抗肝癌基因疫苗内毒素去除方法的建立

目的建立去除抗肝癌基因疫苗pVAX1-aLCGV内毒素的方法。方法采用Triton X-114液相分离法去除抗肝癌基因疫苗中的内毒素,鲎试剂法检测疫苗中的内毒素含量,家兔法复试,ELISA法检测内毒素入血前后血浆中细胞因子TNF-α和IL-8的含量及疫苗的免疫原性,并比较处理前后的基因疫苗转染SMMC-7721和HepG2细胞的转染效率。结果经Triton X-114处理后,基因疫苗的内毒素含量及热原检测均合格;内毒素入血后,血浆中TNF-α和IL-8的含量均明显升高;疫苗的免疫原性与处理前比较,差异无统计学意义;处理后疫苗质粒的细胞转染效率增高。结论 Triton X-114能有效去除基因疫苗pVAX1-aLCGV中的内毒素,为其临床应用奠定了基础。

Development of A Method for Removal of Endotoxin from DNA Vaccine against Liver Cancer

Objective To develop a method for removal of endotoxin from DNA vaccine against liver cancer.Methods The endotoxin was removed from DNA vaccine pVAX1-aLCGV against liver cancer by Triton X-114 liquid phase separation,and the result was verified by pyrogen test in rabbits.The endotoxin content in vaccine was determined by limulus amoebocyte lysate(LAL)test.The TNF-α and IL-8 contents in plasma of rabbits before and after injection as well as serum antibody titers of rats after injection with the DNA vaccine were determined by ELISA.SMMC-7721 and HepG2 cells were transfected with the DNA vaccine before and after treatment with Triton X-114 separately,and the transfection efficacies were compared.Results The endotoxin content in DNA vaccine after treatment with Triton X-114 met the relevant requirements,and the vaccine was qualified in pyrogen test.The TNF-α and IL-8 contents in plasma of rabbits after injection with the DNA vaccine before treatment with Triton X-114 were significantly higher than those after treatment.However,the immunogenicity of DNA vaccine after treatment with Triton X-114 showed no significant difference with that before treatment,while the transfection efficacy of the former was higher than that of the latter.Conclusion Triton X-114 removed the endotoxin in DNA vaccine pVAX1-aLCGV effectively,which laid a foundation of clinical application of the vaccine.