产肠毒素大肠杆菌多价肠毒素疫苗的研究

发布时间：2018-02-20 23:41

本文关键词： 产肠毒素大肠杆菌肠毒素腹泻疫苗融合蛋白　出处：《大连理工大学》2011年博士论文　论文类型：学位论文

【摘要】：产肠毒素大肠杆菌(ETEC)能够引起多种幼龄动物以及婴幼儿的胃肠道感染与严重腹泻,给各国的畜牧养殖业及人类健康带来极大的危害。疫苗接种是预防ETEC感染的有效手段,它避免了抗生素滥用所带来的药物残留及细菌产生耐药性等问题,是多年来重要的研究方向。耐热性肠毒素(ST)是ETEC的重要毒力因子,它是免疫原性很弱的小分子半抗原,如何将ST与大分子载体偶联使其具备良好的免疫原性并消除其天然毒性是ETEC疫苗研究中的难点。现有ETEC疫苗中均不含有ST组分,不能诱导有效的抗毒素免疫,因此免疫保护效果有待提高。为了获得更有效的ETEC疫苗,本论文的研究目的是将编码两种ST分子(STa、STb)的基因与不耐热肠毒素B亚基(LTB)的基因进行融合,进而制备3价融合ETEC肠毒素的DNA疫苗与蛋白疫苗,其特征是含有3种ETEC重要肠毒素毒力因子(分别为LTB、STa, STb)的抗原表位,能够引起抗3种肠毒素的特异性抗体反应,为机体提供全面的抗毒素免疫,从而提高ETEC疫苗的保护率。本论文主要工作如下： (1)多价肠毒素DNA疫苗的研究：应用重叠延伸PCR技术扩增获得了鸡胰岛素信号肽的基因ins和鸡尿激酶型血纤维蛋白溶酶原激活剂的信号肽基因upa,将肠毒素基因LTB、STa、STb及信号肽基因进行连接得到多价融合基因,在相邻的肠毒素基因之间设计了编码5-7个氨基酸的连接肽序列,将融合基因插入真核表达载体pCI,获得了3种多价肠毒素真核表达质粒pCI-ins-SLS、pCI-upa-SLS、pCI-SLS。经DNA测序证明,所构建的表达载体目标基因序列正确。利用柱层析法纯化DNA疫苗,经脂质体介导转染体外培养的Hela细胞进行瞬时表达实验,免疫荧光实验的结果表明所构建的真核表达质粒能够表达目的重组蛋白。用3种DNA疫苗免疫产蛋母鸡,ELISA测定表明获得了LTB特异性抗体,但STa及STb特异性抗体的效价较低；用其中不带信号肽的DNA疫苗pCI-SLS免疫小鼠,获得了高水平的抗LTB、STa及STb的特异性血清抗体,该抗血清具有中和STb毒性的活性,但没有STa的中和活性,在小鼠攻毒保护实验中提供了50%的保护率,与对照组相比有显著差别(P0.05) (2)多价肠毒素蛋白疫苗的研究：首先利用PCR法扩增获得了三种肠毒素LTB、STa、STb的编码基因,应用“限制性内切酶酶切-连接酶连接”等基因工程手段将肠毒素基因LTB、STa、STb进行连接得到三价融合基因LTB-STa-STb,每两段相邻的基因之间设计了编码7个氨基酸的连接肽序列。又以质粒pCI-SLS为模板扩增得到了三价融合基因STa-LTB-STb。将融合基因克隆入原核表达载体pET-30a,获得了2种三价肠毒素原核表达质粒pET30-LSS和pET30-SLS,转化入宿主菌E. coli BL21(DE3)中后经诱导剂IPTG诱导,聚丙烯酞胺凝胶电泳及蛋白质免疫印迹检测实验表明成功的表达了重组三价肠毒素融合蛋白LSS和SLS,分子量均为21 kDa。使用镍离子琼脂糖凝胶色谱柱进行亲和层析,获得了纯化的重组三价肠毒素蛋白。用两种三价肠毒素蛋白疫苗免疫母鸡及小鼠,通过ELISA法检测抗体效价,结果表明在两种动物都获得了较高水平的抗LTB、STa及STb免疫反应,多价抗毒素能够中和STa及STb的毒性,在小鼠攻毒保护实验中提供了50-70%的保护率,与对照组相比有显著差别(P0.05)。综上所述,本论文构建的2种三价肠毒素重组蛋白LSS和SLS,同时具有ETEC的三种肠毒素(LTB、STa及STb)免疫原性,免疫动物可引发全面的保护性抗毒素免疫反应,从而能对所有菌毛类型的ETEC致病菌株产生有效的免疫保护,为广谱性ETEC疫苗的研究打下基础。在本论文构建的三价肠毒素DNA疫苗中,STa及STb的免疫原性有所提高,但低于相应的蛋白抗原。
[Abstract]:Heat - resistant enterotoxin ( ST ) is an important virulence factor of ETEC . It is an important virulence factor for ETEC . How to couple ST with macromolecule carrier makes it possess good immunogenicity and to eliminate its natural toxicity is a difficult problem in ETEC vaccine research . In order to obtain a more effective ETEC vaccine , the aim of this paper is to fuse the genes encoding two ST molecules ( STa , STb ) with non - heat - labile Enterotoxin B subunit ( LTB ) gene . ( 1 ) Study on the DNA Vaccine of Multi - valent Enterotoxin : The signal peptide gene upa of chicken insulin signal peptide and plasminogen activator of chicken urokinase type were amplified by overlapping extension PCR . ( 2 ) The research of multivalent Enterotoxin protein vaccine : Firstly , the coding genes of three kinds of enterotoxins LTB , STa and STb were amplified by PCR method . The genes LTB , STa and STb were ligated to obtain three - valence fusion gene LTB - STa - STb . coli BL21 ( DE3 ) was transformed into E . coli BL21 ( DE3 ) , induced by IPTG . The results showed that the recombinant three - valent Enterotoxin fusion protein was successfully expressed , and the molecular weight was 21 kDa . Two kinds of three - valent Enterotoxin protein vaccine were used to immunoaffinity chromatography . The results showed that the two kinds of three - valent Enterotoxin protein vaccine were used to test the antibody titer . The results showed that both the two animals had higher levels of anti - LTB , STa and STb immune responses . In conclusion , two kinds of three - valent Enterotoxin recombinant proteins ( LTB , STa and STb ) , which were constructed by this paper , were immunogenic , and the immunized animals could induce a comprehensive protective immune response to ETEC vaccine . The results showed that the immunogenicity of STa and STb increased , but lower than the corresponding protein antigens .

【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

【引证文献】