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目的探索一种利用离子交换层析技术制备高纯度卡介菌基因组DNA(genomic DNA frommycobacterium bovis Bacillus calmette-Guerin,BCG-DNA)的方法,并探讨其免疫调控活性。方法运用超声、加热、胰酶消化、离子交换层析、凝胶过滤层析及冻干法制备高纯度BCG-DNA;小鼠脾细胞与BCG-DNA共同培养72h,采用ELISA方法检测细胞培养上清中干扰素γ(interferon-γ,IFN-γ)的水平。结果所制备的BCG-DNA纯度较高(DNA 95.3%),片断大小均匀一致,集中于200～250bp;BCG DNA可显著诱导小鼠脾细胞产生IFN γ(P<0.01),且IFN-γ水平与BCG-DNA浓度呈正相关。结论利用层析法制备BCG-DNA,高效、重复性好。所制备的BCG-DNA纯度较高,具有较强的免疫调控作用。
Objective To explore a method for preparing genomic DNA from mycobacterium bovis bacillus calmette-Guerin (BCG-DNA) using ion exchange chromatography and investigate its immunological regulation activity. Methods High purity BCG-DNA was prepared by sonication, heating, trypsin digestion, ion exchange chromatography, gel filtration chromatography and freeze-drying. The spleen cells were incubated with BCG-DNA for 72 hours. Interferon-γ (interferon-γ, IFN-γ) levels. Results The purity of BCG-DNA was 95.3%. The size of BCG-DNA was about 200-250bp. BCG DNA could induce the production of IFN-γ (P <0.01) And BCG-DNA concentration was positively correlated. Conclusion The preparation of BCG-DNA by chromatography has high efficiency and reproducibility. The prepared BCG-DNA has high purity and strong immunomodulatory effect.