野生型及突变型人Toll样受体23'非编码区基因重组质粒的构建及鉴定  

Construction and identification of wild/mutant type recombinant plasmid with 3'-terminal untranslated region of Toll-like receptor 2

作　　者：包丽丽 孙传凯 李晓玫 任向宇 殷兆丽 孙晓琳 福泉[2] 纳仁高娃 BAO Li-li;SUN Chuan-kai;LI Xiao-mei;REN Xiang-yu;YIN Zhao-li;SUN Xiao-lin;FU Quan;NAREN Gao-wa(Department of Basic Medicine,Inner Mongolia Medical University,Hohhot 010059,Inner Mongolia Autonomous Region,China;不详)

机构地区：[1]内蒙古医科大学基础医学院,内蒙古呼和浩特010059 [2]内蒙古医科大学附属医院,内蒙古呼和浩特010050

出　　处：《中国生物制品学杂志》2022年第5期557-561,共5页Chinese Journal of Biologicals

基　　金：内蒙古自治区自然科学基金(2015MS0888、2019MS08113);内蒙古自治区高等学校科学技术研究项目基金(NJZZ20128);内蒙古自治区蒙医药协同创新培育中心项目基金(MYYXT201908,MYYXT202004);内蒙古医科大学“致远”人才计划项目(ZY0201020);内蒙古医科大学“大创计划”项目(202110132004);内蒙古自治区卫生健康科技计划项目(20221197)。

摘　　要：目的构建野生型及突变型人Toll样受体2(Toll-like receptor 2,TLR2)3'非编码区(3'UTR)基因重组质粒,并进行鉴定。方法提取293T细胞基因组DNA,以其为模板进行PCR扩增,获得TLR2的3'UTR基因片段,连接至载体pmiR-RB-REPORTTM,转化感受态E.coli DH5α,于LB(Amp+)固体培养基中培养过夜,菌落PCR筛选阳性克隆,并测序。根据TLR23'UTR与miR-122的预测结合靶点,设计突变型TLR23'UTR扩增引物,以构建的野生型TLR23'UTR重组质粒为模板,PCR扩增突变序列,相同方法进行载体连接及转化,挑取菌落,测序。结果野生型TLR23'UTR重组菌经菌落PCR鉴定,可见1000 bp的目的基因片段,大小与预期相符;测序结果表明,插入序列与TLR23'UTR序列完全一致。突变型TLR23'UTR重组质粒测序鉴定结果表明,靶序列CACTCC已更改为GTGAGG。结论成功构建了野生型及突变型人TLR23'UTR重组质粒,本实验为TLR2功能的深入研究提供了实验依据。Objective To construct and identify the wild/mutant type recombinant plasmid with 3'-terminal untranslated region(UTR)of Toll-like receptor 2(TLR2).Methods Genomic DNA was extracted from 293T cells and used as a template for amplification of TLR23'UTR gene fragment by PCR.The PCR product was inserted into vector pmiR-RB-REPORTTM,and the constructed recombinant plasmid was transformed to E.coli DH5αand cultured overnight in LB(Amp+)solid medium.The positive colonies were screened by PCR and sequenced.According to the predicted binding target of mi R-122and TLR23'UTR,the primers for amplification of mutant type TLR23'UTR was designed.The mutant sequence was amplified by PCR using the constructed wild type recombinant plasmid with TLR23'UTR as template,inserted into the vector and transformed into DH5α,and the positive colonies were screened and sequenced.Results The PCR of wild type TLR23'UTR showed the target gene fragment at a length of 1000 bp which was consistent with that expected.The sequencing result showed that the inserted sequence was completely consistent with that of TLR23'UTR.However,the sequencing result of mutant type recombinant plasmid with TLR23'UTR showed the change of target sequence from CACTCC to GTGAGG.Conclusion Wild/mutant type recombinant plasmid with TLR23'UTR was constructed successfully,which provided an experimental basis for further study on the function of TLR2.

关 键 词：TOLL样受体2 3'非编码区 基因重组 

分 类 号：R334[医药卫生—人体生理学]