Whole-cell Subtractive Screening The Peptides Binding T Lymphocytes With The Phage Peptide Library Of Mycobacterium Tuberculosis

Objective:Whole-cell subtractive screening the peptides specifically bind to T lymphocytes with phage peptide library of the mycobacterium tuberculosis. Looking for the biologically active peptide that can activate T lymphocytes, and provid some experimental basis for developing the tuberculosis new peptide vaccines.Methods:T lymphocyctes were isolated with nylon wool column method. The positive clones were screened by whole-cell subtractive screening with the phage peptide library of mycobacterium tuberculosis. The T lymphocytes of the immunization Balb/c mice were used as the target cells and the T lymphocytes of the normal Balb/c mice were used as the negative adsorption cells. The time of the phage peptide library of mycobacterium tuberculosis incubated with target cells respectively was 2h,1.5h, 1h, and with normal negative adsorption cells respectively was 2h,2.5 h,3h. The concentration of the eluent respectively was 1%, 3%,5%. The positive clones were obtained with white selection. The positive clones single-strand DNA was extracted and purified, and identified by electrophoresis. Observe the effect of the positive clones on T lymphocytes transformation of mice.Results:The recovery rate of T cells was 30% andthe living cells ratio was 93%. The high activity T lymphocytes were obtained. The level of the serum anti PPD IgG in immune Balb/c mice was 3,200 at the sixth week. The difference between the control group and the immune group was significant at the first 2 weeks, the first 4 weeks and the first 6 weeks (p< 0.05); the difference between the control group and the immune group was very significant in addition to the first 2 weeks (p< 0.01). This indicated that we successfully obtained the immune mice. The positive clones were enriched after three rounds of screening. The recovery rate was increased from 4.0×10-5 to 52. The positive clones were obtained with white selection. The electrophoresis strip of the positive cloning single-strand DNA was consistent. The proliferation and transformation of T lymphocytes induced by positive phage in the immunized mice was significantly greater than other irritants, and the maximum SI was 3.00.Conclusion:The recovery rate of T lymphocytes separated by nylon wool column was 30%, and the living cells ratio was 93%. The positive clones were obtained after three rounds of screening, and the titer of the positive clones was 1.3×104/ml. The positive clonings could effectively stimulat the activation of T lymphocytes.