肠出血型大肠埃希菌O157:H7 Tir-C在大肠杆菌和嗜酸乳杆菌中的表达研究

发布时间：2018-04-26 12:46

本文选题：肠出血型大肠埃希菌O157:H7 + 生物信息学分析　；参考：《南方医科大学》2014年硕士论文

【摘要】：一、研究背景和目的肠出血型大肠埃希菌(enterohaemorrhagic E.coli, EHEC) O157:H7是一种主要经消化道传播的病原菌,可引起出血性肠炎(haemorrhagic colitis, HC)、溶血性尿毒综合症(hemolytic uremic syndrome,HUS)、血栓性血小板减少性紫癜(thrombotic thromobocytopenie porpura, TTP)等并发症,重者可致死亡,其感染的病死率为0%-10%。自从Riley1983年首次报道由EHEC O157:H7引起的出血性肠炎暴发以来,该菌引起的暴发流行不断发生并呈上升趋势,已成为全球性公共卫生问题。因此,预防和控制EHEC O157:H7感染十分重要。目前,针对EHEC O157:H7感染尚缺乏有效的防治方法,一般采取对症治疗。尽管EHEC O157:H7对大多数抗生素比较敏感,但其被抗生素杀死后,可释放志贺毒素,加剧患者并发HUS的危险性。因此,对使用抗生素治疗应采取慎重态度。鉴于EHEC O157:H7暴发流行的严重性和抗生素治疗上的困难,疫苗的研究极为重要。 EHEC O157:H7的致病性主要体现在细菌的黏附定植力和毒素两个方面,而转位紧密黏附素受体(Translocation intimin receptor, Tir)是0157：H7的一种重要毒力蛋白,经EHEC Ⅲ型分泌系统(Type Ⅲ secretion system,TTSS)分泌并转位进入宿主细胞,定位于细胞膜上,与紧密黏附素结合后,可引起宿主细胞多聚肌动蛋白聚合,造成黏附局部微绒毛刷状缘破坏,形成特征性的黏附和擦拭损伤(attaching and effacing lesion, A/E lesion),即Tir在EHEC O157:H7的定植感染过程中起着重要的作用。因此,阻断EHEC O157:H7感染的黏附初始阶段,可避免黏附和擦拭性损伤的发生,从而终止感染。本研究从NCBI网站GenBank上查到EHEC O157:H7标准株EDL933转位紧密黏附素受体完整的编码区(cds)基因序列(Accession number NC002655.2)和氨基酸序列(protein id NP290261.1),利用生物信息学在线工具和抗原表位分析软件,预测分析其蛋白结构和抗原特性,从Tir基因全长中选取抗原表位分值高的基因进行研究(命名为Tir-C);设计特异性引物,扩增目的基因Tir-C;将目的基因连至PET-30a(+)载体上,构建重组原核表达质粒PET-30a(+)-Tir-C,转化至大肠埃希菌BL21/DE3,诱导表达和纯化重组蛋白Tir-C;将目的基因克隆入乳酸菌高效组成型表达质粒PMG36e中,获得重组质粒PMG36e-Tir-C,经电穿孔法将该重组质粒转化至嗜酸乳杆菌中,构建表达EHEC0157:H7Tir-C基因的重组嗜酸乳杆菌活载体疫苗；进一步用原核表达的Tir-C蛋白、表达EHEC O157:H7Tir-C基因的重组嗜酸乳杆菌、重组嗜酸乳杆菌结合原核表达的Tir-C蛋白分别免疫小鼠,观察亚单位疫苗和重组载体疫苗的免疫原性及免疫保护性,探讨与比较预防EHEC O157:H7感染最有效的疫苗,为发展新型疫苗提供实验依据。二、研究方法 1.EHEC O157:H7Tir基因的生物信息学分析从NCBI网站GenBank上获得EHECO157:H7标准株EDL933Tir完整的编码区基因序列和氨基酸序列；运用InterProScan分析Tir氨基酸序列；同时使用两种B细胞表位在线分析软件(http://www.imtech.res.in/raghava/abcpred/与http://www.cbs.dtu.dk/services/bepipred/)预测Tir蛋白的B细胞表位。 2.目的基因Tir-C的原核表达 2.1目的基因Tir-C克隆载体的构建根据Tir基因生物信息学分析的结果,选取Tir基因全长中的一段基因作为目的片段(Tir-C)进行研究,根据该片段的基因序列设计两条引物,通过PCR,从EHEC O157:H7广州株882364染色体DNA中扩增出Tir-C基因,克隆入T载体pMD19-T中,构建和鉴定重组克隆载体pMD19-T-Tir-C。 2.2目的基因Tir-C表达载体的构建利用限制性内切酶Nde I和Mo I,双酶切重组克隆载体pMD19-T-Tir-C,回收纯化目的基因Tir-C,与经过Nde I和Xho I双酶切的空质粒PET-30a(+)用T4DNA连接酶进行连接,通过PCR、双酶切及测序鉴定重组表达载体PET-30a(+)-Tir-C。 2.3目的基因Tir-C的表达、纯化和鉴定将重组质粒PET-30a(+)-Tir-C转化至BL21/DE3中,在异丙基-p-D-硫代半乳糖苷(IPTG)诱导下表达重组蛋白Tir-C,用12%SDS-PAGE鉴定,进一步利用Ni-Agarose His标签蛋白纯化试剂盒纯化蛋白。以抗6×His单克隆抗体稀释液为一抗,辣根过氧化物酶标记的羊抗小鼠IgG稀释液为二抗,通过Western blot方法,初步鉴定重组蛋白的免疫反应性。 3.表达目的基因Tir-C的重组嗜酸乳杆菌菌株的构建 3.1目的基因Tir-C的扩增根据Tir-C基因的碱基组成及乳酸菌高效组成型表达质粒PMG36e的酶切位点,设计特异性引物,PCR扩增目的基因Tir-C。 3.2构建重组质粒PMG36e-Tir-C 将目的基因克隆入乳酸菌高效组成型表达质粒PMG36e中,获得重组质粒PMG36e-Tir-C。 3.3重组嗜酸乳杆菌菌株的构建将重组质粒PMG36e-Tir-C,经电穿孔法转化至嗜酸乳杆菌中,构建表达EHEC0157:H7Tir-C的重组嗜酸乳杆菌,经革兰染色、菌落PCR、质粒PCR、双酶切、SDS-PAGE及Western blot进行鉴定。三、结果 1.EHEC O157:H7Tir基因的生物信息学分析 Tir基因全长1,677bp,起始密码子为ATG,终止密码子为TAA,共编码558个氨基酸；InterProScan分析该氨基酸序列含有三个结构和功能域,即Tir的N端、C端和与Intimin结合的M区；Bepipred分析预测有20个B细胞线性表位肽段,ABCPred分析预测有26个B细胞线性表位肽段,综合分析结果,拟取C端,即Tir基因全长中的1000bp-1674bp作为目的片段进行研究。 2.Tir-C基因的原核表达从EHEC O157:H7广州株882364染色体DNA中扩增出Tir-C基因,大小为675bp,与预期一致,重组质粒pMD19-T-Tir-C及重组表达质粒PET-30a(+)-Tir-C经质粒VCR、Nde I和Xho I双酶切和测序鉴定,证实构建成功；目的蛋白诱导表达成功,经SDS-PAGE电泳显示与理论预测的融合蛋白分子质量相符,经纯化后获得目的蛋白Tir-C。Western blot鉴定结果显示,纯化的融合蛋白在24KDa左右处出现一条特异性条带,与预期一致。 3.表达目的基因Tir-C的重组嗜酸乳杆菌菌株的构建成功扩增出目的基因Tir-C,克隆入表达质粒PMG36e中,获得重组质粒PMG36e-Tir-C,经电穿孔法将该重组质粒转化至嗜酸乳杆菌ATCC4356中,经镜下观察细菌形态学的变化、菌落PCR、质粒PCR、双酶切、SDS-PAGE电泳及Western blot鉴定,证实表达EHEC O157:H7Tir-C基因的重组嗜酸乳杆菌菌株构建成功。四、结论 1.选取的Tir-C基因抗原表位预测分值较高,大小为675bp。 2.成功构建重组克隆载体pMD19-T-Tir-C和PET-30a(+)-Tir-C原核表达载体。细菌培养物经IPTG诱导表达出重组融合蛋白,纯化得到重组融合蛋白,分子量大小为24KD左右。Western blot初步鉴定该融合蛋白有一定的免疫反应性。 3.表达EHEC O157:H7Tir-C基因的重组嗜酸乳杆菌菌株证实构建成功。
[Abstract]:First, research background and purpose
Enterohaemorrhagic E.coli (EHEC) O157:H7 is a major pathogenic bacteria transmitted through the digestive tract, which can cause hemorrhagic enteritis (haemorrhagic colitis, HC), hemolytic uremic syndrome (hemolytic uremic syndrome, HUS), thrombotic thrombocytopenic purpura (thrombotic) The mortality rate is 0%-10%..
Since the outbreak of hemorrhagic enteritis caused by EHEC O157:H7 for the first time in Riley1983, the outbreak of this bacterial outbreak has been increasing and rising, which has become a global public health problem. Therefore, the prevention and control of EHEC O157:H7 infection is very important. At present, there is still a lack of effective prevention and control methods for EHEC O157:H7 infection. Take symptomatic treatment. Although EHEC O157:H7 is more sensitive to most antibiotics, it can release Shiga toxin and exacerbate the risk of patients with HUS after being killed by antibiotics. Therefore, a prudent attitude should be taken for the use of antibiotics. In view of the severity of the outbreak of EHEC O157:H7 and the difficulties in the treatment of antibiotics, the study of the vaccine is extremely important. It's important.
The pathogenicity of EHEC O157:H7 is mainly reflected in two aspects of bacterial adhesion colonization and toxin, and the transposition close adhesion receptor (Translocation intimin receptor, Tir) is an important virulence protein of 0157:H7, which is secreted and transposition into the host cells through the EHEC III secretory system (Type III secretion system, TTSS) and is located in the cell. On the membrane, after binding with the close adhesion element, it can cause Polyactin polymerization of host cells, resulting in adhesion to local microhair brush border damage, and forming characteristic adhesion and wiping damage (attaching and effacing lesion, A/E lesion), that is, Tir plays an important role in the process of colonization and infection of EHEC O157:H7. Therefore, the EHEC O157:H7 is blocked. The initial stage of infection can avoid the occurrence of adhesion and wiping injury, thereby terminating infection.
In this study, the complete coding region (CDS) gene sequence (Accession number NC002655.2) and amino acid sequence (protein ID NP290261.1) of the EDL933 translocated close adhesion receptor (CDS) gene (Accession number NC002655.2) and amino acid sequence (protein ID NP290261.1) of the EHEC O157:H7 standard strain EHEC O157:H7 standard strain were predicted and analyzed by bioinformatics online tool and antigen epitope analysis software to predict and analyze the protein structure and antigen specificity. The gene of high antigen epitopes (named Tir-C) was selected from the full length of the Tir gene, and specific primers were designed to amplify the target gene Tir-C; the target gene was linked to the PET-30a (+) vector, and the Recombinant Prokaryotic expression plasmid PET-30a (+) -Tir-C was constructed and transformed into Escherichia coli BL21/DE3 to induce and purify the recombinant protein Tir-C; The target gene was cloned into the high efficient expression plasmid PMG36e of lactic acid bacteria, and the recombinant plasmid PMG36e-Tir-C was obtained. The recombinant plasmid was transformed into Lactobacillus acidophilus by electric perforation, and the recombinant Lactobacillus acidophilus live vector vaccine expressing EHEC0157:H7Tir-C gene was constructed, and the expression of Tir-C protein expressed in the prokaryotic cell was used to express EHEC O157:H7Tir-C. Recombinant Lactobacillus acidophilus, recombinant Lactobacillus acidophilus combined with prokaryotic expression Tir-C protein to immunize mice respectively, to observe the immunogenicity and immunogenicity of subunit vaccine and recombinant vector vaccine, to explore and compare the most effective vaccine against EHEC O157:H7 infection, and to provide experimental basis for the development of new vaccines.
Two, research methods
Bioinformatics analysis of 1.EHEC O157:H7Tir gene
A complete coding region gene sequence and amino acid sequence of EHECO157:H7 standard strain EDL933Tir were obtained from the NCBI site GenBank; Tir amino acid sequence was analyzed with InterProScan; and two B cell epitopes online analysis software (http://www.imtech.res.in/raghava/abcpred/ and http: //www.cbs.dtu.dk/services/bepipred/) was used to predict Tir The B cell epitopes of the protein.
Prokaryotic expression of 2. target gene Tir-C
Construction of 2.1 target gene Tir-C cloning vector
According to the results of Tir gene bioinformatics analysis, a segment of the whole length of Tir gene was selected as the target fragment (Tir-C), and two primers were designed according to the gene sequence of the fragment. The Tir-C gene was amplified from the DNA of the 882364 chromosome of EHEC O157:H7 Guangzhou strain, and was cloned into the T carrier pMD19-T to construct and identify the recombinant gram. Long carrier pMD19-T-Tir-C.
Construction of 2.2 target gene Tir-C expression vector
Using restriction endonuclease Nde I and Mo I, double enzyme cut recombinant cloning vector pMD19-T-Tir-C was used to reclaim the purified target gene Tir-C. The recombinant plasmid PET-30a (+) was linked with Nde I and Xho I, and the recombinant expression vector (+) was identified by double enzyme digestion and sequencing.
Expression, purification and identification of 2.3 target gene Tir-C
The recombinant plasmid PET-30a (+) -Tir-C was transformed into BL21/DE3, and the recombinant protein Tir-C was expressed under the induction of isopropyl -p-D- Thioglucoside (IPTG). The recombinant plasmid was identified by 12%SDS-PAGE, and the purified protein purified by the Ni-Agarose His label protein purification kit was further used. The anti 6 * His monoclonal anti body diluent was one resistance and horseradish peroxidase labelled sheep resistance. Mouse IgG diluent was the two antibody. The immunoreactivity of the recombinant protein was preliminarily identified by Western blot.
3. construction of recombinant Lactobacillus acidophilus strain expressing target gene Tir-C
Amplification of 3.1 target gene Tir-C
According to the base composition of Tir-C gene and the restriction site of high effective expression plasmid PMG36e of lactic acid bacteria, specific primers were designed, and the target gene Tir-C. was amplified by PCR.
3.2 construction of recombinant plasmid PMG36e-Tir-C
The target gene was cloned into an efficient expression plasmid PMG36e of Lactobacillus, and the recombinant plasmid PMG36e-Tir-C. was obtained.
Construction of 3.3 recombinant Lactobacillus acidophilus strain
Recombinant plasmid PMG36e-Tir-C was transformed into Lactobacillus acidophilus by electroporation, and recombinant Lactobacillus acidophilus expressing EHEC0157:H7Tir-C was constructed by Gram staining, colony PCR, plasmid PCR, double enzyme digestion, SDS-PAGE and Western blot.
Three, the result
Bioinformatics analysis of 1.EHEC O157:H7Tir gene
The Tir gene is full length 1677bp, the starting codon is ATG, the terminating codon is TAA, which encodes 558 amino acids. InterProScan analyses the amino acid sequence containing three structures and functional domains, namely, the N end of Tir, the C end and the M region with Intimin, and the Bepipred analysis predicts that there are 20 linear epitopes of the B cells, and the analysis predicts 26 lines of cell lines. Based on the analysis of the sex epitopes, the C ends, the 1000bp-1674bp of the full length of the Tir gene, were taken as the target fragments.
Prokaryotic expression of 2.Tir-C gene
The Tir-C gene was amplified from the 882364 chromosome DNA of the EHEC O157:H7 Guangzhou strain. The size of the gene was 675bp. The recombinant plasmid pMD19-T-Tir-C and the recombinant expression plasmid PET-30a (+) -Tir-C were identified by plasmid VCR, Nde I and Xho restriction double enzyme digestion and sequencing, which confirmed the success of the egg white induced expression. The molecular weight of the fusion protein was consistent. After purification, the target protein Tir-C.Western blot identification results showed that the purified fusion protein appeared a specific band around 24KDa, which was the same as expected.
3. construction of recombinant Lactobacillus acidophilus strain expressing target gene Tir-C
The target gene Tir-C was amplified and cloned into the expression plasmid PMG36e to obtain the recombinant plasmid PMG36e-Tir-C. The recombinant plasmid was transformed into Lactobacillus acidophilus ATCC4356 by electric perforation. The morphological changes of bacteria were observed under the microscope, colony PCR, plasmid PCR, double enzyme cutting, SDS-PAGE electrophoresis and Western blot identification, and the EHEC O157:H7Tir-C was confirmed. The recombinant Lactobacillus acidophilus strain was constructed successfully.
Four. Conclusion
1. the predicted epitopes of Tir-C gene were higher than those of 675bp..
2. the recombinant clone vector pMD19-T-Tir-C and PET-30a (+) -Tir-C prokaryotic expression vector were successfully constructed. The recombinant fusion protein was expressed by IPTG, and the recombinant fusion protein was purified. The molecular weight of the fusion protein was about 24KD.Western blot, and the fusion protein was preliminarily identified as a certain immunoreactivity.
3. the recombinant Lactobacillus acidophilus strain expressing EHEC O157:H7Tir-C gene was successfully constructed.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

【参考文献】