人β-干扰素和大肠杆菌O157：H7表面抗原EspA在乳酸乳球菌食品级诱导表达体系中表达及相关免疫活性分析

【作者】 庄重；

【导师】 王鹏；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 乳酸乳球菌是公认安全的食品级微生物(Generally Regard As Safe),对人畜无害,长期以来被广泛地用于各类乳制品发酵和生产中。近年来对乳酸乳球菌的研究日益受到重视。目前其全基因组测序工作业已完成,同时随着一些内部调控元件的发现,相继开发出一系列克隆、表达和整合载体,促进了乳酸乳球菌在分子生物学方面的大力发展,其中以NICE(Nisin Controlled Expression System)为代表的食品级诱导表达系统成为现今研究的热点。本文利用乳酸乳球菌食品级诱导表达系统重组表达人β-干扰素和大肠杆菌O157:H7表面抗原EspA蛋白,在这两方面开展相关研究工作。本文旨在利用乳酸乳球菌食品级诱导表达系统表达与疾病相关的免疫因子和抗原,从而开发相应的口服制剂,为预防和治疗疾病提供新途径。第一部分:人β-干扰素在乳酸乳球菌中分泌表达及生物活性测定炎症性肠病(inflammatory bowel disease,IBD)是一组病因不明的慢性肠道炎症性疾病,包括溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn’s disease,CD)。其发病原因一般认为主要与遗传免疫、环境因素和细胞因子等多方面因素有关。人β-干扰素作为最先发现的细胞因子之一,具有抗病毒、抗增殖、免疫调控等一系列生物学效应,能够促进IL-10表达。Katakura等人发现,在先天免疫中,外源细菌入侵肠道时,其特异性的DNA会被体内树突状细胞Toll样受体-9所识别,激活一系列信号传导途径,最终诱导产生Ⅰ型干扰素(IFN-α/β),对肠道内环境平衡起到保护性作用。本实验首次利用乳酸乳球菌食品级诱导表达系统,将成功点突变改造之后的ifn-β1b(17 Ser取代Cys)基因连接入乳酸菌分泌表达载体pSec:Nuc中,同时设计另一对引物,在ifn-β基因前插入一段促进蛋白分泌的九个氨基酸的前导肽(LEISSTCDA),并对前导肽中部分密码子进行了优化,分别电转入Lactococcus lactis NZ9000中。在成功筛选到重组菌株后,加入Nisin诱导目的基因的表达,Western blot鉴定了重组蛋白的正确表达。ELISA定量实验发现,含有LEISSTCDA前导肽的重组菌株目的蛋白分泌量提高了三倍,分泌比率达到95%,最大表达量为20μg/L。进一步对于重组蛋白生物活性检测发现,重组菌株胞内外重组蛋白生物活性均达到了1×10~7 i.u./mg。基于乳酸乳球菌食品级诱导表达系统的诸多优势,体内递呈人β-干扰素不仅可以避免消化道对其的降解,而且乳酸乳球菌是非肠道内定植菌株,只能在肠道内存活一段时间,保证了其表达的细胞因子不会长时间刺激机体,减少了细胞因子的毒性,以上结果表明利用重组菌株治疗肠道炎症有望成为现有的治疗方法的有益补充。第二部分:大肠杆菌O157:H7 espA在乳酸乳球菌中的表达及小鼠免疫分析大肠杆菌O157:H7是一种危害严重的病原体,能够引起水样腹泻进而发展为出血性腹泻,易引起并发性溶血性尿毒综合症(HUS)和血栓形成性血小板减少性紫癜,病死率极高。粘附是大肠杆菌O157:H7感染的第一步。EspA蛋白在其粘附定植中起到关键性作用。它通过形成纤丝样管状结构,在细菌和宿主细胞之间架起一座桥梁,把效应蛋白转移至宿主细胞表面,对细菌粘附至宿主内皮细胞具有关键性启动作用。基于EspA具有良好的免疫原性及免疫反应性,可以作为候选免疫原,用于大肠杆菌O157的疫苗研制,在消除定植带来A/E(attaching/effacing)损伤,缩短感染周期方面具有较大的应用价值。本实验利用乳酸乳球菌食品级诱导表达系统首次将根据乳酸菌偏好密码子优化过的espA基因连接入乳酸乳球菌分泌表达载体,构建重组菌株。经过Nisin诱导之后,Westernblot实验证明了重组蛋白的正确表达。之后用重组菌株免疫小鼠,经过三次灌胃免疫之后,间接ELISA检测发现,与对照组相比,使用重组菌株免疫小鼠血清中特异性抗EspA IgG水平有显著提高,说明能够诱发小鼠体内特异性免疫应答,这一结果为将来利用重组菌株作为活疫苗来预防大肠杆菌O157:H7感染打下基础。更多还原

【Abstract】 Lactococcus lactis is a Gram-positive,nonpathogenic,noninvasive,nonclonizing and generally regard as safe bacterium.It is mainly used for the production of fermented milk products and daily food industries for a long time.In the last 25 years,impressive progress has been made in the development of genetic engineering tools and the molecular characterization of lactococci.The tools include transformation,the availability of many different vectors,gene intergration,gene knock out,conjugation,and constitutive and regulated gene systems.At present the genome information of at least three strains of L.lactis is elucidated and publicly available.The availability of an easy-to-operate and strictly controlled food-grade expression system-nisin controlled expression system（NICE）has been developed and used for many application.This article is divided into two parts.It is mainly about expression of recombinant human interferon beta 1b and Escherichia coli O157:H7 EspA antigen in recombinant L.lactis strain using food-grade expression system.We aim to in situ delivery of heterologous cytokines and antigens by recombinant strains for preventing and treatment of related diseases.PartⅠsecretion of human interferon-beta 1b in Lactococcus lactis for intestinal immunotherapyInflammatory bowel disease（IBD）is caused by excessive and tissue damaging chronic inflammatory responses in the gut wall,including Crohn’s disease and ulcerative colitis.The cause of IBD involves genetic,enviromental and immune factors.Human interferon beta was one of the first discovered cytokines.It exerts many biological functions,such as antivirus,anti-proliferation and immuno-regulatory. Many evidences show that IFN-α/βis relevant to the protection against bacterial and parasitic infections.In 2005,Katakura et al.had discovered a protective role of IFN-α/βin a RAG1^-/-murine model of experimental colis,through the activation of a Toll-like receptor 9-dependent signal pathway.Moreover,IFN-βhas also been evaluated in pilot clinical trials in active ulcerative colitis.In this study,we successfully secreted bioactive human IFN-βlb in L.lactis using food-grade expression system.The site-direct mutagenesis was successfully introduced into the recombinant ifn-β1b gene in which 17 Cys was changed to Ser. The PCR product was double digested by restriction enzymes and cloned it into pSec:Nuc vector.In order to increase recombinant protein secretion,a nine-residue propeptide was inserted between usp45 signal peptide and ifn-β1b gene.The two expression plasmids were transferred by electroporation into L.lactis NZ9000 respectively.The recombinant IFN-βwas expression in the recombinant strains and the expression level and biological activity of recombinant proteins were both confirmed.The production of secreted form of IFN-βfrom recombinant strain L. lactis NZ9000/pSec-LEISS-IFNb and pSec-IFNb in culture was about 20μg L^-1and 6.2μg L^-1.Moreover,about 95%of total recombinant protein from the L.lactis NZ9000/pSec-LEISS-IFNb was secreted into the culture medium.The biological activities of recombinant IFN-β1b in both strains were determined to be 1×10⁷ i.u. mg^-1.Since the in situ delivery of recombinant strains has many advantages on low cost,safety and fewer side effects,the use of L.lactis to deliver IFN-βwill provide an alternative or complementary option for the treatment of ulcerative colitis in future.PartⅡexpression of Escherichia coli O157:H7 espA gene in Lactococcus lactis and oral administration to induce systemic anti-EspA response in miceEnterohemorrhagic Escherichia coli O157:H7（EHEC）is an important zoonotic pathogen of humans,causing severe diarrhea（hemorrhagic colitis）and in a small percentage of cases,haemolytic-uremic syndrome（HUS）.Unfortunately,antibiotics increase the risk of HUS,and there are currently no therapies for EHEC human infection other than general supportive measures.EspA,a protein also expressed from the LEE（locus of enterocyte effacement）pathogenity island and essential for A/E lesion formation,is one of the TTSS（TypeⅢsecretion system）translocator proteins and a major if not the only component of a filamentous structure which extends from the basic needle complex of the secretion apparatus and connects the pathogen to the plasma membrane of the host cell.Many experiments found that EspA protein can cause immune response in vivo.In this study,we successfully expressed the recombinant EspA protein in L.lactis using food-grade expression system.The espA gene was amplified by polymerase chain reaction（PCR）from the genomic DNA of E. coli O157:H7.The espA gene was optimized by lactic acid bacteria bias codons and cloned into pSec:Nuc vector.Western blot analysis demonstrated that the EspA protein was expressed in the L.lactis transformant.The oral administration of the transformants into mice significantly induced the anti-EspA antibody IgG in serum. These results suggest that recombinant L.lactis which expressed EspA can cause specific immune response and may be applicable as an oral vaccine to induce protective immunity against E.coli O157:H7 infection.更多还原