Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli.
- Author:
Ang LI
1
;
Hong-yan XU
;
Jian-feng SHI
;
Chun-hui ZHU
;
Guo-zhou RAO
;
Jian-zhong GOU
Author Information
- Publication Type:Journal Article
- MeSH: Cells, Cultured; Cloning, Molecular; Cloning, Organism; Escherichia coli; Genetic Vectors; Glyceraldehyde; Oxidoreductases; Phosphates; Polymerase Chain Reaction; Porphyromonas gingivalis
- From: West China Journal of Stomatology 2011;29(2):199-202
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli.
METHODSGAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density.
RESULTSDNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed.
CONCLUSIONThe GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.
