Korean J Lab Med.  2006 Apr;26(2):98-102. 10.3343/kjlm.2006.26.2.98.

Production of Antibody against Helicobacter pylori HP0231

• Lee J
• Lee SY
• Lee JH

• KMID: 2238898
• DOI: http://doi.org/10.3343/kjlm.2006.26.2.98

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.

Keyword

Helicobacter pylori HP0231; Stool Antigen Detection; Antibody; Specificity; Sensitivity

MeSH Terms

Antibodies
Biopsy
Blotting, Western
Cell Extracts
Chromatography, Affinity
Clone Cells
Databases, Nucleic Acid
Diagnosis
DNA
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Gastritis
Genetic Variation
Helicobacter pylori*
Helicobacter*
Humans
Immunization
Peptic Ulcer
Polymerase Chain Reaction
Rabbits
Sensitivity and Specificity
Stomach
Antibodies
Cell Extracts
DNA

Figure

• 

  Fig. 1. Induction of HP0231 protein (35 kDa) after IPTG addition. Lane 1, Molecular weight marker; Lane 2, before Induction; Lane 3, 1 hr after induction; Lane 4, 2 hr after induction; Lane 5, 3 hr after induction; Lane 6, 4 hr after induction; Lane 7, 5 hr after induction.

• 

  Fig. 2. Purification of overexpressed HP0231 protein using Ni-NTA column. Lane 1, Whole cell lysate; Lane 2, Flow-through fraction from Ni-NTA column; Lane 3, 1st Wash fraction from Ni-NTA column; Lane 4, 2nd Wash fraction from Ni-NTA column; Lane 5, 1st Elution fraction from Ni-NTA column; Lane 6, 2nd Elution fraction from Ni-NTA column; Lane 7, 3rd Elution fraction from Ni-NTA column; Lane 8, 4th Elution fraction from Ni-NTA column.

• 

  Fig. 3. Western Blot Analysis for Specificity of Rabbit Anti-HP0231 Antibody. Lane 1, E. coli BL21 (DE3); Lane 2, H. pylori cell extract; Lane 3, Stomach biopsy tissue extract; Lane 4, HP0231 protein.

• 

  Fig. 4. Sensitivity of Rabbit Anti-HP0231 Antibody.

Reference

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