融合表达禽致病性大肠杆菌fima-ompC基因及微皱褶细胞和树突状细胞靶向肽重组乳酸菌的鉴定及免疫学评价

摘要

禽大肠杆菌病是由致病性禽大肠杆菌(APEC)引发的一种可导致鸡胚和成年鸡死亡的传染病。该病可引发禽类的急性致死性肠炎、腹膜炎、输卵管炎、亚急性心包炎、大肠杆菌性肉芽肿以及脓毒败血症。该病是一种常见的禽病，给世界养禽业造成了重大经济损失。虽然某些血清型的APEC并不引起发病，但多数APEC均能引发肠道外感染。其中，O1、O2和O78是引发大肠杆菌病的主要血清型。APEC可通过呼吸道和胃肠道进入血液造成系统感染。
　　近期研究表明，由于APEC血清型众多，抗原结构复杂，大肠杆菌病很难治愈。因此，通过抗原刺激机体天然黏膜免疫系统诱导机体黏膜免疫显得尤为重要。本论文在含有锚定蛋白PgsA的乳酸菌L.casei(Lc393)和L.saerimneri(M11)中融合表达O78血清型APEC的fimA、ompC基因以及微褶皱细胞和树突状细胞靶向肽(M-DC)基因，通过加入T7g10增强子以及M-DC靶向肽提高目的蛋白的表达量，增强目的蛋白的免疫活性。以O78型大肠杆菌基因组为模板，分别用特异性引物A1,A2，C1和C2扩增fimA基因(528bp)和ompC基因(1074 bp)。通过融合PCR链接fimA和ompC基因，构建重组载体pMD19-T-OmpC-fimA。双酶切OmpC-fimA后(Apa1和Sac1)链接M-DC，构建表达载体pPG-T7g10-PPT-OmpC-fimA和pPG-T7g10-PPT-OmpC-fimA-M-DC。将重组质粒电转化入L.casei(Lc393)和L.saerimneri(M11)中，成功构建四株重组菌pPG-T7g10-PPT-OmpC-fimA/M11(POF/M11)、pPG-T7g10-PPT-OmpC-fimA-M-DC/M-11(POFm-dc/M11)、pPG-T7g10-PPT-OmpC-fimA/Lc393(POF/Lc393)和ppG-T7g10-PPT-OmpC-fimA-M-DC/Lc393(POFm-dc/Lc393)。Western blot结果表明分子量分别为101KDa和104.5KDa的外源蛋白成功获得表达。重组菌在低PH值、高胆汁盐浓度的强刺激条件下定植于肠道并从小肠黏膜层排出病原体。
　　本研究在不同时间间隔检测重组菌在回肠、盲肠和结肠的定植能力和吸附能力，在强刺激条件下检测表型特征。结果表明，四株重组菌均能吸附于肠道上皮细胞，相比于POF/Lc393和POF/M11，POFm-dc/Lc393和POFm-dc/M11显著提高了鸡抵抗肠道疾病病原的免疫力，并且显著提高了鸡的生长水平。同时，本研究检测了重组菌POFm-dc/Lc393和POFm-dc/M11在不同段小肠的生理化学特性、黏附能力和定植能力。重组菌作为口服疫苗具有良好的免疫原性，能够对机体产生有效的免疫保护;并且作为益生菌和治疗制剂，能够调节免疫系统功能。并且，L.casei重组菌的融合蛋白表达量及其黏附能力均高于L.saerimnerL重组菌。表达靶向肽和融合蛋白的重组乳酸菌是未来开发针对大肠杆菌病有效口服疫苗的候选菌株，同时为研究重组乳酸菌的治疗制剂和生长调节因子功能指明了新的方向。

目录

声明

摘要

Abstract

1 Introduction

1．1 Pathogenicity of Avian Pathogenic Escherichia coli(APEC)

1．1．1 Epidemiology of Avian Colibacillosis

1．1．2 Virulence factors of(APEC)

1．1．3 Prevention of broiler chickens from APEC infection

1．2 Lactobacillus mueosal immunity progress against cofibacillosis

1．2．1 Mechanism of mucosal immunity

1．2．2 Lactobacillus use as delivery vehicle in vaccination

1．2．3 Expression strategies of recombinant proteins on the cell surface of lactic acid bacteria

1．2．4 Advantages of mucosal immunization

1．3 Specific aims and experimental strategies of the study

2 Materials and Methods

2．1 Matedals

2．1．1 Bacterial strains and plasmids

2．1．2 Primers

2．1．3 Experimental Animals

2．1．4 Chemical reagents

2．1．5 Instrument and equipment

2．2 Methods

2．2．1 Activation of bacteria

2．2．2 E.coli challenge to broiler chickens

2．2．3 Genome Extraction from O78 serotypes(APEC)

2．2．4 FimA Gene Amplification

2．2．5 DNAAgarose Gel Electrophoresis

2．2．6 OmpC Gene Amplification

2．2．7 Ligation of fimA and ompC gene with PMDl9-TS simple vector

2．2．8 Heat shock transformation of plasmid into TG1

2．2．9 Screening and identiffcation of recombinant strains

2．2．10 Development of recombinant lactobaciilus

2．2．11 Extraction of limA and ompC gene from pMDl9-TS

2．2．12 Fusion of fimA and ompC gene with PMDl9-TS

2．2．13 Extraction of positive recombinant plasmids

2．2．14 Confirmation of recombinant plasmid by single and double digestion

2．2．15 Fusion of pMDl9-fimA-ompC gene with M-DC targeting peptide

2．2．16 Confirmation of recombinant plasmid with M-DC cells by single and double digestion

2．2．17 Engineering of expression vector and development of recombinant lactobacillus

2．2．18 Preparation of competent cells from Lactobacillus strains

2．2．19 Electroporation protocol assay

2．2．20 Identification of recombinant lactobacillus strains

2．2．21 Expression and identification of recombinant protein by SDS-PAGE and Western blot

2．2．22 Recombinant Lactobacillus colonization characteristies in the Chicken GI tract

2．2．23 (Pathogenicity of O78 E．coli)

2．2．24 Effect of recombinant laetobacillus against O78 E．coli

3 Results

3．1 Construction of fusion genes FimA and OmpC from O78 E．coli

3．1．1 PCR amplification result of target FimA and OmpC gene

3．1．2 Identification of recombinant plasmid pMDl9-TS-FimA by single and double digestion

3．1．3 PCR Identification of pMDl9-TS-FimA plasmid

3．1．4 Identification of recombinant plasmid pMDl9-TS-OmpC by single and double digestion

3．1．5 PCR Identification of pMDl9-T-OmpC plasmid

3．1．6 Fusion and identification of OmpC gene with PMDl9-TS-FimA by single and double digestion

3．1．7 Confirmation of pMDl9-T-fimA-ompC by PCR amplification

3．2 Construction of recombinant plasmid by fusion of FimA-OmpC with M-Dc cells

3．2．1 PCR of targeting peptide M-DC with PMD19-T-FimA-OmpC genes

3．2．2 Identification of PMD19-TS-OmpC-FimA-M-DC by single and double digestion

3．3 Development and analyzing recombinant plasmid by single and double digestion

3．3．1 PCR identification of recombinant lactobacillus(pPG-T7g10-PPT-OmpC-FimA／M11 and pPG-T7g10-PPT-OmpC-FimA／Lc393)

3．3．2 PCR identification of recombinant lactobacillus(pPG-T7g10-PPT-OmpC-FimA-M-DC／M11 and pPG-T7g10-PPT-OmpC-FimA-M-DC／Lc393)．

3．3．3 Confirmation of empty vector into lactobacillus strains

3．3．4 Protein expression of pPG-T7g10-PPT-OmpC-FimA／Lc393 and pPG-T7g10-PPT-OmpC-FimA／M-11 recombinant lactobacillus with PgsA

3．3．5 Identification of protein expressed by recombinant LAB(with the first antibody of Flag tag)

3．3．6 Protein expression of pPG-T7910-PPT-OmpC-FimA-M-DC／Lc393and pPG-T7910-PPT-OmpC-FimA-M-DC／M-11 recombinant lactobacillus

3．3．7 Identification of protein expressed by recombinant LAB(with the anti Flag tag)

3．4 Analysis of Colonization characteristics of recombinant lactobacillus Within chicken GI tract

3．4．1 Recombinant lactobacillus colonization ability in the chicken GIT

3．5 Recombinant lactobacillus impact on chicken growth production

3．6 Pathogenicity of Oral and Nasal challenge with 078 E．coli

3．6．1 Lesions with E．coli challenge

3．7 Protection Efficacy against O78 E．coli

3．7．1 Analysis of protection Efiicacy of Recombinant Lactobaciilus

4 Discussions

4．1 Physiological characteristics of isolated lactobacillus

4．2 Selection of protective antigen genes

4．3 Electroporation of fusion genes into competent cells

4．4 Recombinant Lactobacillus-based oral vaccine induce systemic immune responses

4．5 Expression of fusion protein(FimA-OmpC)with(M-DC)in Lactobacillus

4．6 Comparison of fusion proteins(FimA-Ompc)and(FimA-OmpC-M-Dc)

4．7 Recombinant Lactobacillus colonization capacity and Growth performance

5 Conclusions

Acknowledgment

References

Papers published in the periods of PhD education