本文選題：腸出血型大腸埃希菌O157:H7 + 生物信息學(xué)分析　； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：一、研究背景和目的 腸出血型大腸埃希菌(enterohaemorrhagic E.coli, EHEC) O157:H7是一種主要經(jīng)消化道傳播的病原菌,可引起出血性腸炎(haemorrhagic colitis, HC)、溶血性尿毒綜合癥(hemolytic uremic syndrome,HUS)、血栓性血小板減少性紫癜(thrombotic thromobocytopenie porpura, TTP)等并發(fā)癥,重者可致死亡,其感染的病死率為0%-10%。 自從Riley1983年首次報(bào)道由EHEC O157:H7引起的出血性腸炎暴發(fā)以來,該菌引起的暴發(fā)流行不斷發(fā)生并呈上升趨勢,已成為全球性公共衛(wèi)生問題。因此,預(yù)防和控制EHEC O157:H7感染十分重要。目前,針對(duì)EHEC O157:H7感染尚缺乏有效的防治方法,一般采取對(duì)癥治療。盡管EHEC O157:H7對(duì)大多數(shù)抗生素比較敏感,但其被抗生素殺死后,可釋放志賀毒素,加劇患者并發(fā)HUS的危險(xiǎn)性。因此,對(duì)使用抗生素治療應(yīng)采取慎重態(tài)度。鑒于EHEC O157:H7暴發(fā)流行的嚴(yán)重性和抗生素治療上的困難,疫苗的研究極為重要。 EHEC O157:H7的致病性主要體現(xiàn)在細(xì)菌的黏附定植力和毒素兩個(gè)方面,而轉(zhuǎn)位緊密黏附素受體(Translocation intimin receptor, Tir)是0157：H7的一種重要毒力蛋白,經(jīng)EHEC Ⅲ型分泌系統(tǒng)(Type Ⅲ secretion system,TTSS)分泌并轉(zhuǎn)位進(jìn)入宿主細(xì)胞,定位于細(xì)胞膜上,與緊密黏附素結(jié)合后,可引起宿主細(xì)胞多聚肌動(dòng)蛋白聚合,造成黏附局部微絨毛刷狀緣破壞,形成特征性的黏附和擦拭損傷(attaching and effacing lesion, A/E lesion),即Tir在EHEC O157:H7的定植感染過程中起著重要的作用。因此,阻斷EHEC O157:H7感染的黏附初始階段,可避免黏附和擦拭性損傷的發(fā)生,從而終止感染。 本研究從NCBI網(wǎng)站GenBank上查到EHEC O157:H7標(biāo)準(zhǔn)株EDL933轉(zhuǎn)位緊密黏附素受體完整的編碼區(qū)(cds)基因序列(Accession number NC002655.2)和氨基酸序列(protein id NP290261.1),利用生物信息學(xué)在線工具和抗原表位分析軟件,預(yù)測分析其蛋白結(jié)構(gòu)和抗原特性,從Tir基因全長中選取抗原表位分值高的基因進(jìn)行研究(命名為Tir-C);設(shè)計(jì)特異性引物,擴(kuò)增目的基因Tir-C;將目的基因連至PET-30a(+)載體上,構(gòu)建重組原核表達(dá)質(zhì)粒PET-30a(+)-Tir-C,轉(zhuǎn)化至大腸埃希菌BL21/DE3,誘導(dǎo)表達(dá)和純化重組蛋白Tir-C;將目的基因克隆入乳酸菌高效組成型表達(dá)質(zhì)粒PMG36e中,獲得重組質(zhì)粒PMG36e-Tir-C,經(jīng)電穿孔法將該重組質(zhì)粒轉(zhuǎn)化至嗜酸乳桿菌中,構(gòu)建表達(dá)EHEC0157:H7Tir-C基因的重組嗜酸乳桿菌活載體疫苗；進(jìn)一步用原核表達(dá)的Tir-C蛋白、表達(dá)EHEC O157:H7Tir-C基因的重組嗜酸乳桿菌、重組嗜酸乳桿菌結(jié)合原核表達(dá)的Tir-C蛋白分別免疫小鼠,觀察亞單位疫苗和重組載體疫苗的免疫原性及免疫保護(hù)性,探討與比較預(yù)防EHEC O157:H7感染最有效的疫苗,為發(fā)展新型疫苗提供實(shí)驗(yàn)依據(jù)。 二、研究方法 1.EHEC O157:H7Tir基因的生物信息學(xué)分析 從NCBI網(wǎng)站GenBank上獲得EHECO157:H7標(biāo)準(zhǔn)株EDL933Tir完整的編碼區(qū)基因序列和氨基酸序列；運(yùn)用InterProScan分析Tir氨基酸序列；同時(shí)使用兩種B細(xì)胞表位在線分析軟件(http://www.imtech.res.in/raghava/abcpred/與http://www.cbs.dtu.dk/services/bepipred/)預(yù)測Tir蛋白的B細(xì)胞表位。 2.目的基因Tir-C的原核表達(dá) 2.1目的基因Tir-C克隆載體的構(gòu)建 根據(jù)Tir基因生物信息學(xué)分析的結(jié)果,選取Tir基因全長中的一段基因作為目的片段(Tir-C)進(jìn)行研究,根據(jù)該片段的基因序列設(shè)計(jì)兩條引物,通過PCR,從EHEC O157:H7廣州株882364染色體DNA中擴(kuò)增出Tir-C基因,克隆入T載體pMD19-T中,構(gòu)建和鑒定重組克隆載體pMD19-T-Tir-C。 2.2目的基因Tir-C表達(dá)載體的構(gòu)建 利用限制性內(nèi)切酶Nde I和Mo I,雙酶切重組克隆載體pMD19-T-Tir-C,回收純化目的基因Tir-C,與經(jīng)過Nde I和Xho I雙酶切的空質(zhì)粒PET-30a(+)用T4DNA連接酶進(jìn)行連接,通過PCR、雙酶切及測序鑒定重組表達(dá)載體PET-30a(+)-Tir-C。 2.3目的基因Tir-C的表達(dá)、純化和鑒定 將重組質(zhì)粒PET-30a(+)-Tir-C轉(zhuǎn)化至BL21/DE3中,在異丙基-p-D-硫代半乳糖苷(IPTG)誘導(dǎo)下表達(dá)重組蛋白Tir-C,用12%SDS-PAGE鑒定,進(jìn)一步利用Ni-Agarose His標(biāo)簽蛋白純化試劑盒純化蛋白。以抗6×His單克隆抗體稀釋液為一抗,辣根過氧化物酶標(biāo)記的羊抗小鼠IgG稀釋液為二抗,通過Western blot方法,初步鑒定重組蛋白的免疫反應(yīng)性。 3.表達(dá)目的基因Tir-C的重組嗜酸乳桿菌菌株的構(gòu)建 3.1目的基因Tir-C的擴(kuò)增 根據(jù)Tir-C基因的堿基組成及乳酸菌高效組成型表達(dá)質(zhì)粒PMG36e的酶切位點(diǎn),設(shè)計(jì)特異性引物,PCR擴(kuò)增目的基因Tir-C。 3.2構(gòu)建重組質(zhì)粒PMG36e-Tir-C 將目的基因克隆入乳酸菌高效組成型表達(dá)質(zhì)粒PMG36e中,獲得重組質(zhì)粒PMG36e-Tir-C。 3.3重組嗜酸乳桿菌菌株的構(gòu)建 將重組質(zhì)粒PMG36e-Tir-C,經(jīng)電穿孔法轉(zhuǎn)化至嗜酸乳桿菌中,構(gòu)建表達(dá)EHEC0157:H7Tir-C的重組嗜酸乳桿菌,經(jīng)革蘭染色、菌落PCR、質(zhì)粒PCR、雙酶切、SDS-PAGE及Western blot進(jìn)行鑒定。 三、結(jié)果 1.EHEC O157:H7Tir基因的生物信息學(xué)分析 Tir基因全長1,677bp,起始密碼子為ATG,終止密碼子為TAA,共編碼558個(gè)氨基酸；InterProScan分析該氨基酸序列含有三個(gè)結(jié)構(gòu)和功能域,即Tir的N端、C端和與Intimin結(jié)合的M區(qū)；Bepipred分析預(yù)測有20個(gè)B細(xì)胞線性表位肽段,ABCPred分析預(yù)測有26個(gè)B細(xì)胞線性表位肽段,綜合分析結(jié)果,擬取C端,即Tir基因全長中的1000bp-1674bp作為目的片段進(jìn)行研究。 2.Tir-C基因的原核表達(dá) 從EHEC O157:H7廣州株882364染色體DNA中擴(kuò)增出Tir-C基因,大小為675bp,與預(yù)期一致,重組質(zhì)粒pMD19-T-Tir-C及重組表達(dá)質(zhì)粒PET-30a(+)-Tir-C經(jīng)質(zhì)粒VCR、Nde I和Xho I雙酶切和測序鑒定,證實(shí)構(gòu)建成功；目的蛋白誘導(dǎo)表達(dá)成功,經(jīng)SDS-PAGE電泳顯示與理論預(yù)測的融合蛋白分子質(zhì)量相符,經(jīng)純化后獲得目的蛋白Tir-C。Western blot鑒定結(jié)果顯示,純化的融合蛋白在24KDa左右處出現(xiàn)一條特異性條帶,與預(yù)期一致。 3.表達(dá)目的基因Tir-C的重組嗜酸乳桿菌菌株的構(gòu)建 成功擴(kuò)增出目的基因Tir-C,克隆入表達(dá)質(zhì)粒PMG36e中,獲得重組質(zhì)粒PMG36e-Tir-C,經(jīng)電穿孔法將該重組質(zhì)粒轉(zhuǎn)化至嗜酸乳桿菌ATCC4356中,經(jīng)鏡下觀察細(xì)菌形態(tài)學(xué)的變化、菌落PCR、質(zhì)粒PCR、雙酶切、SDS-PAGE電泳及Western blot鑒定,證實(shí)表達(dá)EHEC O157:H7Tir-C基因的重組嗜酸乳桿菌菌株構(gòu)建成功。 四、結(jié)論 1.選取的Tir-C基因抗原表位預(yù)測分值較高,大小為675bp。 2.成功構(gòu)建重組克隆載體pMD19-T-Tir-C和PET-30a(+)-Tir-C原核表達(dá)載體。細(xì)菌培養(yǎng)物經(jīng)IPTG誘導(dǎo)表達(dá)出重組融合蛋白,純化得到重組融合蛋白,分子量大小為24KD左右。Western blot初步鑒定該融合蛋白有一定的免疫反應(yīng)性。 3.表達(dá)EHEC O157:H7Tir-C基因的重組嗜酸乳桿菌菌株證實(shí)構(gòu)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R392

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