Prokaryotic expression and antigenicity of the CagA gene in Helicobacter pylori.
10.3969/j.issn.1672-7347.2010.08.012
- Author:
Xiong CHEN
1
;
Canxia XU
;
Fen WANG
;
Shourong SHEN
;
Yan JIA
Author Information
1. Department of Gastroenterology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Antigens, Bacterial;
genetics;
immunology;
Bacterial Proteins;
genetics;
immunology;
Base Sequence;
Genetic Vectors;
Helicobacter pylori;
genetics;
immunology;
Molecular Sequence Data;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
immunology
- From:
Journal of Central South University(Medical Sciences)
2010;35(8):847-853
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To synthesize the specific CagA gene segment of the gastric cancer idiotype Helicobacter pylori (H. pylori), establish the prokaryotic expression system and identify the antigenicity sequence of recombination signals.
METHODS:We selected the CagA fragment which was related to gastric cancer in our earlier research. The CagA gene segment was optimized and synthesized. The synthesized CagA gene was cut from the pUC57-CagA plasmid and then was carried by expression vector pET32a to be transformed into the host bacterium BL21 (DE3). The positively cloned pET32a-CagA was selected by receptivity of aniline and colony PCR. The host bacterium with pET32a-CagA was induced by IPTG to express fusion protein. The expression of CagA protein was analyzed by SDS-PAGE gel electrophoresis and the antigenicity of fusion protein was examined by Western blot.
RESULTS:CagA gene segment was designed and synthesized. The sequence of synthesis CagA gene segment was the same as the one designed before (AF289435). We successfully constructed the plasmid of prokaryotic expression of the pET32a-CagA. Homology of the target CagA proteinum was 100%, the same as AAG09884. The host bacterium BL21 (DE3) containing pET32a-CagA could express CagA fusion protein after the IPTG induction. SDS-PAGE gel electrophoresis showed that the molecular weight of fusion protein was the same as expected (45 kD). Western blot showed that the fusion protein could be combined with the antibody of the whole bacterium of anti-H. pylori.
CONCLUSION:The synthesized CagA fusion protein from the prokaryotic expression system has antigenicity. We hope to set the foundation for selecting the strain in H. pylori correlated to gastric cancer and corresponding therapy in clinical practice.
