[Gene cloning of cdtB from Actinobacillus actinomycetemcomitans and bioactivity test of the recombinant protein CdtB in vitro].
OBJECTIVE
To construct the prokayotic expression vector pET-15b-cdtB containing the cdtB gene from Actinobacillus actinomycetemcomitans (Aa) and to test the bioactivity of this recombinant CdtB in vitro.


METHODS
The toxic cytolethal distending toxin (CDT) subunit encoding gene cdtB was amplified by PCR. Through restriction endonuclease digestion, gene cdtB and vector pET-15b were ligated to form pET-15b-cdtB expression system which was transformed into competent cells Escherichia coli BL21 (DE3). Protein expression was induced by isopropyl-beta-D-thiogalactoside and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Supercoiled plasmid pET-32a DNA was incubated with purified recombinant CdtB protein in vitro to view any changes in the electrophoretic mobility of the plasmid pET-32a DNA band.


RESULTS
PCR testing results of pET-15b-cdtB transformed cells demonstrated that all strains contained cdtB gene. The DNA sequence was blast with cdtB gene from GenBank and 99% homology was obtained. Both of SDS-PAGE and Western blotting confirmed that recombinant CdtB was obtained. After incubated with the purified recombinant CdtB in vitro, the supercoiled plasmid pET-32a DNA was observed relaxing by 1% agarose gel electrophoresis test.


CONCLUSIONS
The recombinant plasmid pET-15b-cdtB was successfully constructed and the recombinant CdtB protein which has the DNaseI-like activity was obtained.
Institute of Stomatology, Nanjing Medical University & Department of Periodontology, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing 210029, China.
Lu
L
Li
Jun-lan
JL
Duan
Xiao-qian
XQ
Wang
Mi-fang
MF
Yang
Yan
Y
Xu
D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins
D001427:Bacterial Toxins; D011994:Recombinant Proteins; C067044:cytolethal distending toxin; C447141:cytolethal distending toxin, Actinobacillus actinomycetemcomitans