| Periodontitis is one of the most common chronic progressive infectious diseases of humans. Localized aggressive periodontitis (LAP) can invade and destroy the periodontal tissue, progress more rapidly,lead to destruction of the periodontal connective tissue. Actinobacillus actinomycetemcomitans (A.a) is considered to be the most important microorganism in LAP. LAP is a disease destructing the periodontal ligament and alveolar bone, resulting in tooth loss in permanent incisors and first molars. The therapy of medicine and mechanism are used to treat periodontitis at present.The curative effect is not very good. It is the trend to treat peridontitis by antibody against bacteria. The mechanisms behind the pathogenicity of A. actinomycetemcomitans are not known. A. actinomycetemcomitan possesses a mumber of virlence factors, but colonization and penertration across gingival tissue of this microorganism are critical first steps in periodontal disease. Previous stufies have demonstrated its ability to attach to epithelial cells. The attachment behavior of the microorganism play an important role in initiating and developing periodontal disease. So we attempted to inhibit the adhesion of A. actinomycetemcomitans to prevent the periodontitis. Fimbrial associated protein (fap) is the major fimbrial subunit,it can induce rabbit to produce antibody against A. actinomycetemcomitan. LTB is E.coli heat-labile enterotoxin,it also is a kind of mucosal immunoadjuvant to enhance the immune response of co-antigen which were applied at the same time.We want construct a fusion protein of ltb and fap by a linker to immune organism to inhibit the adhesion of A. actinomycetemcomitans. The fields of this research is following: 1 The e.coli heat-labile enterotoxin B subunit gene was amplified from the plasmid EWD299 by the method of PCR,and cloned into the expressing vector pET28a.The recombinant plasmid was then transformed into the component cell BL21(DE3)plyss.Induced with IPTG,these cells expressed the recombinant protein LTB,identified by SDS-PAGE and Western-blot.The amount of the expressed recombinant protein was about 38% of the total cells scaled by gel thin scanner instrument. CS-9000. 2 The gene of toxB encoding e.coli heat-labile enterotoxin B subunit in the plasmid EWD299 and the fap gene of Actinobacillus actinomycetemcomitans(A.a) were amplified from EWD2992 and A.a, respectively ,the two gene were linked with a linker by the way of PCR and cloned into the expressing vector pET28a,the recombinant plasmid was then transformed into BL21(DE3)plyss.Induced with IPTG,the recombinant bacterium could express recombinant protein LTB-(Gly4Ser)2-FAP,identified by SDS-PAGE and Western-blot.The total amount of the protein was about 15% of the total cell protein determined by gel thin scanner. 3 The recombinant plasmids were transformed into BL21(DE3)plyss.The fusion protein LTB-FAP of inclusion bodies was acquired. The urea concentration to wash and purify protein was confirmed. The recombinant protein LTB-FAP was purified with DEAE Sophaced amino exchange resin and Sephrase 6B.4 The purpose of this study is to certify the ability of the recombinant protein LTB-FAP ,which could induce rabbit to produce an anti-fimbrial significant antibody and inhibit adhesion of Actinobacillus actinomycetemcomitans. The antigen of the recombinant protein LTB-FAP was used to immunize rabbits. The titter of antibody in rabbits for the recombinant protein LTB-FAP were tested by enzyme-linked immnosorbent assay(ELISA). A rabbit antiserum was used to inhibit the attachment of A.actinomycetemcomitans to buccal epithelial cells. We got the rabbit antiserum to the recombinant protein LTB-FAP with significant titter, This antiserum strongly inhibited the attachment of fimbriated A.actinomycetemcomitans 310-a to buccal epithelial cells. The antigen of recombinant protein LTB-FAP may be effective in inducing antibodies which could inhibit the adhesion and subsequent colonization of A.actinomycetemcomitans.It is significant to the dev...
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