Expression of Helicobacter pylori AlpA protein and its immunogenicity.
OBJECTIVE
To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA.


METHODS
Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 mug/mL ampicillin overnight at 37 degrees. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity.


RESULTS
The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of beta-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected.


CONCLUSIONS
Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.
Xue
Jing
J
PLA Institute for Digestive Medicine, Nanfang Hospital, The First Military Medical University, Guangzhou 510515, Guangdong Province, China. xj0302@fimmu.com
Bai
Yang
Y
Chen
Ye
Y
Wang
Ji-De
JD
Zhang
Zhao-Shan
ZS
Zhang
Ya-Li
YL
Zhou
Dian-Yuan
DY
D018829:Adhesins, Bacterial / Q000235:genetics* / Q000276:immunology*; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001428:Bacterial Vaccines / Q000235:genetics* / Q000276:immunology; D016481:Helicobacter Infections / Q000517:prevention & control* / Q000628:therapy; D016480:Helicobacter pylori / Q000235:genetics* / Q000276:immunology; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D018829:Adhesins, Bacterial; C470179:AlpA protein, Helicobacter pylori; D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins