[논문]Immunization with a Genetically Engineered Uropathogenic Escherichia coli Adhesin-Escherichia coli Enterotoxin Subunit A2B Chimeric Protein

Lee, Yong-Hwa; Kim, Byung-O; Pyo, Suhk-Neung

Immunization with a Genetically Engineered Uropathogenic Escherichia coli Adhesin-Escherichia coli Enterotoxin Subunit A2B Chimeric Protein 원문보기

The journal of applied pharmacology : the official journal of the Korean Society of Applied Pharmacology, v.13 no.2, 2005년, pp.101 - 106

Lee, Yong-Hwa (Division of Immunopharmacology, College of Pharmacy SungKyunKwan University) , Kim, Byung-O (College of Life Science and Natural Resources, Sanju National University) , Pyo, Suhk-Neung (Division of Immunopharmacology, College of Pharmacy SungKyunKwan University)

Abstract ▼ AI-Helper

The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.

주제어

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문제 정의

The expression, purification, and characterization of a recombinant protein containing Adhesin/LTXA2B is described. This study also demonstrates that peroral administration of this chimeric imm_unogen in mice elicited a high level of secretory IgA and serum IgG antibodies to the UPEC adhesin.

제안 방법

Five female BABL/c mice in each group were immunized three times at 10-day intervals by oral adminstration with 100 jig and 25 gg of the puriHed adhesin/LTXA2B chimeric protein, 30 gg of the purified adhesin alone, and 25 μg of purified ETXA2B alone. The control mice were only given sterile PBS.
The primers used to amplify the ltxa2b gene were 5-AGCTGGATCCGAAGAGCCGTGGATT-3 (underline containing BamHI site) and 5 -CCCCAAGCTTCTAGTTTTC- CATACTGATTGC-3 (underline containing Hindill). The amplified fimH and ltxa2b genes digested with BamUl and HindHI and inserted into the expression vector pMAL-p2E to construct the pMALfimH and pMALlixa2b, ,respectively. pMALltxa2b was digested with HindHI and BamHI, then amplified 599 bp DNA fragment was ligated into the pMAL/zw/f vector.
coli TB1 carrying pMALfimH/ltxa2b. The cell lysate proteins (lane 2-10) from E. coli TB1 carrying eMKLfimH/ltxa2b after 0, 2, 4, 6, 8, 10, 12, 24, and 48 hours of induction with 0.01 mM of IPTG were resolved by electrophoresis on 7% (A) and 15% (C) polyacrylamide gels. The gels were stained with Coomassie blue.

데이터처리

coll adhesin and LTXB in vaginal wash and serum samples taken from individual vaccinated mice were meas_ured by ELTSA. Levels of the slgA antibody response to E. coll adhesin with adhesin/LTXA2B were greater than those found with adhesin alone or LTXA2B alone (P<0.05 by Student's t test) (Fig. 3). A minimal effect was observed when adhesin alone was used.
The differences in the antibody levels in immunized and non-immunized mice were evaluated using the Student's t test. The differences were considered significant for P<0.

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