Studies On Bioactivity And Safety Of Freeze-drying RBCG-hIFN-a-2B

Objective: we attempted to evaluate the feasibility of freeze drying genetically engineered microorganisms—rBCG-hIFN-a-2b.we analysis the effect of lyophillization on rBCG-hIFN-a-2b about viability , form and growth curvesand expression of exogenous protein------interferon-a-2b.To study the degree ofartificially transconjugated plasmid activity and stability occurring subsequent to freeze-drying and storage. We also carry out safety test of freeze-dried rBCG-hIFN-a-2b.Method: Freeze drying and conserve rBCG-hIFN-a-2b, screening optimal technical parameter and draw freeze-drying curves. Compare viable count before and after lyophillization using plate count, calculate survival rate. After acid-fast staining , observe micro form of freeze-dried rBCG-hIFN-a-2b. Serially determined optical density of rBCG-hIFN-a-2b before and after lyophillization, draw growth curves. 48 freeze dried monoclone colony were cultured in Middlebrook 7H9 containing kanamycin, then hIFN-a-2b gene segment were amplification using PCR and separated by 1% agarose gel electrophoresis. To determine the expression of extrinsic protein—interferon-a-2b by enzyme linked immunosorbent assay (ELISA). 8 guinea pigs which PPD test were negative, were respectively subcutaneous inoculated using before and after freeze dried rBCG Observed their mental state, action and body weight, after 6 weeks killed them and macroscopic observation each organ, made pathological section , HE stain and test under microscop.Result: Successfully freeze drying rBCG-hIFN-a-2b, screen out optimal technical parameter. Viable count was 6.5×10~6cfu/ml,survival rate was about65%.The form and growth curves have no obviously change. 4 lost kanamycin resistance in 48 freeze dried monoclone colony. Randomly selected 5 out of 44 remaining kanamycin resistance freeze dried monoclone colony, hIFN-a-2b gene segment could be detected. Then, Plasmid remain rate was 91.7%, lost rate about 8.3%. The expression of IFN-a-2b have no significant difference before and after freeze drying rBCG. Every guinea pigs have no action abnormality and their body weight obviously increase. Each organ have no tubercler or cheesy necrosis. There are epithelioid cell aggregation occasionally in injection region and lung.Conclusion: The test demonstrated that genetically engineered microorganisms: rBCG-hIFN-a-2b could be storaged by lyophillization and the method was feasible. Viable count satisfy immunotherapy request which need viable count greater than 1 million. Their form, growth, expression of interferon-a-2b have no change after lyophillization. Although there are small amounts recombinant plasmid loss, about 8%, we think recombinant plasmid could resist harmful environment of freeze drying process. Freeze-drying rBCG-hIFN-a-2b were consistent with rule of safety test.