治疗性双质粒HBV DNA疫苗的纯化与检定

目的研究双质粒HBVDNA疫苗(pS2.S和pFP)的纯化工艺,并建立质控标准。方法首先对两工程菌(DH5α/pS2.S和DH5α/pFP)的高效发酵菌体采用碱裂解法进行质粒初提;然后通过三步柱层析(依次为分子筛层析、亲和层析、阴离子层析)进行质粒纯化,并检测质粒含量、超螺旋比例、内毒素等,最后对终产品质粒溶液进行全面质量检定。结果质粒初提液通过分子筛层析后,去除了大量的RNA、宿主DNA、内毒素等,再经亲和柱纯化后获得了高比例的超螺旋质粒DNA,达95%,最后经阴离子柱层析有效去除内毒素,并浓缩了质粒,质粒得率为0.9～1.1mg/g菌,质粒总回收率达78%。三批终产品全面质量检定均符合规定。结论建立了稳定的双质粒HBVDNA疫苗(pS2.S和pFP)的纯化工艺及质量控制标准,为进一步研究奠定基础。

Purification and Quality Control of Two Recombinant Plasmids as Therapeutic HBV DNA Vaccine

Objective To develop a procedure for purification and a standard for quality control of two recombinant plasmids(pS2.S and pFP)as therapeutic HBV DNA vaccine.Methods Extract plasmids from recombinant E.coli DH5α/pS2.S and DH5α/pFP by alkaline Lysis respectively and purify by three steps of chromatography,i.e.molecular sieve,affinity and anion exchange chromatography.Perform control tests on the purified plasmids.Results Most of foreign matters such as RNA,host DNA and endotoxin were removed from the crude extract of plasmids by molecular sieve chromatography.After affinity chromatography,the proportion of supercoiled plasmid DNA reached 95%.After anion exchange chromatography,endotoxin was removed effectively,and the plasmids were concentrated.The yield and total recovery rate of plasmids reached 0.9～1.1 mg/g bacteria and 78% respectively.All the quality control indexes of three batches of final products of plasmids met the relevant requirements.Conclusion A stable procedure for purification as well as a standard for quality control of recombinant plasmids pS2.S and pFP as therapeutic HBV DNA vaccine were developed,which laid a foundation for further study.