BCG-CpG-DNA制备、理化特性及免疫刺激活性的初步研究

BCG-CpG-DNA isolation,physicochemical characterization and the function of immunostimulation

采用机械破碎、CTAB沉淀、酚:氯仿:异戊醇抽提法从卡介菌(BCG)中制备BCG-CpG-DNA,经化学方法确定其理化成分,通过双抗体夹心ELISA法检测小鼠IgM水平检测其免疫刺激活性.结果显示每克半干重的卡介菌提取BCG-CpG-DNA约0.9mg.提取物主要成分为BCG-CpG-DNA,分子量大小在3 000～15 710 bp,含少量的多糖和蛋白;紫外分光扫描在260nm有最大吸收;对特异性识别DNA长链的CCGG中的CpG位点的限制性内切酶HpaⅡ高度敏感;能够活化小鼠B细胞产生IgM.所制备的BCG-CpG-DNA含较多的未甲基化CpG基序,具有免疫刺激活性功能.

Using the following methods including mechanical disruption, precipitated with CTAB, extracted with Phenol:chloroform:isoamyl alcohol, BCG-CpG-DNA was prepared from Mycobacterium bovis strain BCG. In detection of immunostimulatory function, the double antibodies sandwich ELISA was taken to determine mouse IgM secretion. Results showed that BCG-CpG-DNA is the main component of the DNA fraction with low amount of protein and polysaccharide. BCG-CpG-DNA is about 3 000～15 710bp, showing an absorption maximum at 260nm and an absorption minimum at 230nm. The restriction enzyme Hpa II recognizes the sequence CCGG, and cuts this site when the inner cytidine is unmethylated. BCG-CpG-DNA is sensitive to Hpa II digestion by cleaved as 100～250bp from 3 000～15 710 bp, and it can activiate IgM secretion from mouse B cells. This indicated that there are plenty of unmethylated CpG motifs in BCG-CpG-DNA, which have immunostimulatory functions.