基于mRNA 5’端TIR区二级结构优化提高重组sTNFαRI在大肠杆菌中的表达水平  

Increasing the Expression Level of Soluble Tumor Necrosis Factor Type Ⅰ Based on Optimization of Secondary Structure of mRNA 5’ Terminal TIR

作　　者：秦娇荣 赵兆[1] 罗心梅 李春阳[1] QIN Jiao-rong ZHAO Zhao LUO Xin-mei LI Chun-yang(Chengdu Institute of Biological Products Co. Ltd, Chengdu 610023 ,Chin)

机构地区：[1]成都生物制品研究所有限责任公司

出　　处：《中国生物工程杂志》2018年第3期62-69,共8页China Biotechnology

摘　　要：目的：通过优化PET11b-s TNFαRI 5＇mRNA翻译起始区（TIR）二级结构从而提高可溶性肿瘤坏死因子I型受体（sTNFαRI）在大肠杆菌[E.coli BL21（DE3）]中的表达水平。方法：通过对PET11b-s TNFαRI mRNA 5＇端TIR区二级结构的自由能及核苷酸位置熵分析,设计相应的引物对mRNA 5＇翻译起始区（TIR）相应密码子进行突变,从而使核糖体结合位点（RBS）及起始密码子（AUG）暴露于发夹结构之外,此外将p ET11b核糖体结合位点由GAAGGAGA突变为GAAGAA,以利于翻译复合体的组装以及翻译起始。通过基因克隆的方法将5＇端TIR区优化后的序列与s TNFαRI序列一起克隆到p ET11b载体中,并转化大肠杆菌BL21（DE3）,阳性转化子经IPTG诱导表达,SDS-PAGE和Western blot检测。结果：通过对PET11b-s TNFαRI 5＇TIR mRNA二级结构优化,经SDS-PAGE和Western blot分析表明重组s TNFαRI的表达水平较优化前提高50%~60%。结论：通过对重组载体翻译起始区（TIR）mRNA序列的二级结构优化可以有效提高目的蛋白的表达水平,对进一步工业化生产具有重要的应用价值。Objective： To increase the expression level of soluble tumor necrosis factor type-I receptor（ s TNFαRI） in E. coli BL21（ DE3） by optimizing the secondary structure of PET11 b-s TNFaRI translation initiation region（ TIR）. Methods： The free energy and nucleotide position entroy of the secondary structure of the translational initiation region was analyzed as the first step,and the primers were designed to mutate the codons of TIR of the PET11 b-s TNFαRI in order to exposure of ribosome binding site and start codon to the outside of hairpin structure,in addition to mutating the p ET11 b ribosome binding site from GAAGGAGA to GAAGAA in order to facilitate assembly of the translational complex and initiation of translation. The optimized sequence of 5＇terminal TIR was cloned into PET11 b vector and transformed into E. coli BL21（ DE3）. The positive transformants were induced by IPTG and analyzed by SDS-PAGE and Western blot. Results： SDS-PAGE and Western blot analysis showed that the expression of Recombinant s TNFαRI was increased by 50% ~ 60%,after optimizing the secondary structure of 5＇ terminal TIR of PET11 b-s TNFαRI. Conclusion： The optimization of the secondary structure of the translation initiation region（ TIR） mRNA of recombinant vector can effectively increase the expression level of the target protein,which is of great value for further industrialized production.

关 键 词：sTNFαRI MRNA二级结构 翻译起始区 自由能 

分 类 号：Q819[生物学—生物工程]