[Construction of  eukaryotic co-expression vector   of Porphyromonas gingivalis outer membrane protein ragB and glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL)   and its immunogenic analysis].
OBJECTIVE
To construct eukaryotic co-expression vector of  Porphyromonas gingivalis outer membrane protein ragB and mouse glucocorticoid-induced tumor necrosis factor receptor ligand (mGITRL)   and to analyze its immunogenicity in vivo.


METHODS
The ragB gene was obtained from pMD18-T-ragB,  and then cloned into the eukaryotic expression vector pIRES and pIRES-mGITRL,  respectively. The eukaryotic expression vectors:  pIRES-ragB and pIRES-ragB-mGITRL were identified by double enzyme digestion and DNA sequencing,  then transfected into COS7  cells by Lipofectamine(TM);2000. The expressions  of ragB or mGITRL in COS7 cells were  detected by Western blotting. The mice were immunized with the recombinant pIRES-ragB-mGITRL plasmid. The serum  antibody level was determined by ELISA.


RESULTS
pIRES-ragB and pIRES-ragB-mGITRL plasmids were successfully constructed. Western blotting showed that the targeted gene was over-expressed in COS7 cells and skeletal muscle cells,  respectively. The high titers of antibodies against RagB were detected in mouse serum.


CONCLUSIONS
The construction of pIRES-ragB-mGITRL co-expression vector provides the experimental basis for Porphyromonas gingivalis vaccine research,  prevention and treatment of periodontitis.
Jiangsu University, Zhenjiang, China. kfz108@sohu.com
Fan-zhi
FZ
Kong
Dong
D
Zheng
Zhao-liang
ZL
Su
Peng
P
She
Xin-xin
XX
Xu
Sha-sha
SS
Tian
Pan
P
Zhang
Jian-guo
JG
Chen
Hua-Xi
HX
Xu
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D023061:Gene Order; D051379:Mice; D010957:Plasmids / Q000235:genetics; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000276:immunology; D014162:Transfection; D048069:Tumor Necrosis Factors / Q000235:genetics* / Q000276:immunology*
D001426:Bacterial Proteins; C484346:RagB protein, bacteria; C478966:Tnfsf18 protein, mouse; D048069:Tumor Necrosis Factors