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| Preparation and identification of monoclonal antibody against Helicobacter pylori CagL protein |
| ZHU Hong， CHEN Cheng， LING Feng， SHAO Chen， YU Ming， SHAO Shi-he |
| School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang Jiangsu 212013, China |
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Abstract Objective: To prepare and characterize the monoclonal antibody(mAb) against Helicobacter pylori (H.pylori) CagL protein. Methods: The CagL fusion protein was induced to express by IPTG in E.coli BL21(DE3) which contains recombinant expression vector pET-28a (+) and then purified by Ni2＋-NTA resin. Purified CagL fusion protein was used to immunize 6week BALB/c mice, and then the spleen cells were fused with myeloma SP2/0 cells; the positive hybridoma cell lines were obtained by means of several rounds of subclone. Immunoglobulin isotype of mAb was identified by ELISA method. The titer of mAbs in cell culture supernatant and ascites, as well as the relative affinity of these antibodies, were measured by indirect ELISA method. The antigenic epitopes of mAbs were evaluated with ELISA additivity test. The specificity for antibody was determined by Western blotting. The stability of hybridoma cell lines specifically secreting mAbs was tested by cryopreservation and resuscitation. Results: Three hybridoma cell lines which could secrete stably mAbs against CagL were successfully obtained. The isotype of two mAbs was classified as IgM, another IgG2a, and the light chain subset of three mAbs was classified as type κ. The titers of three mAbs in cell culture supernatant were 1∶64～1∶128, while those in ascites reached more than 1∶16 000. The affinities of mAbs were found to be high. Three different antigenic epitopes might be recognized by the mAbs. Western blotting analysis showed that all three mAbs could bind to the CagL fusion protein; however, only two of them had specific reaction with the whole protein extracted from H.pylori strains. After cryopreservation, resuscitation and subculture, the titers of mAbs secreted by three hybridoma cell lines were still high. Conclusion: The monoclonal antibody against CagL protein was successfully prepared, which would play a significant role for the further study in the mechanisms of CagL as well as H.pylori type Ⅳ secretion system.
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Received: 05 April 2013
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2013, Vol. 23 
