The Study Of Immunological Protection About Helicobacter Pylori OipA DNA Vaccine

Objective To investigate the effect and mechanism of oipA DNA vaccine ofHelicobacter pylori(Hp)on protecting against the infection of helical and coccoidforms of Hp.Methods After the successful expression of oipA had been detected in vitro,extracted and purified the recombinant plasmid pcDNA3.1-oipA andpcDNA3.1 .Then the recombinant plasmid had been inoculated to BABL/C micethrough particle gun at 0,14 days(5 μg each mouse). 1) Investigated theimmunological protection. Eight weeks after last immunization,the other part ofimmunized mice had been given orogastric challenged with helical and coccoid formsof Hp Sydney strain four times ( 0.5 ×10~9/0.5ml each mouse) at 0,3,6,9 days. Fourweeks after challenge, mice had been sacrificed. Histological change and thecolonization of Hp in the gastric mucosa had been detected by urease test, the cultureof Hp, electronic microscopy and so on. 2) Detected the immunological index ofimmunized mice.The serum of immunized mice were collected at 2,8,12 weeks afterthe last immunization. Titer of anti Hp OipA IgA,IgG ,IgG1 antibodies had beendetected by ELISA to understand the level of humoral immunity generated. Twelveweeks after last immunization,the eyeballs of a part of immunized mice had beenremoved to collect blood,and then sacrificed by shifing its'cervical vertebrae. ①Thesplenic cells were taked and detected the contents of IFN-γ,IL-2 by ELISA(serumand splenic cells). ②The ratio of CD4~+/CD8~+ T lymphocyte by flow cytometyr weredetected to understand the level of cellular immunity generated. 3)Then, the vectorpVAX1 Which was more suitable to human vaccine was replaced by the vectorpcDNA3.1 to construction the recombinant plasmid. Intramuscular injection Whichwas more feasible in human body was used instead of particle gun. PVAX1-oipA hadbeen injected into BABL/C mice through muscles of right leg once each week forthree weeks (100μg each mouse). Titer of antibodies had been detected by ELISAeight weeks after the last immunization,then mice had been given orogastricchallenged with live Hp Sydney strain (dose as before) . Four weeks after challenge,mice had been sacrificed. Gasters of mice were taked and treated as before andanalysised the immunological protection. Rusult 1)Investigated the immunological protection of oipA DNA vaccine.After challenged with bacterium, the positive rate of urease test of gastric mucosa wasas follow: the experimental group challenged with helical forms of Hp 20%(2/10);the experimenta group challenged with coccoid forms of Hp 10%(1/10),lower thanthe control group(p<0.05); The positive rate of cultures of Hp was as follow: theexperimental group challenged with helical forms of Hp 60% ( 6/10 ), theexperimenta group challenged with coccoid forms of Hp 50%(5/10), lower than thecontrol group(p<0.05). The diffent degree of erosion had been observed in controlgroup, but 50%(5/10)of gastric mucosa were normal in immunized mice challengedwith helical forms of Hp, 60%(6/10)of gastric mucosa were normal in immunizedmice challenged with coccoid forms of Hp(p<0.05). 2) Detected the immunologicalindex of immunized mice. BABL/C mice inoculated pcDNA3.1-oipA DNA vaccinehad been induced anti-oipA IgA,IgG ,IgG1 antibodies, their titer of antibodies is twotimes higher than control group. The contents of IFN-γ,IL-2 of culture supernatantof splenic cell is obviously higher than the control group(p<0.05). The ratio ofCD4+/CD8+ T lymphocyte was higher than the control group(0.05