| Objects: Since Marshall et al separate Helicobacter pylori from human gastric mucosa membrane in 1983, many studies showed that it is implicated in the development of chronic gastritis, peptic ulceration and listed as the first cancer factor by WHO. Hp infection rate of human is as heavy as 50%. With antibiotics abusing and the increasing unsensitive strains the issue on anti-Hp infection has turned to immune prevention and therapy. Since some proteins of milk can improve stabilization of IgG in stomach and milk is safe and an ordinary drink, it might be the new way for oral antibodies application to utilize immune milk against Helicobacter infection. So we studied the influence of bovine antibodies against Hp growth and colonization in vivo and vitro. Methods:1 Bacteria and antigen preparation: Hp (SS1 and NCTC11637) was grown on blood agar plates at 37℃ for 48~72h in 10% CO2 and 85% N2 and 5% H2. It is identified on shape with Gram's staining and on biologic character with urease assay and catalase assay. The cells were washed threetimes in sterile saline and the sonicated antigens was determined protein-concentration by CBBR-assay and aliquors were frozen at -20℃ until used.2 Production, isolation, purification and identification of bovine specific antibodies: Cows were immunized with Hp sonicated antigen. After finishing immunization schedule an average of 1~2 month was the likely interval, which can maintain the titer of bovine serum a high level during a long period. Two-day diffusion agar assay offered a standard that until 1:32 titer of bovine serum it can be collected, the same as milk. The serum proteins (81.4mg/ml) were isolated and purified by 50%,30%,30% saturated ammonium sulfate and DEAE-32. With that the terminal collecting rate is 3.14%. The matter was analyzed by SDS-PAGE with the result of 55KD and 25KD polypeptides and the purification of 90% or so. Aliquors were frozen at -20℃ until used.3 SDS-PAGE of Hp antigens and Western blot of bovine antigen-specific antibodies: Hp sonicate supernant was analyzed by SDS-PAGE with the conditions of isolated-gel 12%, concentrated gel 5%, sample given 20ul. During Western blotting progress the current of transferring was 1.6mA/cm2 and the time was 1.5h. After coated with 5% nonfat dry milk NC film was incubated with bovine samples or with HRP-rabbit anti-bovine IgGs for 4h at room temperature. Immune complexes were detected by reaction with a solution containing DAB and a time of 5~10 minutes. 4 Neutralization of bovine specific IgG against the proliferation-inhibiting action of Hp to Hela cell: Hela cell growth curve was determined to select the right cell density. MTT assay was adapted to evaluate the proliferation- inhibiting action of SS1 and NCTC11637 antigens to Hela cell. Bovine anti-Hp IgGs were incubated with SS1 or NCTC11637 respectively at 37℃ for 2h then added into solution including Hela cell. 5 Prevention and therapy study of bovine IgG against Hp infection in mouse model: 5.1 model of mice: BALB/c mice were no feeding 16h then inoculated with suspension of SS1, which had been harvested directly from 48h cultures into brucella broth (109CFU/ml), once every two days till the fifth finished.5.2 Assessment of Hp infection: several mice were killed at 4w,6w,8w respectively after inoculation to be detected as follows: 1.collect blood by removing eyeball. 2. intact mouse stomach was opened along the greater curvature to release the gastric contents and was washed with sterile saline. The presence of Hp infection in mice was determined by biopsy urease, isolating culture and histological analyses. One longitudinal piece (1/2) was homogenized to inoculate selective agar (Vancomycin 4.0mg, Polymyxin 100mg, Trimethoprim 2.0mg) for 3~5 day. One piece (1/4) was fixed in 10% formalin and stained by the hematoxylin-eosin and Warthin-Starry silver staining. The presence of inflammatorycell infiltrates, erosive lesions, edema, hyperplasia and gland atrophy was assessed by HE staining and bacterial...
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