无内毒素污染的基因工程鼠源SINE RNA的制备  

Preparation of endotoxin-free genetically engineered murine SINE RNA

作　　者：杨爽爽 曹洪益 闫丽芳 KHAN Murad 吉宁 刘鑫[2] 宋志学 张东明[2] 王秀芳[2] 吕占军[2] YANG Shuang-shuang;CAO Hong-yi;YAN Li-fang;KHAN Murad;JI Ning;LIU Xin;SONG Zhi-xue;ZHANG Dong-ming;WANG Xiu-fang;LV Zhan-jun(The College of Pharmacy,Hebei Medical University,Shijiazhuang 050017,Hebei Province,China;不详)

机构地区：[1]河北医科大学药学院,河北石家庄050017 [2]河北医科大学河北省实验动物重点实验室遗传研究室,河北石家庄050017

出　　处：《中国生物制品学杂志》2020年第11期1292-1300,共9页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81771499);河北省自然科学基金(H2018206099);河北省大学生创新性实验计划(USIP2017007)。

摘　　要：目的研究从大肠埃希菌宿主提取基因工程RNA同时去除内毒素的条件。方法将串联的小鼠短散布核元件(short interspersed nuclear elements,SINE)B1插入pET-28a(pET)构建pET-B1×16反序质粒(pET-B1as),转化大肠埃希菌BL21(DE3),获得pET-B1as-DE3菌。用TRlzol法、SDS-热酚法、SDS-NaCl离心法和SDS-NaCl过滤法从p ET-B1as-DE3菌制备基因工程RNA,Triton X-114相分离法进一步去除内毒素,检测制备的RNA中内毒素含量及RNA产量、纯度、完整性,并进行小鼠体内毒性试验和家兔热源试验。结果TRlzol法制备的RNA产量低;SDS-热酚法制备的RNA中内毒素含量高;SDS-NaCl过滤法去除内毒素的效果优于SDS-NaCl离心法,同时具有较高的RNA产量及较好的RNA完整性。小鼠静脉注射用SDS-热酚法和SDS-NaCl过滤法制备的RNA,前者引起小鼠不良反应,后者未引起小鼠不良反应。用Triton X-114相分离能进一步去除由SDS-NaCl过滤法制备的RNA中的内毒素,家兔热源试验证明SDS-NaCl过滤法联合Triton X-114相分离法制备的RNA,其热原符合动物体内试验要求。结论建立了用SDS-NaCl过滤法联合Triton X-114相分离法制备基因工程RNA的方法,可有效去除RNA中污染的内毒素。Objective To explore the condition for removing endotoxin while extracting genetically engineered RNA from E.coli host.Methods The tandem short interspersed nuclear elements(SINE)B1 of mice were inserted into vector pET-28α(pET).The constructed antisense plasmid pET-B1×16(pET-B1 as)was transformed to E.coli BL21(DE3)to obtain recombinant E.coli pET-B1 as-DE3.TRlzol,SDS-hot phenol,SDS-NaCl centrifugation and SDS-NaCl filtration methods were used to prepare genetically engineered RNAs from pET-B1 as-DE3.Triton X-114 phase separation was used to further remove endotoxin.The prepared RNA was determined for endotoxin content as well as yield,purity and integrity,and subjected to in vivo toxicity test in mice and pyrogen test in rabbits.Results The yield of RNA prepared by TRlzol method was low,while the endotoxin content of that prepared by SDS-hot phenol method was high.SDS-NaCl filtration method was superior to SDS-NaCl centrifugation in removing endotoxin,which resulted in high RNA yield and good RNA integrity.The RNA for intravenous injection prepared by SDS-hot phenol method caused adverse reactions in mice,while that by SDS-NaCl filtration caused no adverse reactions.The endotoxin in RNA prepared by SDS-NaCl filtration was further removed by Triton X-114 phase separation.Rabbit experiments proved that the RNA prepared by Triton X-114 combine with SDS-Na Cl filtration met the requirements of nucleic acid reagent for in vivo experiments in animals.Conclusion An experimental method for removing endotoxin from genetically engineered RNA by SDS-NaCl filtration combined with Triton X-114 phase separation was developed,which was sutiable for effectively removal of enotoxin contaminated in RNAs.

关 键 词：基因工程RNA 鼠短散布核元件 反义RNA 内毒素 

分 类 号：R915[医药卫生—微生物与生化药学]