动物用质粒DNA内毒素去除方法的建立及其质量检验

Establishment of endotoxin removal for animal-used plamsid and the quality detection

用自行改进设计的TritonX-114方法去除动物用重组质粒DNA中的内毒素,鲎试剂检测方法确定每毫克质粒DNA中内毒素的含量为50 EU。将3种不同方法制备的重组质粒pEGFPN1包裹后转染COS-7、MDCK和Marc145细胞,结果表明,经TritonX-114去除内毒素后的质粒DNA(A)与无热源质粒提取试剂盒制备的质粒DNA(B)的转染效率相当,比未经内毒素去除的质粒DNA(C)的转染效率高。将上述3种方法制备的pcDNAlacZ经PEI包裹后尾静脉注射小鼠,注射后每隔2 d扑杀小鼠,取其心、肝、脾、肺和肾组织,定量检测β-半乳糖苷酶的表达和持续时间,结果显示报告基因仅在小鼠的心、脾和肝组织中表达至第8天,A与B的表达水平接近,且明显高于C制备的pcDNALacZ的表达水平,提示经TritonX-114去除内毒素后的质粒DNA可提高转染效率和表达水平。多种动物体内注射重组质粒后,无体温升高等异常临床表征,进一步说明了使用经TritonX-114去除内毒素的质粒DNA具备良好的安全性。

The endotoxin in animal-used plasmid was removed by self-designed TritonX-114 method, the endotoxin content was 50 endotoxin unit detected by Limulus amoebocyte lysate. Following coated with transfected reagent, the pEGFPN1 prepared by three ways was transfected into eukaryotie cell COS-7, MDCK and Marc-145. The fluorescence calculation results showed that the transfection efficiency of plasmid purified by the TritonX-114 （A） and EndoFree Plasmid Mega Kit （B）were similar,and the efficiency was higher than the plasmid without endotoxin removal（C）, the pcDNAlacZ prepared as mentioned above was coated with PEI and injected into mice via tail vein, then the mice were killed every 2 days, the heart, liver, spleen, lung and kidney were sampled, and the β-glactosidase expression and persistence time were detected quantitatively. The results showed that the report gene was only expressed in the heart, spleen and liver in the recombinant plasmid injected-mice to 8 days, the expression level of A and B was similar,and was obvious higher than C,which indicated the plasmid with endotoxin removal by TritonX-114 could boost the transfection efficiency and expression level in vitro and vivo. The plasmid after endotoxin removal was injected into animals and no temperature and abnormality were observed, further explained the safety of the application of plasmid after purification with TritonX-114.