[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.
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ABSTRACT: To examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylori (H pylori) infection to two H pylori outer membrane proteins (OMPs) (Mr18,000 and Mr 26,000) acquired by gene recombinant technique, and to determine the diagnostic significance of serological tests derived from these OMPs. Recombinant vectors encoding the two H pylori OMPs were used to transform and express in BL21 (DE3) E.coli. After purification with Ni2+-NTA agarose resin, colloid gold kits were prepared with purified recombinant proteins to detect H pylori infection and H pylori-associated diseases by the immunity-marker technology. We selected 150 patients with H pylori infection and digestive symptoms without previous treatment, including chronic gastritis (n = 60), duodenal ulcer (n = 30), gastric ulcer (n = 30), and gastric cancer (n = 30). As controls, 33 H pylori-negative healthy volunteers were also recruited. Serum samples were collected from all subjects, and the antibodies to specific proteins of H pylori were tested with the colloid gold test kits. The sensitivity, specificity and accuracy of the colloid gold tests were evaluated, by using the combination of standard diagnostic methods (13C urea breath test and bacteria culture) and classic enzyme-linked immunosorbent assay (ELISA) as reference. After purification with Ni2+-NTA agarose resin, the purity of recombinant fusion proteins was about 95%. The recombinant fusion proteins were recognized by the specific monoclonal antibodies against the two H pylori OMPs, as demonstrated by the ELISA. Of the 150 serum samples from patients infected with H pylori 141 (94.0%) responded positively to the recombinant protein with Mr 26,000, while the seropositive rates were 95.0%, 96.7%, 96.7% and 90.0% for patients with H pylori-associated chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer respectively. The sensitivity, specificity, and accuracy of the colloid gold kit with Mr 26,000 protein were 94.0%, 97.0%, and 94.5%, respectively. Compared with the classic ELISA, bacteria culture and 13C urea breath test results in detecting H pylori-infection, there was no significant difference (P>0.05). For the colloid gold kit with Mr 18,000, the seropositive rates were 52.0%, 40.0%, 40.0%, 53.3% and 86.7%, respectively, in H pylori-infected patients, and those with H pylori-associated chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer. There was a significant difference (P<0.05) in seropositivity between patient with gastric cancer (86.7%) and those with other diseases (43.3%). The two colloid gold kits derived from the recombinant OMPs are useful tools either for detecting H pylori infection, or for, predicting H pylori-associated gastric malignancy.World Journal of Gastroenterology 12/2004; 10(23):3464-9. · 2.43 Impact Factor