Deletion of cagA gene of Helicobacter pylori by PCR products

Abstract

Aim: 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).

Conclusion: 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.

Figure 1

Strategy for constructing fragments with chloramphenicol resistance gene and gene allelic replacement by PCR. Primers P1-P2, P3-P4 and C1-C2 were amplified and each PCR products were purified. Each three PCR products were mixed and amplified by primer P1 and P4. Then, the PCR product P1-4 was acquired containing three fragments.

Figure 2

PCR amplification for constructing fragments with chloramphenicol resistance gene and gene allelic replacements. The length of cagA/P1-4, a 1.7-kb fragment, is equal to the sum of the length of cagA/P1-2, cagA/P3-4, and cam.

Figure 3

A: Result of PCR amplified for diagnosing homologous recombination strains of cagA gene. Lanes 1-12 are 12 clones selected from chloramphenicol-resistant plates. Control is 26695 wild type. Twelve clones were all negative for diagnostic primers B1-2 while control is positive, suggesting that the flanking region of cagA gene of all the selected clones was deleted; B: Result of PCR amplified for positive control of homologous recombination strains of cagE gene. Lanes 1-12 are 12 clones selected from chloramphenicol-resistant plates. Control is 26695 wild type. Twelve clones were all positive of cagE gene; C: Result of Southern blot for diagnosing homologous recombination strains of cagA gene. Lanes 1-12 are 12 clones selected from chloramphenicol-resistant plates. Control is 26695 wild type. All the 12 clones except 3, 5, and 9 were positive hybridization with probes of chloramphenicol-resistant gene while control was negative.

Figure 4

Mechanism of allelic fragments recombination used by suicide plasmid. The recombination consists of two steps. In the first step, suicide plasmid with PCR products to be exchanged recombined with one allelic fragment and formed a loop. Then, the suicide plasmid recombined with another allelic fragment.