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High-level Expression of Recombinant Human Interferon-α2b in Escherichia Coli BL21 by Mutating Its 5’ End

Author: WangCanJun
Tutor: SongLiHua；ZouWenYi
School: Anhui Medical University,
Course: Biochemistry and Molecular Biology
Keywords: Human Interferon-α2b site-directed mutagenesis rare codon mRNA secondary structure gene expression Escherichia coli
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 81
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Abstract


Objective To construct the prokaryotic expression system of recombinant human interferon-α2b(rhIFN-α2b) and improve the expression level of rhIFN-α2b in Escherichia coli (E.coli) by mutating its 5’ end and selection of optimal host strains from E.coli DH5α, E.coli BL21, E.coli Rosetta-gami B (DE3). Methods The 5’ end sequences of hIFN-α2b genes were mutated by site-directed mutagenesis and the sequences of hIFN-α2b genes were amplified with PCR according to the frequency table of gene codon in E.coli without changing their amino acid sequences, then cloned into the cloning vector pUC19, and pUC19-rhIFN-α2b’ vector was transformed into competent cell of E.coli DH5α. The positive clones were identified by blue-white screen and DNA sequencing, and rhIFN-α2b’ was cloned into the temperature-controlled expression vector pJW2, the recombinant pJW2-rhIFN-α2b’ vector was identified by NdeⅠ/BamHⅠdigest, PCR identification and DNA sequencing. pJW2-rhIFN-α2b and pJW2-rhIFN-α2b’ were transformed into E.coli DH5α, E.coli BL21, E.coli Rosetta-gami B (DE3) competent cells respectively, the positive recombinant pJW2-rhIFN-α2b and pJW2-rhIFN-α2b’ clones were screened and expressed under 42℃temperature-induced, and the expression productions were investigated by SDS-PAGE, the optimal host bacteria was selected from E.coli DH5α, E.coli BL21and E.coli Rosetta-gami B (DE3). The positive pJW2-rhIFN-α2b’/BL21 clones were screened and expressed under 42℃temperature-induced, and the expression productions were investigated by SDS-PAGE, Western blot analysis and WISH-VSV system to indentify anti-virus vitalism. Resμlts The resμlt of NdeⅠ/BamHⅠdigestion, PCR identification and DNA sequencing showed that the recombinant pJW2-rhIFN-α2b’ vector was successfμlly constructed; The analysis of SDS-PAGE showed that the expression of rhIFN-α2b in pJW2-rhIFN-α2b’/BL21 was much high than that in pJW2-rhIFN-α2b’/DH5αor pJW2-rhIFN-α2b’/Rosetta-gami B (DE3), and it also indicated that the rhIFN-α2b was expressed in inclusion bodies in E. coli BL21 with the yield accounting for 23.6% of total bacteria proteins. Western blot analyses showed that the expression productions had the immunogenicity of hIFN-α2b and WISH-VSV verified the specific activitity of purified rhIFN-α2b was able to obtain 1.2×108 IU/mg similar to that of no-mutated engineered strain. Conclusion The expression level of rhIFN-α2b in E.coli coμld be increased by mutating its 5’ end, and the efficient expression system of rhIFN-α2b in E.coli BL21 was constructed successfμlly.

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