A Combination Strategy for Construction of Peptide-β2m-H-2Kb Single Chain with Overlap Extension PCR and One-Step Cloning

Tao Xu; Xiaoe Li; You Wu; Khawar Ali Shahzad; Wei Wang; Lei Zhang; Chuanlai Shen

doi:10.4014/jmb.1606.06038

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자료유형: 학술저널

저자정보: Tao Xu (Medical School Southeast University) Xiaoe Li (Medical School Southeast University) You Wu (Medical School Southeast University) Khawar Ali Shahzad (Medical School Southeast University) Wei Wang (Medical School Southeast University) Lei Zhang (Medical School Southeast University) Chuanlai Shen (Medical School Southeast University)

저널정보: 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제26권 제12호

발행연도: 2016.12

수록면: 2,184 - 2,191 (8page)

DOI: 10.4014/jmb.1606.06038

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The time-consuming and high-cost preparation of soluble peptide-major histocompatibility complexes (pMHC) currently limits their wide uses in monitoring antigen-specific T cells. The single-chain trimer (SCT) of peptide-β2m-MHC class I heavy chain was developed as an alternative strategy, but its gene fusion is hindered in many cases owing to the incompatibility between the multiple restriction enzymes and the restriction endonuclease sites of plasmid vectors. In this study, overlap extension PCR and one-step cloning were adopted to overcome this restriction. The SCT gene of the OVA257-264 peptide-(GS4)3-β2m-(GS4)4-H-2Kb heavy chain was constructed and inserted into plasmid pET28a by overlap extension PCR and one-step cloning, without the requirement of restriction enzymes. The SCT protein was expressed in Escherichia coli, and then purified and refolded. The resulting H-2Kb/OVA257-264 complex showed the correct structural conformation and capability to bind with OVA257-264-specific Tcell receptor. The overlap extension PCR and one-step cloning ensure the construction of single-chain MHC class I molecules associated with random epitopes, and will facilitate the preparation of soluble pMHC multimers.

#Overlap extension PCR #One-step cloning #recombinant gene #MHC tetramer #antigen-specific T cell

참고문헌 (16)

참고문헌 신청

Altman JD / 1996 / Phenotypic analysis of antigen-specific T lymphocytes / Science 274:94~96

Bryksin AV / 2010 / Overlap extension PCR cloning : a simple and reliable way to create recombinant plasmids / Biotechniques 48:463~465

Chen GJ / 2000 / Restriction site-free insertion of PCR products directionally into vectors / Biotechniques 28:498~500

Grozdanov PN / 2015 / Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly / J. Vis. Exp 96:e52235

Hansen T / 2009 / Current Protocols in Immunology / John Wiley & Sons, Inc :17.5.1~17.5.17

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