【作者】 尤建嵩；

【导师】 徐永平； Tim A. McAllister；

【作者基本信息】 大连理工大学 ， 生物化工， 2011， 博士

【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(P<0.05)(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(P<0.05)。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。更多还原

【Abstract】 Enterotoxigenic Escherichia coli (ETEC) strains remain important pathogens causing diarrhea in children and a wide variety of newborn animals. Vaccination is an effective approach to control ETEC-induced infections which avoids the problem of antibiotics residue and antibiotics resistance. Currently available ETEC vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). Heat-stable enterotoxin a (STa) and b (STb) are important virulence fators of ETEC, but they are pooly immunogenic due to their small size. Preparation of ST-carrier toxoids which are immunogenic and devoid of toxicity continues to be a challege for ETEC vaccines development. However, the induction of antitoxic responses against STa and STb has merit, as these two poorly immunogenic toxins are frequently associated with ETEC strains.In order to obtain more effective ETEC vaccines, the aim of this study was to fuse the three coding genes of enterotoxins LTB, STa, and STb, and construct recombinant trivalent enterotoxin DNA vaccines and protein vaccines, in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. The main work of this thesis was as follows:(1) The study of polyvalent enterotoxin DNA vaccines:chicken insulin signal peptide gene ins and chicken urokinase type plasminogen activator signal peptide gene upa were amplified by splicing by overlap extension (SOE) PCR. Coding genes for LTB, STa, and STb and signal peptide gene (ins or upa) were fused to yield the polyvalent enterotoxin fusion gene with or without signal peptide gene. Two carefully selected proline-rich natural oligopeptides, PPASP and SASTTPP, were included in the fusion to link the adjacent toxins. Fusion genes were then cloned into eukaryotic expression vector pCI to obtain 3 polyvalent enterotoxin DNA vaccines pCI-ins-SLS、pCI-upa-SLS、pCI-SLS. The DNA sequences of the recombinant plasmids were verified by DNA sequencing. DNA vaccines were purified by column chromatography and used to transfect Hela cells using liposome, then indirect immunofluorescence experiments confirmed the expression of polyvalent enterotoxin in Hela cells by the recombinant plasmids. Immunization of chickens with the 3 DNA vaccines elicited low antibody level to LTB and very weak antibody responses to STa or STb, but the DNA vaccine pCI-SLS elicited a significant elevation in serum antibodies to LTB, STa, and STb in mice and the mice anti-sera were able to neutralize the toxicity of STb but not STa. Mice receiving this DNA vaccine had a survival rate of 50% in the vaccination-challenge study which is significantly higher (P<0.05) than that in the control group.(2) The study of polyvalent enterotoxin protein vaccines:Coding genes for LTB, STa, and STb were obtained by PCR and fused to yield the polyvalent enterotoxin fusion gene LTB-STa-STb by traditional "restriction enzyme digestion-ligation" method. Two similar 7-amino-acid oligopeptides were included in the fusion to link the adjacent toxins. The other fusion enterotoxin gene of STa-LTB-STb was amplified from plasmid pCI-SLS. These two fusion genes were then cloned into vector pET-30a to produce 2 polyvalent enterotoxin prokaryotic expression plasmids. Fusion enterotoxin proteins, LSS and SLS, with the molecular weight of 21 kDa, were expressed in E. coli BL21 which was confirmed by SDS-PAGE and Western blot using anti-His tag antibodies. Fusion proteins were then purified by nickel affinity chromatography. Immunization of chickens or mice with 2 protein vaccines both elicited a high level of antibody responses to LTB, STa, and STb. The IgY or the mice anti-sera were able to neutralize the toxicity of STa and STb. Mice receiving the protein vaccines had a survival rate of 50-70% in the vaccination-challenge study, which was significantly higher (P<0.05) than that in the control group.In conclusion, the two recombinant trivalent enterotoxin proteins, LSS and SLS, were immunogenic for all the three enterotoxin components (i.e., LTB, STa, and STb) and were able to induce protective antitoxins in animals. Therefore, they could be included in a multivalent vaccine as a potent component to provide broad-spectrum protection against diarrhea caused by ETEC expressing various fimbriae. Though the immunogenicity of STa and STb was enhanced in the trivalent enterotoxin DNA vaccines constructed in this work, it was still weaker than that in the corresponding recombinant protein.更多还原