Gene cloning of cdtB from Actinobacillus actinomycetemcomitans and bioactivity test of the recombinant protein CdtB in vitro / 中华口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>The recombinant plasmid pET-15b-cdtB was successfully constructed and the recombinant CdtB protein which has the DNaseI-like activity was obtained.</p>