Construction of the markless clpP-deletion mutant of Streptococcus mutans / 中华微生物学和免疫学杂志

Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

Construction of AT2R gene recombinant adenovirus by an efficent method / 医学研究生学报

Objective:To construct the recombinant adenovirus of AT2R by using the method of homogenous recombination in bacteria. Methods:AT2R gene was get from the vector of PUHD-AT2R by PCR ,and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/AT2R.Then it was linearized with PmeⅠ and transformed into Adeasier-1 cell. The DNA of identified recombinant plasmid was digested with PacⅠ and transfected to 293cells to package adenovirus. The PCR technique was used to detect target gene . The titre of the Ad-AT2R was measured with the aid of GFP expression. Results:PCR test indicated each the recombinant adenovirus contained the insert of AT2R. The titre of purified recombinant adenovirus was 1.5?10 12pfu/ml. Conclusion:The method of homologous recombination in bacteria is more convenient and efficient compared with that in cell .The prepared Ad-AT2R paves a sound foundation for further study.