Abstract:

Abstract:Objective To screen mimic peptide epitopes of Mycobacterium tuberculosis from 12-mer phage display random peptide library,and to explore its immune activity.Methods  A 12-mer phage display random peptide library was screened for 3 rounds by using the polyclonal antibody IgG which was purified from tubercle positive serum of deers according to such a procedure as adsorbing,eluting and amplification,and 20 positive clones were selected randomly,confirmed by its specificity.The BALB/c mice were immuned with mixture of the positive clones as test group,BCG group as a positive control,TBS group as negative control group;Three weeks later,ELISA was used to detect the anti-PPD antibody titer in the immunized mice sera. The lymphocytes were separated from the immunized mice and the stimulating index (SI) was determined to further evaluate protective immune response to MTB.All the immunized mice were challenged with 10⁶ CFU BCG to study the protection conferred by the positive clones.Results After 3 rounds of effective bio-panning,15 of 20 phage clones were identified as positive clones by competent ELISA which could bind to polyclonal antibody IgG,the positive rate was 75%. A significant difference in valence of antibody in test group was observed compared with BCG group(P<0.01).In mouse spleen lymphocyte proliferation test,after stimulated by ConA,LPS,PPD,the SI values in test group were higher than those in BCG group,but there was no significant difference(P>0.05);the SI values were significantly higher than those in TBS group(P<0.01). The pathological changes of mouse lung section were observed in test group and BCG group, the alveolar tissue appeared to be intact with significantly less lymphocyte infiltration.In TBS group the pulmonary parenchyma developed severe fibrosis .The lotus bacterium (log CFU·mg^-1) 　in test group（4.080±0.035）was lower than that in TBS group（4.360±0.110）(P<0.01),but was higher than that in BCG group（3.980±0.023）. Conclusion  Mycobacterium tuberculosis mimic epitopes of positive clones have been successfully screened.They can induce mouse to produce specific immunity and protective effect.This result laid a further experiment foundation for development of  new tuberculosis vaccine of Mycobacterium tuberculosis.

Key words: Mycobacterium tuberculosis;phage display peptide library;mimotope;immunoassay