Production of Antibody against Helicobacter pylori HP0231 / 대한진단검사의학회지

ABSTRACT

BACKGROUND: Stool antigen detection kits for diagnosis of infection of Helicobacter pylori have been widely used for their convenience, but are mostly imported. Since Helicobacter pylori strains show a distinctive genetic diversity, it is important to find a protein that is a common antigen among various strains and shows a strong immunogenicity for the development of a stool antigen detection kit. HP0231 protein strongly reacts with the sera of patients suffering from gastritis and peptic ulcer. Therefore, HP0231 is an excellent candidate as a target gene for this study. METHODS: Chromosomal DNA from H. pylori was isolated. HP0231 gene was amplified by PCR, cloned into pET28a(+) vector, and overexpressed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). HP0231 protein was purified by Ni-NTA affinity chromatography followed by electroelution after SDS-PAGE. Rabbits were immunized with the purified HP0231 protein for the production of antibodies. Rabbit anti-HP0231 antibody was partially purified and tested for the sensitivity and specificity using ELISA and Western Blot Analysis. RESULTS: The sequence of the cloned HP0231 gene was identical with the gene sequence from Genbank (AA216016). HP0231 gene was overexpressed and HP0231 protein was purified. Rabbit anti-HP0231 antibody produced after immunization with the purified HP0231 protein reacted with the purified HP0231 protein, cell extracts from cultured H. pylori, and stomach biopsy tissue from patients, but not with cell extracts from cultured E. coli used as a negative control. After 1 million fold dilution, rabbit anti-HP0231 antibody still reacted with 1 microgram of HP0231 protein. CONCLUSIONS: Rabbit anti-HP0231 antibody was produced to detect HP0231 protein of H. pylori and will be tested for the development of a stool antigen detection kit for H. pylori.