关键词: 牙龈卟啉单胞菌, 菌毛蛋白A, 融合表达

Abstract:

Objective To clone the FimA gene of fimbriae from Porphyromonas gingivalis（P.gingivalis） and to construct prokaryotic expression vector which was induced in E.coli BL21（DE3）pLyS in the form of fusion protein expression and to identify, purify the product of its expression. Methods To clone the FimA gene of fimbriae from P.gingivalis and to construct prokaryotic expression vector pET15b-FimA vector which was transformed into the competent cells of BL21（DE3）pLyS. The expression of fusion protein was induced by isopropyl β-D-1-thiogalactopyranoside（IPTG）. With anti-6×His Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co2+-NTA affinity chromatography. Results Cloned FimA gene sequencesand inserted into expression vector of the FimA sequences were related to the sequence in GenBank database showed 100% homology. IPTG induced and then identified by Western blot showed a fragment of 4.1×104 has been expressed. Co2+-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein. Conclusion The recombinant prokaryotic expression vector of pET15b-FimA was constructed and was expressed and purified successfully in E.coli BL21（DE3）pLyS. This study laid the experimental foundation to further prepare for monoclonal antibodies of fimbriae of P.gingivalis and to develop the subunit protein vaccine of prevention of periodontitis.

Key words: Porphyromonas gingivalis, FimA, fusion expression