Deletion of cagA gene of Helicobacter pylori by PCR products.
OBJECTIVE
Cytotoxin-associated protein (antigen) A (CagA) plays an important role in Helicobacter pylori (H pylori) pathogenesis. Our aim was to obtain cagA- mutant strains by a new mutation method so as to better understand the mechanism of CagA in epithelial cells.


METHODS
In contrast with the traditional method using suicide plasmid, we constructed cagA- mutant strains directly with PCR products. The constructed mutant clones grew on selective media and allelic exchange was confirmed by Southern blot. Furthermore, two different transformation methods, electroporation, and natural transformation, were compared with regard to the efficiency of recombination.


RESULTS
The mutation by PCR products could be completed within 3-5 d, and the recombination rate by electroporation and natural transformation was 4.02 x 10(-8) and 1.03 x 10(-9) respectively. Mutation rate by electroporation (4.02 x 10(-8)) was far higher than by natural transformation (1.03 x 10(-9)) (P = 0.000<0.005).


CONCLUSIONS
cagA- mutant strains have been constructed, which is important for further study on the function of CagA in epithelial cells. A mutation method by directly using PCR products has been proved successful with a much higher mutation rate, and is easier, especially when in combination with electroporation. This method could be widely used in gene deletion of H pylori.
Zeng
Xun
X
Department of Diagnostics, National Institute of Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, PO Box 5, Changping, Beijing 102206, China.
He
Li-Hua
LH
Yin
Yan
Y
Zhang
Mao-Jun
MJ
Zhang
Jian-Zhong
JZ
D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D017353:Gene Deletion; D016480:Helicobacter pylori / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C080866:cagA protein, Helicobacter pylori