Gene cloning of cdtB from Actinobacillus actinomycetemcomitans and bioactivity test of the recombinant protein CdtB in vitro
- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF STOMATOLOGY
- Issue:
- 8
- DOI:
- 10.3760/cma.j.issn.1002-0098.2010.08.010
- Key Word:
- 放线杆菌,伴放射菌;毒素类,生物学;脱氧核糖核酸酶Ⅰ;基因克隆;Actinobacillus actinomycetemcomitans;Toxins,biological;Deoxyribonuclease Ⅰ;Gene cloning
Abstract： 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-pelyacrylamide 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% ngarose gel electrophoresis test. Conclusions The recombinant plasmid pET-15b-cdtB was successfully constructed and the recombinant CdtB protein which has the Dnase Ⅰ -like activity was obtained.
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