[Gene cloning and expression of outer membrane protein of Helicobacter pylori].
ABSTRACT To construct a recombinant vector which expresses the 18 kDa outer membrane protein (OMP) from Helicobacter pylori (Hp), and exploit the possibility of obtaining Hp vaccine and diagnostic reagent kit for rapid diagnosis of Hp infection.
The gene encoding the structural 18 kDa outer membrane protein of Hp was amplified from Hp chromosomal DNA by PCR. The purified PCR product and identified plasmid pQE30 underwent restriction enzyme (Hind III, BamHI, BgLI) digestion and purified by PCR purification reagent kit, and then linked in the proportion of 4:1 (molar weight). The recombinant vector was sequenced with T7 as seqiencing primer. Homology of the determined DNA sequence was analyzed by DNA analysis software. The recombinant vector was selected and identified by restriction enzyme digestion, and then transformed into DH5 a (PREP4) Escherichia coli strain which was cultured and induced by isopropylthio-beta-D-galactosideso as to determine its expressed products.
The gene segment inserted into the recombinant vector was identified as the gene experssing the OMP of HP with a molecular mass of 18 fDa. As compared with previously reports, 2% of the gene was mutated, 1.68% of the amino acid residues was changed, and the homogeneity was about 98%. The level of soluble expression products was about 18% of total cellular protein. ELISA results showed that this objective protein could be recognized by anti-serum against Hp.
The product expressed by Hp OMP gene clone has good antigenicity. The recombinant vector expressing 18 kDa OMP may be a potential source for effective protein vaccine against Hp infection and reagent kit of Hp infection diagnosis.