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«Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in bacterial strains HOANG VAN ...»

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Expression of recombinant T-cell epitopes of major

Japanese cedar pollen allergens fused with

cholera toxin B subunit in bacterial strains

HOANG VAN VINH

A dissertation submitted to

Kochi University of Technology

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

The Graduate School of Engineering

Kochi University of Technology

Kochi, Japan

September, 2014

-1Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in bacterial strains HOANG VAN VINH

TABLE OF CONTENTS

Abstract Chapter I. General introduction Chapter II. Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli

2.1. Introduction

2.2. Materials and Methods 2.2.1. Bacterial strains, plasmid, and reagents 2.2.2. Construction of cry j 1 epi and cry j 2 epi using ctb as a scaffold 13 2.2.3. Construction of fusion gene BamHI-ctb-linker-cry j 1 epi-cry j 2 epi-flag-HindIII 2.2.4. Construction of the recombinant plasmid for E. coli 2.2.5. SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting 18 2.2.6. Expression and purification of antigen peptides

2.3. Results 2.3.1. Selection of major T-cell epitopes from Cry j 1 and Cry j 2 2.3.2. Construction of the fusion gene BamHI-ctb-linker-cry j 1 epi-cry j 2 epi-flag-HindIII 2.3.3. Expression and purification of the fusion antigen peptide 2.3.4. Western blot analysis of the fusion antigen peptide

2.4. Discussion Chapter III. Expression of recombinant T-cell epitopes of major Japanese cedar

-3pollen allergens fused with cholera toxin B subunit in Brevibacillus choshinensis

3.1. Introduction

3.2. Materials and Methods 3.2.1. Bacterial strains, plasmid, and reagents 3.2.2. Construction of fusion genes by PCR amplification 3.2.2.1. Construction of BamHI-BLA-stop-terminator-HindIII 3.2.2.2. Construction of fusion gene BamHI-ctb-linker-cry j 1 epi-cry j 2 epi-flag-stop-terminator-HindIII 3.2.2.3. Construction of fusion gene BamHI-BLA-linker-ctb-linker-cry j 1 epi-cry j 2 epi-flag-stop-terminator-HindIII 3.2.3. Construction of recombinant plasmids and transformation 3.2.4. Expression of target proteins by Brevibacillus recombinants 3.2.5. SDS-PAGE 3.2.6. Western blotting

3.3. Results 3.3.1. Construction of fusion genes 3.3.2. Expression and purification of fusion antigen peptides 3.3.2.1. Expression of CTB-Linker-Cry j 1 epi-Cry j 2 epi-Flag 3.3.2.2. Expression and purification of BLA

–  –  –

3.3.3. Western blot analysis of the fusion antigen peptide 50

3.4. Discussion Chapter IV. Conclusions References Acknowledgement Appendix

–  –  –

Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant and carrier for the effective delivery of antigen peptide, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem by overlap extension PCR. Then, the fusion gene was expressed in two expression systems using Escherichia coli strain BL21(DE3) and Brevibacillus choshinensis, respectively.

PART-I Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli Fusion gene of BamHI-ctb-linker-cry j 1 epi-cry j 2 epi-flag-HindIII was constructed by linking five sequences, including BamHI-ctb, linker, cry j 1 epi, cry j 2 epi, and flag-HindIII, using stepwise and overlap extension PCR methods. Then, the fusion gene was introduced into a pET-28a(+) vector for expression in an Escherichia coli system that exhibits various advantages such as potentially high expression levels, low cost, simple culture conditions, rapid growth, and scalability. In addition, it is known that E. coli expression system exhibited almost no proteolytic activity. Thus, the structural stability of expressed recombinant protein is guaranteed. The aim of this study using E. coli as host was to investigate expression of the recombinant antigen peptide in bacterial expression system as well as to examine property and antigenicity of the recombinant

–  –  –

Expression of the recombinant fusion antigen peptide induced with 1 mM IPTG continued for 3-5 h, and then the concentration of the expressed protein decreased gradually. Recovery of the recombinant protein was approximately 120 mg/L of culture. The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant antigen peptide also proved antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Thus it was shown that the whole recombinant protein was expressed and existed stably in E. coli expression system.





The present study indicates that production of sufficient amounts of recombinant protein for immunotherapy may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.

PART-II Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Brevibacillus choshinensis E. coli expression system is the most commonly used organism for heterologous protein production. However, there is a disadvantage for therapeutic use of expressed recombinant protein in E. coli due to the presence of endotoxin, lipopolysaccharide (LPS) which locates in the outer membrane of Gram-negative bacteria. Endotoxin that is well-known as a pyrogen causes fever, shock and other various symptoms in humans and animals. Therefore, recombinant proteins produced in E. coli must be purified until they become endotoxin-free.

In contrast to E. coli, the expression of recombinant antigen peptide in Brevibacillus choshinensis (Bacillus brevis) seems to be an interesting alternative system. Gram-positive bacterium B. choshinensis is a well-established host-vector system for the production of foreign proteins, especially secretory proteins. Thus, the expressed recombinant protein can be easy

–  –  –

extracellular proteases. Therefore, the secreted and produced recombinant protein is not degraded by the protease activity and the yield of the recombinant protein can be maintained stably. These unique characteristics of B. choshinensis make it one of the most promising hosts for the production of recombinant proteins.

In this study, B. choshinensis and pNC-HisT were used as a host for overproduction and as an expression-secretion vector, respectively. Fusion antigen gene of BamHI-ctb-linker-cry j 1 epi-cry j 2 epi-flag-stop-terminator-HindIII was constructed by stepwise PCR and overlap extension PCR methods to investigate the expression of the fusion antigen peptide CTB-Linker-Cry j 1 epi-Cry j 2 epi-Flag. The recombinant B. choshinensis was grown under various culture conditions. However, expression of the recombinant fusion antigen peptide was not detected by SDS-PAGE analysis.

Therefore, to utilize the ability of secretion of Bacillus licheniformis α-amylase (BLA) in B. choshinensis, the fusion antigen was linked to BLA to design a genetically engineered fusion antigen gene BamHI-BLA-linker-ctb-linker-cry j 1 epi-cry j 2 epi-flag-stop-terminator-HindIII.

Then, the fusion antigen genes were introduced into pNC-HisT vectors and expressed in B.

choshinensis. Unexpectedly, only BLA was secreted to the culture medium, suggesting that BLA was cleaved from the recombinant fusion protein by proteolytic activity of unidentified protease(s). When the recombinant B. choshinensis was cultured in the presence of protease inhibitors, whole recombinant protein and degradated antigen peptide were detected in the bacterial cells by western blot with anti-CTB and anti-FLAG antibodies.

These results indicate that the recombinant protein could not exist stably in intracellular space of B. choshinensis due to cellular proteolytic activity. In addition, it is suggested that unique proteolytic cleavage between BLA and CTB might occur during the secretion process of the recombinant protein.

–  –  –

Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common IgE-mediated type I allergies in Japan, causing allergic rhinitis, conjunctivitis, and asthma as clinical symptoms. Approximately 27% of the Japanese population is afflicted by this disease from February to April each year [1,2]. Two major allergenic proteins have been isolated and characterized from CJ pollen, Cry j 1 [3,4] and Cry j 2 [5-7]. In a clinical study, IgE antibodies specific to Cry j 1 and Cry j 2 in sera were detected in 134 of 145 (92%) patients suffering from CJ pollinosis, while the remainder contained IgE reactive to one of the two major antigens [8].

This study suggested that both Cry j 1 and Cry j 2 play important roles in the pathogenesis of CJ pollinosis.

A promising approach to prevent and treat allergies is desensitization by vaccination with peptides derived from allergens. To avoid allergic reactions due to the presence of allergen-specific IgE-binding sites in the whole antigen, novel antigens have been developed that lack epitopes reactive to IgE. The application of peptides only containing T-cell epitopes that induce T-cell tolerance is a safe treatment strategy to control allergies [9,10]. However, a major obstacle with this approach is the diversity of MHC class II molecules among individuals, leading to patients with different MHC class II molecules responding to unique allergen-derived peptides [11]. Therefore, as many T-cell epitopes of allergens as possible should be included to achieve sufficient efficacy in a larger population of sensitized patients [12]. T-cell epitopes in Cry j 1 and Cry j 2 were determined by epitope mapping using synthetic peptides covering their amino acid sequences, followed by proliferation assays using these synthetic peptides and peripheral blood mononuclear cells (PBMCs) from CJ pollinosis sufferers [13-15].

Thus far, recombinant peptides consisting of multiple linked T-cell epitopes from Japanese cedar allergenic proteins have been developed, and basic immunological studies have revealed

–  –  –

transgenic organisms that express recombinant allergens including T-cell epitopes from Japanese cedar pollen. Takaiwa and colleagues reported transgenic rice seeds containing T-cell epitopes in Cry j 1 and Cry j 2, which were designed as an oral (edible) vaccine [17-19]. Using a different approach, egg white containing T-cell epitopes has been produced by transgenic chickens [20].

Another report has described Lactobacillus plantarum producing Cry j 1 and its prophylactic effect in vivo [21]. However, few T-cell epitopes of Cry j 1 and Cry j 2 have been included in previously developed immunotherapeutic peptides. In this study, we selected four major T-cell epitopes from Cry j 1 and six from Cry j 2 based on the ability of the epitopes to stimulate a strong proliferative response in T-cell lines [12,15].

Cholera toxin (CT) is secreted by the gram-negative bacterium Vibrio cholera. CT is an oligomeric protein with molecular weight of 84 kDa containing a single A subunit (CTA), and five B subunits (CTBs). CTA with molecular weight of 27 kDa is composed of the toxic domain CTA1 and a short sequence CTA2 which are generated by proteolytic cleavage of CTA between residues 192 and 195. These two peptide chains are still connected by a disulfide bond between residues 187 and 199. Before CTA1 enter the cytosol of host cells, the disulfide bond must be cleaved. CTB is a peptide of 11.5 kDa and forms a stable pentamer through non-covalent interaction. The CTB pentamer binds ganglioside GM1 on the plasma membranes [22]. Then the CTB-GM1 complex carries the CTA1 into the endoplasmic reticulum [23]. The three-dimensional structure of CT is shown in Fig. 1.1

–  –  –

CTB has been used as an efficient carrier molecule to generate mucosal immune responses and induce T-cell tolerance to antigens linked to CTB [25-27]. CTB binds with high affinity to the ganglioside GM1 that is found in membrane microdomains on the plasma membrane of host cells, and is able to cluster five GM1 molecules at once [28]. Owing to this property of CTB, it can be employed as an adjuvant and transporter for effective delivery of antigens as a mucosal vaccine with reduced toxicity and high efficacy [27].

–  –  –

2.1. Introduction Production of edible vaccines by plants is a significant innovation because plant-derived vaccines offer advantages such as low cost and easy control of production scale [17,29,30].

However, a long growth period and the possibility of gene diffusion to surrounding plants by pollination limit the application of antigen-producing transgenic plants. Therefore, expression of vaccines using E. coli is one of the most attractive alternatives because its genetics and physiology are well understood and recombinant proteins can be produced at up to 50% of the total cellular protein [31].

CJ pollinosis is one of the major allergic diseases in Japan [1,2]. Allergen-specific immunotherapy is safer and more effective than conventional immunotherapy for the treatment of IgE-mediated allergic diseases [32,33]. Peptide vaccines using T-cell epitopes would be an effective and safe immunotherapy for allergic diseases, because recombinant antigen peptides can be designed by selecting T-cell epitopes that lack IgE-binding activity [9,10].



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