[Construction of prokaryotic expression vector of FimA gene from Porphyromonas gingivalis, fusion expression and purification in E. coli BL21(DE3)pLyS].
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 beta-D-1-thiogalactopyranoside (IPTG). With anti-6xHis Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co(2+)-NTA affinity chromatography.


RESULTS
Cloned FimA gene sequences and 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 x 10(4) has been expressed. Co(2+)-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein.


CONCLUSIONS
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.
Li
Ang
A
Research Center for Stomatology, College of Stomatology, Xi'an Jiaotong University, Xi'an 710004, China.
Xie
Hong-Guon
HG
Liang
Ping
P
Zhu
Chun-Hui
CH
Shi
Jian-Feng
JF
Rao
Guo-Zhou
GZ
Gou
Jian-Zhong
JZ
D003001:Cloning, Molecular; D004926:Escherichia coli; D016966:Porphyromonas gingivalis; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins