| Objective: The present study was intended to prepare a kind of specific monoclonal antibody( McAb) against the expressed vacuolating segment of VacA gene in Helicobacter pylori by the cell engineering method, and to try its application primarily. It would lay a foundation for the development of diagnoses and treatment for the VacA + H.pylori infection,and the elucidation of H. pylori virulence mechanisms. Methods : This study includes two parts: purification and identification of the VacA antigen, then study of the monoclonal antibody (McAb) and its preliminary application . The main research contents as follows: The first part is purification and identification of the recombinant VacA aquired by over-cluturing and inducing the engineered bacteria E.coli . The expressional product was analyzed by SDS-PAGE and its antigenicity was confirmed by Western blot. Ni2+-NTA agarose purified the recombinant protein,SDS –PAGE assayed its purity,Bradford measured its protein concentration.To identify antigenicity of the recombinant protein, we established indirect ELISA . The second part is the study of VacA monoclonal antibody and its application primarily. We take the purified recombinant protein VacA(rVacA) as immunogen.The BALB/c mouse was immunized with VacA for four times.The immune interval was two weeks. And the mouse was attacked by the vacuolating cytotoxin A inclusion body protein(VacA)three days before the hybridization. The spleen cells of the immunized mouse were hybridized with SP2/0 cells by 50%PEG. The hybridoma cells were selected by HAT culture medium . The antibody-secreting hybridoma cells were primary detected by the indirect ELISA using the purified rVacA as the antigen,and coloned by limited dilution to obtain the hybridoma cell lines which can stably secret the McAb against rVacA. The stability of the McAb-positive cell lines were detected by thirty subcultures in vitro,being frozen and thawed repeatedly for 6 times and thawed after frozen for 1.5 months. The Ig class of McAb was identified by the Mouse Mab Isotyping Test Kit (10 assay, b.v). The antibody titers of ascites McAb were measured. The ascites McAb was purified by the saturated ammonium sulfate. The indirect ELISA was established with the purified monoclonal antibody,and used to detect the VacA protein in the gastric mucosa . Results: The pQE30-V-DH5α was extracted from the genetic engineering E.coli successfully . SDS-PAGE showed that the recombinant protein's Mr was about 27000 represented 30% total protein of E.coli; Western blot indicated that the recombinant protein could be recognized by antiserum against Hp; SDS-PAGE showed that the recombinant protein most existed in the precipitation of the lysed bacteria broken by ultrasonic wave, and its purity is about 93% after purified with Ni2+-NTA agarose. The mouse was immunized successfully using antigen.Sixty-eight wells of antibody-secreting hybridoma cells against rVacA were primary obtained by cell hybridation,HAT selection and antibody detection. Four hybridoma cell lines can secret the McAb against rVacA continuously aftercoloning.The McAb can bind to the purified rVacA protein . The hybridoma cell lines were named zhA2,zhB5,zhC8 and zhD4,and one of them, the zhD4 has the most best stability.The McAb can be secreted by zhD4 after thirty subcultures in vitro,being frozen and thawed repeatedly for 6 times and thawed after frozen for 1.5 months. The Ig class of zhA2,zhB5,zhC8 and zhD4 McAb was IgG2b,IgM,IgG1 and IgG1,respectively and all the light chains were κ.The titers of the ascites McAb-zhD4 was l:12800. The McAb could specially bind to the rVacA. Conclusions: The recombinant protein was successfully aquired from the genetic engineering E.coli .Four hybridoma cell lines secreting specific monoclonal antibody against VacA in Helicobacter pylori were established. McAbs against VacA were successfully obtained and purified. It might play an important role in further preparing the VacA test kit and could afford data in exploring the mechanisms of the VacA cytotoxinity .
|
PDF Full Text Request