| Helicobacter pylon (Hp), a kind of gram-negative bacteria which colonizes in the huanin stomach, has been identified as a major pathogen associated with the development of chronic gastritis and gastroduodenal ulcer disease as well as gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. The infection rate of Hp is very high. They can colonize in human stomach nearly all the time because human immun system can not eradicate them. Vacuolating cytotoxin A (VacA) is the unique secreted toxin of Hp that induces vacuolation of epithelial cell and is closely related to the incidence of disease in a population infected with Hp. The six segments (A~ B~ C. D E~ F) of vacA gene were amplified by polymerase chain reaction using the total DNA isolated from Hp strain NCTC 11638 as the template. The nucleotide sequences of the six segments of vacA gene were consistent with the papers reported. Then they were inserted into the prokaryotic fusion expression vector pRSETA respectively 7 to construct recombinant expression plasmids pRSET-vacA. The SDS-PAGE showed E ccli BL2 1 DE3 (pLysS) containing pRSET-vacA expressed five His6-fusion proteins (B~ C. D. E. F) which existed as inclusion bodies. Their relative molecular mass (Mr) of fusion proteins were 33000, 20240, 26300, 27500 and 32450. The expression levels of the five pRSET-VacA segments were about 47.8%, 31.8%, 43.6%, 43.2% and 22.7%. Inclusion bodies of His6-B and His6-D were dissolved under denaturing condition (8molIL urea) and purified by a single-step using Ni2~ chelating resin. Western-blot assay results indicated that His6-B and His6-D could recognize the polyclonal antibody against VacA. Then we prepared the antiserum of rabbit against His6-B and His6-D respectively by immunizing rabbits with the PAGE gel of bacteria induced by IPTG. The rabbits were immunized for four times. The titer of antiserum against His6-B was I : iooo梚 :10000 and that of His6-D was i 10000梸- :100000. We test the antibody against VacA in the serum of health people and patients using the purified and refolded protein (His6-B) as antigen. Sera of 77 (26.9%) of 285 samples were positive, as well as 47 (28%) of 168 health people, 2 (14%) of 14 patients with duodenal ulcer, 2 (22%) of 9 patients with gastric ulcer, 3 (3 8%) of 8 patients with gastric gastritis, 6 (3 3%) of 18 patients with gastric carcinoma, 1 (14%) of 7 patients with hemorrhage of upper digestive tract. Finally, we redesigned four pairs of primers according to the restriction sites in the MCS of eukaryotic expression plasmid pIRES2-EGFP. The different segments of vacA gene, that were NB .~ NC~ ND~ NE~ NG and NH, were amplified and their nucleotide sequences were consistent with the paper reported. Then they were inserted into pIRES2-EGFP plasmid respectively to construct recombinant plasmids pIRES2-NvacA with the GFP-tag. The 8 recombinant plasmids were transfected into HeLa cell respectively to test their vacuolating cytotoxin activity. With the help of electron microscope, we gottheresultstbatsegmentsNBandNGbadtheactivityandNBwasthe shortest vacuolatmg activity segment in our study. ELISA assay is the primary method to test Hp infection epidemiologically. Oral immunization is an effective approach for the prevention of...
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