不同试剂盒提取质粒中内毒素水平的比较
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摘要
目的比较几种不同质粒提取试剂盒提取质粒中内毒素水平。方法选用6种市售商业化试剂盒:QIAGEN质粒小提试剂盒Plasmid Mini Kit(12123),QIAGNE无内毒素质粒大提试剂盒EndoFree PlasmidMaxi Kit(12362),康为世纪高纯质粒中提试剂盒(CW0502),A厂家无内毒素质粒中提试剂盒,B厂家无内毒素质粒小提试剂盒,C厂家无内毒素质粒小提试剂盒(BSC01S2D)。分别提取质粒DNA,应用鲎试剂显色基质法和凝胶法检测所提质粒中内毒素水平。结果6种试剂盒提取质粒得率、A260/A280及A260/A230值均较好,但内毒素水平各不相同,其中各厂家标明无内毒素试剂盒提取质粒中内毒素水平相对较低,QIAGEN无内毒素质料大提试剂盒提取的质粒中内毒素小于0. 125 EU/μg。结论6种质粒提取试剂盒使用时应根据实际情况进行选择。
Objective To compare the endotoxin levels in plasmid purification with different plasmid DNA extraction kits. Methods Six commerical kits(QIAGEN Plasmid Mini Kit(12123),QIAGENEndoFree Plasmid Maxi Kit(12362),CWBIO PurePlasmid Midi Kit(CW0502),EndoFree PlasmidMidi Kit,Endo-free Plasmid Mini Kit,Endo-free Plasmid DNA Mini Extraction Kit)were applied topurify plasmid,endotoxin levels were determined with the gel-clot technique and Limulus amebocytelysate chromogenic method,respectively. Results The yields,A260/A280 and A260/A230 values of plasmidDNA extracted with the six kits were appropriate,the endotoxin levels were different,and theendotoxin levels extracted with endotoxin-free kits were relatively much lower. With QIAGENEndoFree Plasmid Maxi Ki(t12362),the endotoxin level was less than 0. 125 EU/μg. Conclusion Theendotoxin levels in plasmid DNA extraction with six kits were significantly different,which shall beselected according to the actual situation.
Objective To compare the endotoxin levels in plasmid purification with different plasmid DNA extraction kits. Methods Six commerical kits(QIAGEN Plasmid Mini Kit(12123),QIAGENEndoFree Plasmid Maxi Kit(12362),CWBIO PurePlasmid Midi Kit(CW0502),EndoFree PlasmidMidi Kit,Endo-free Plasmid Mini Kit,Endo-free Plasmid DNA Mini Extraction Kit)were applied topurify plasmid,endotoxin levels were determined with the gel-clot technique and Limulus amebocytelysate chromogenic method,respectively. Results The yields,A260/A280 and A260/A230 values of plasmidDNA extracted with the six kits were appropriate,the endotoxin levels were different,and theendotoxin levels extracted with endotoxin-free kits were relatively much lower. With QIAGENEndoFree Plasmid Maxi Ki(t12362),the endotoxin level was less than 0. 125 EU/μg. Conclusion Theendotoxin levels in plasmid DNA extraction with six kits were significantly different,which shall beselected according to the actual situation.
引文
[1]李卫玲,易初丽.质粒DNA纯化及内毒素去除策略研究进展[J].江汉大学学报(自然科学版),2018,46(1):62-66.
[2] BEUTLER B,RIETSCHEL E T. Innate immune sensing and its roots:the story of endotoxin[J]. Nat Rev Immunol,2003,3(2):169-176.
[3]国家药典委员会.中华人民共和国药典[S]. 2015年版.北京:中国医药科技出版社,2015.
[4]胡春生,张庆林,张通.基因治疗用质粒DNA生产面临的问题及研究进展[J].中国生物工程杂志,2011,31(4):119-123.
[5] PI W,SUN C,SONG Z,et al. Purification of plasmid DNA using anion-exchange chromatography and removal of endotoxin[J]. Se Pu,2007,25(6):809-813.
[6] SOUSA F,PRAZERES D M,QUEIROZ J A. Improvement of transfection efficiency by using supercoiled plasmid DNA puri?fied with arginine affinity chromatography[J]. J Gene Med,2009,11(1):79-88.
[7]张艳萍.细菌内毒素对基因免疫小鼠存活率和产仔率的影响[J].科技导报,2009,27(15):44-47.
[8] MA R,ZHAO J,DU H C,et al. Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction[J]. AnalBiochem,2012,424(2):124-126.
[9] ONGKUDON C M,HODGES E,MURPHY K,et al. Removal of endotoxins from plasmid DNA:analysis of aggregativeinteraction of mobile divalent metal cations with endotoxins and plasmid DNA[J]. J Sep Sci,2012,35(22):3208-3216.
[10] SAKATA M,UEZONO K,KIMURA K,et al.γ-cyclodextrin-polyurethane copolymer adsorbent for selective removal ofendotoxin from DNA solution[J]. Anal Biochem,2013,443(1):41-45.
[11] ONGKUDON C M,DANQUAH M K. Analysis of selective metal-salt-induced endotoxin precipitation in plasmid DNA purifi?cation using improved Limulus amoebocyte lysate assay and central composite design[J]. Anal Chem,2011,83(1):391-397.
[12] NEUN B W,DOBROVOLSKAIA M A. Detection and quantitative evaluation of endotoxin contamination in nanoparticle for?mulations by LAL-based assays[J]. Methods Mol Biol,2011,697:121-130.
[13] SU W,DING X. Methods of endotoxin detection[J]. J Lab Autom,2015,20(4):354-364.
[14] DULLAH E C,ONGKUDON C M. Current trends in endotoxin detection and analysis of endotoxin-protein interactions[J].Crit Rev Biotechnol,2017,37(2):251-261.
[15]陈祖欢,郭瀛军,章亚男,等. DNA疫苗的细菌内毒素检测[J].中国生物工程杂志,2004,24(1):81-83.
[16]向春和,宋志强,皮文辉.显色基质法定量测定质粒DNA中的细菌内毒素[J].石河子大学学报(自然科学版),2007,25(4):453-455.
[2] BEUTLER B,RIETSCHEL E T. Innate immune sensing and its roots:the story of endotoxin[J]. Nat Rev Immunol,2003,3(2):169-176.
[3]国家药典委员会.中华人民共和国药典[S]. 2015年版.北京:中国医药科技出版社,2015.
[4]胡春生,张庆林,张通.基因治疗用质粒DNA生产面临的问题及研究进展[J].中国生物工程杂志,2011,31(4):119-123.
[5] PI W,SUN C,SONG Z,et al. Purification of plasmid DNA using anion-exchange chromatography and removal of endotoxin[J]. Se Pu,2007,25(6):809-813.
[6] SOUSA F,PRAZERES D M,QUEIROZ J A. Improvement of transfection efficiency by using supercoiled plasmid DNA puri?fied with arginine affinity chromatography[J]. J Gene Med,2009,11(1):79-88.
[7]张艳萍.细菌内毒素对基因免疫小鼠存活率和产仔率的影响[J].科技导报,2009,27(15):44-47.
[8] MA R,ZHAO J,DU H C,et al. Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction[J]. AnalBiochem,2012,424(2):124-126.
[9] ONGKUDON C M,HODGES E,MURPHY K,et al. Removal of endotoxins from plasmid DNA:analysis of aggregativeinteraction of mobile divalent metal cations with endotoxins and plasmid DNA[J]. J Sep Sci,2012,35(22):3208-3216.
[10] SAKATA M,UEZONO K,KIMURA K,et al.γ-cyclodextrin-polyurethane copolymer adsorbent for selective removal ofendotoxin from DNA solution[J]. Anal Biochem,2013,443(1):41-45.
[11] ONGKUDON C M,DANQUAH M K. Analysis of selective metal-salt-induced endotoxin precipitation in plasmid DNA purifi?cation using improved Limulus amoebocyte lysate assay and central composite design[J]. Anal Chem,2011,83(1):391-397.
[12] NEUN B W,DOBROVOLSKAIA M A. Detection and quantitative evaluation of endotoxin contamination in nanoparticle for?mulations by LAL-based assays[J]. Methods Mol Biol,2011,697:121-130.
[13] SU W,DING X. Methods of endotoxin detection[J]. J Lab Autom,2015,20(4):354-364.
[14] DULLAH E C,ONGKUDON C M. Current trends in endotoxin detection and analysis of endotoxin-protein interactions[J].Crit Rev Biotechnol,2017,37(2):251-261.
[15]陈祖欢,郭瀛军,章亚男,等. DNA疫苗的细菌内毒素检测[J].中国生物工程杂志,2004,24(1):81-83.
[16]向春和,宋志强,皮文辉.显色基质法定量测定质粒DNA中的细菌内毒素[J].石河子大学学报(自然科学版),2007,25(4):453-455.