[Construction of eukaryotic expression vector for KGPcd gene from Porphyromonas gingivalis and expression in mammalian cells].
OBJECTIVE
This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.


METHODS
Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.


RESULTS
It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.


CONCLUSIONS
The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.
Zhang
Feng-qiu
FQ
Dept. of Periodontitis, College of Stomatology, The Fourth Military Medical University, Xi'an 710032, China.
Yang
Lian-jia
LJ
Wu
Zhi-fen
ZF
Qin
Hong-yan
HY
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000235:genetics*; D005822:Genetic Vectors; D010957:Plasmids; D016966:Porphyromonas gingivalis / Q000235:genetics*; D014162:Transfection
D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases