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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (3): 62-69    DOI: 10.13523/j.cb.20180308
技术与方法     
基于mRNA 5'端TIR区二级结构优化提高重组sTNFα RI在大肠杆菌中的表达水平
秦娇荣,赵兆,罗心梅,李春阳()
成都生物制品研究所有限责任公司 成都 610023
Increasing the Expression Level of Soluble Tumor Necrosis Factor Type I Based on Optimization of Secondary Structure of mRNA 5' Terminal TIR
Jiao-rong QIN,Zhao ZHAO,Xin-mei LUO,Chun-yang LI()
Chengdu Institute of Biological Products Co. Ltd, Chengdu 610023,China
 全文: PDF(1937 KB)   HTML
摘要： 目的

通过优化PET11b-sTNFαRI 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-sTNFα RI 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pET11b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sTNFαRI序列一起克隆到pET11b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-sTNFα RI 5'TIR mRNA二级结构优化, 经SDS-PAGE和Western blot分析表明重组sTNFαRI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。

关键词： sTNFαRI;  mRNA二级结构;  翻译起始区;  自由能    
Abstract: Objective:

To increase the expression level of soluble tumor necrosis factor type-I receptor(sTNFαRI)in E.coli BL21(DE3)by optimizing the secondary structure of PET11b-sTNFaRI 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 PET11b-sTNFαRI in order to exposure of ribosome binding site and start codon to the outside of hairpin structure, in addition to mutating the pET11b 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 PET11b 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 sTNFαRI was increased by 50%~60%, after optimizing the secondary structure of 5' terminal TIR of PET11b-sTNFαRI.

 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.
Key words: sTNFαRI    mRNA secondary structure    Translation initiation region    Free energy
收稿日期: 2017-12-07 出版日期: 2018-04-04
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秦娇荣
赵兆
罗心梅
李春阳

引用本文:

秦娇荣, 赵兆, 罗心梅, 李春阳. 基于mRNA 5'端TIR区二级结构优化提高重组sTNFα RI在大肠杆菌中的表达水平[J]. 中国生物工程杂志, 2018, 38(3): 62-69.

Jiao-rong QIN, Zhao ZHAO, Xin-mei LUO, Chun-yang LI. Increasing the Expression Level of Soluble Tumor Necrosis Factor Type I Based on Optimization of Secondary Structure of mRNA 5' Terminal TIR. China Biotechnology, 2018, 38(3): 62-69.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180308    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I3/62

名称 序列
rbs1 UUCCCCUCUAGACUAAUAGAUUAAGGAGGUAGUAAAUGGAUAGCGUGUGUCCGCAGGGUAAAUAUAUUCA
rbs2 AUAGGAAGCCCUAUUAAUAGUUAAGGAGGUAGUAGAUGGAUAGCGUGUGUCCGCAGGGUAAAUAUAUUCA
rbs3 UAAGGGAGAUUCAAAAUAGCAUAAGGAGGUAAUCGAUGGAUAGCGUGUGUCCGCAGGGUAAAUAUAUUCA
rbs4 CCCCUCUAGAGUCAAAUAGAUUAAGGAGGUAUAAAAUGGAUAGCGUGUGUCCGCAGGGUAAAUAUAUUCA
Ori-sTNFαRI AUAAUUUUGUUUAACUUUAAGAAGGAGAUAUACAUAUGGAUAGCGUGUGUCCGCAGGGUAAAUAUAUUCA
表1  sTNFα 5'TIR区突变前和突变后序列对比
图1  sTNFαRI 5'TIR区二级结构模拟及总自由能预测
图2  序列相对自由能及位置熵预测
名称 序列
Rbs1 GCAACGTCTAGACTAATAGATTAAGGAGGTAGTAAATGGATAGCGT
Rbs2 GCAACGTCTAGAAACTTTAAGGGAGATTCAAAATAGCATAAGGAGGTAATCGATGGATAGCGT
Rbs3 GCAACGTCTAGATAGGAAGCCCTATTAATAGTTAAGGAGGTAGTAGATGGATAGCGT
Rbs4 GCAACGTCTAGAGTCAAATAGATTAAGGAGGTATAAAATGGATAGCGT
PP6 CGGAGG GGATCCTATTA ATTGAAGCACTGGAACAGG
表2  PCR突变引物
图3  sTNFαRI基因PCR扩增产物电泳图
图4  重组质粒双酶切鉴定电泳图
图5  小试诱导表达SDS-PAGE电泳分析(a)和Western blot 分析(b)
图6  Image Lab 4.0软件分析图
图7  sTNFαRI表达量对比
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