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Screening Of A Single Chain Antibody Fragment Against Helicobacter Pylori With Phage-display Technology

Posted on:2009-11-18Degree:MasterType:Thesis
Country:ChinaCandidate:Q JiFull Text:PDF
GTID:2120360272457096Subject:Microbiology
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Helicobacter pylori ( H. pylori)was seperated from gastric mucosa in 1983 by the Austrilia scientists, Warren and Marshall. H. pylori is a Gram negative helical bacillus, located and reproduced in gastric epithelial cells. Previous research results show that H. pylori predominantly results in chronic gastritis and peptic ulcer, and is also closely related to the occurrence of gastric cancer. H. pylori was listed as I type carcinogenic factor by International Agency for Research on Cancer (IARC). There are many deficiencies in the combined treatments to peptic ulcer, so lots of scientists pay attention to the ongoing researches on the specific antibody of H. pylori.In this paper, the phage-displayed single-chain variable fragment (scFv), which specifically binds to Helicobacter pylori (H. pylori), was screened. Futhermore, its characters were analyzed. The intact H. pylori cells were panned against the human scFv libraries (Tomlinson I+J). 8 positive clones were obtained from 96 random clones after five round screening and ELISA assay. The ELISA value of scFv in round 4 was four times high than that of blank. Finally, only a single clone named JH1, which expresses scFv specifically binding to H. pylori, was selected for no cross reaction with 10 different bacteria. PCRs of VH,VL and scFv gene from JH1 show that the sizes of VH,VL and scFv gene are 527 bp, 368 bp and 935 bp respectively. Sequencing for scFv and then BLAST in NCBI indicate the gene is 96% homologous with a known scFv, which is specific to the replicase of a plant (+) RNA virus.The positive clone JH1 was transferred and expressed in E.coli HB2151, and then analyzed with ELISA. The A540/A630 ratio of JH1 is higher than that of PBS background or mixed bacteria. It indicates that the expressed scFv can bind H. pylori specifically. After the E.coli HB2151 was disrupted with frozen-and-thaw again method, scFv was purified using Ni2+ column and then analyzed with SDS-PAGE. The purity of scFv is high.
Keywords/Search Tags:Helicobacter pylori, phage display, scFv, cell screening, ELISA
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