmid pBS- KS using restrictive enzyme NcoI/BamHI and was inserted into the prokaryotic expression vector pYA3334 containing asd gene and was introduceded into the delta Cya ,delta Crp ,delta Asd attenuated Salmonella typhimurium strain X4550 by electroporation transformations , which is a balanced lethal recombinant. Analyzed the products of expression through SDS-PAGE and Western-blotting . The Immune Effect of Recombinant strain X4550(pYA3334-K88ac-STII) was indicated by the level of antibody and pathological change. Mice were then inoculated with 109 or 1010 cfu recombination strain through a stainless steel gavage tube,as experimentation vaccine, empty vector X4550(pYA3334)and PBS were used as control.Mice of three groups were immunized with each of them and challenged with virulent strain C83915(STⅡ﹢).Result: The fusion gene was coloned into prokaryotic expression vector pYA3334 with the right ORF, and can express the products expected successfully in X4550. The recombinant strain can express fusion protein, which has the same molecular weight as prospective protein.The fusion protein expressed by recombinant strain X4550(pYA3334-K88ac-STII) can be identified by anti-K88ac.No corresponding protein was expressed in X4550 transformed by pYA3334 .The stability of The recombinant strain X4550(pYA3334-K88ac-STII) in vitro asserted The foreign antigen K88ac-STⅡcan be expressed stabily. The mice immunized with recombinant strain X4550(pYA3334-K88ac-STII)vaccine can produce anti- K88ac-STⅡ.Recombinant strain X4550(pYA3334-K88ac-STII) has effective Immune Effect to mice. The recombinant strain is worth considering as a candidate vaccine strain against enterotoxin Escherichia coli disease and Salmonella typhimurium infections.更多还原