Cloning and expression of recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit and Actinobacillus actinomycetemcomitans fimbria associative protein

Objectives:Actinobacillus actinomycetemcomitans fimbria associative protein(fap) has been implicated to play an important role in initiating and developing periodontal disease. Escherichia coli heat-liable enterotoxin B subunit (ltb) is a kind of muscosal adjuvant which can enhance the mucosal immunogenicity. The Objective is to obstain fusion protein Ltb-fap. Tow couple of primers were designed for PCR according to the known sequence of LTB and fap respectively. Methods:The LTB and fap gene were obstained by amplification PCR technique from plasmid EWD299 of E.coli and A.a DNA respectively, and fused by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28altb-fap were transformed into E.coli BL21(DE3) cells and identified by restriction enzyme and sequence. After induced by IPTG at 37°æ, the recombined fusion protein ltb-fap was expressed in inclusion body, and were assayed by SDS-PAGE and Western-blot. Results:The result showed the LTB-fap were obstained successfully. Recombined E.coli fusion protein ltb-fap were obstained. and found to be 20000 of relative molecule weight band in size. The expression fusion protein were identified by SDS-PAGE and Westen-blot,which were about 15% of the total cell protein. Conclusions:The study concluded that recombined E.coli fusion protein ltb-fap can be expressed in E.coli hich is the foundation of the next step of anti-adhesion experiment of A. actinomycetemcomitans.