HCGβ抗肿瘤核酸疫苗的纯化工艺

Purification Process for the Production of HCG β Anti-Tumor DNA Vaccine

对HCGβ抗肿瘤核酸疫苗的预处理和层析纯化工艺进行了研究。用碱裂解法对湿菌体进行预处理,当菌体湿重（g）∶悬浮液体积（mL）∶裂解液体积（mL）∶中和液体积（mL）为1∶2∶2∶2,碱裂解时间为8-12 min时,质粒的产量最高,可达32.94 mg/g。用Sepharose 6 FF凝胶过滤层析去除RNA和蛋白质,再用Plasmidselect亲和层析捕获超螺旋质粒DNA,质粒DNA的得率为48.3%。用L-Histidine-agarose亲和层析可从澄清的碱裂解液中一步分离纯化超螺旋质粒DNA,质粒DNA得率为51.62%。经琼脂糖核酸电泳检测,HCGβ抗肿瘤核酸疫苗超螺旋质粒DNA的纯度为100%电泳纯。

The purification process for the production of HCG β anti-tumor DNA vaccine was studied. The parameters in the process of alkaline lysis were determined. When the ratio of cell weight （g） ： suspension solution （mL） ： lysis solution （mL） ： neutralization solution （mL） was 1 ： 2 ： 2 ： 2, and the lysis time was 8-12 min, the yield of pDNA reached 32.94 mg/g. Sepharose 6 FF gel filtration chromatography was used to remove RNA and protein from the lysates directly, and then Plasmidselect affinity chro matography was used to capture supercoited plasmid DNA. As a result, the final yield of pDNA was 48.3%. Supercoiled plasmid DNA could also be purified from clarified cell lysates with a single L-Histidine-agarose affinity chromatography step, the yield of pDNA was 51. 62%. The purity of the sc pDNA purified by either of the two purification processes was 100% determined by agarose electrophoresis.