| Helicobacter pylon (Hp) is a small, curved, gram-negative bacillus found in the stomach of patients with gastric diseases. Hp is an etiologic agent of chronic active gastritis and gastric and duodenal ulcers. It is also closely related with gastric cancer and MALT type lymphoma. Urease is one of the most abundantly produced proteins by Hp. This enzyme can quickly resolve urea into ammonia and carbon dioxide, and plays an important role in the reside of Hp. The urease of Hp contains nine subunits: ureC, ureD, ureA, ureB, urel, ureE, ureF, ureG and ureH. UreA and ureB are structural genes, and the others are assistant genes. UreA and ureB have always been used as protective antigen against Hp infection. UreC gene has always been used as the target 9f PCR. Three DNA enzyme immunoassays have been developed to detect Hp. They all use biotinylated specific probe based on the ureC gene. There are many available methods for diagnosing the presence of Hp infection. They can be divided into two kinds: invasive methods and noninvasive methods. Invasive methods require examination of a gastric biopsy specimen, for example, histologic examination using special stains, culture for the organism. These methods require endoscopy, which limits its use. Several simple, noninvasive methods have been developed for the detection of Hp infection, such as serologicaltest and urease breath test. ELISA is the most commonly used serological fonnat. It is usually laboratory based, requiring both some technical competence to perform and instruments to measure the optical density of the colored product. DIFGA is an immuno-method developed in recent years. In this method, the red immunogold is used as the marker and nitrocellulose as the substrate, which has the function of absorption, filtration, concentration, and capilliary, and can make the antigen- antibody reaction take place rapidly. In this report, the urease C gene of Hp was cloned and expressed in Eschenichia coli. The recombinant urease C protein was used as the antigen of dot immunogold filtration assay (DIFGA), which was more simple, rapid and convenient than enzyme-linked immunosorbent assay (ELISA). In order to express recombinant gene of Hp ureC, a pair of primers were designed according to the published Hp sequence. A 1 179bp gene fragment was amplified by the polymerase chain reaction. The gene fragment was digested by BamH I and EcoR I and cloned into pGEM-3Zf(-) plasmid. Its sequence was determined by the autosequencing instrument from two directions. The result showed that 95% of the DNA sequence we had got was the same as that of Hp strain M60398. So it was the right gene we wanted. Then the gene fragment was cloned into E.coli expressing plasmid vector--pBV22O, which was regulated by ~ Subsequently the DH5 c~ was transformed by pBV22O/ureC, and a Mr 44000 recombinant protein was induced by heat when temperature reached 42 0C .The molecular mass of the expressed ureC gene was conformed to what we expected. The thin layer chromato gram scanning of the gel showed that the expressed protein accounted for 24.8% of the whole protein of the host bactria. Then the form of the expressed protein was analyzed to learn that, although most of the recombinant proteins were inclusion bodies in host bacteria after translation, there did exist certain quantity of solvable proteins in the supernatant of bacteria lysate. The protein was proved with high antigenicity by Western-blot assay. And the recombinant protein was then used as the antigen of DIGFA to detect Hp infection. |
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