A Combination Strategy for Construction of Peptide-β2m-H-2Kb Single Chain with Overlap Extension PCR and One-Step Cloning
A Combination Strategy for Construction of Peptide-β2m-H-2Kb Single Chain with Overlap Extension PCR and One-Step Cloning
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DOI：
10.4014/jmb.1606.06038
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发表时间：
2016-12-01
影响因子：
2.8
通讯作者：
Shen, Chuanlai
中科院分区：
文献类型：
--
作者：
Xu, Tao;Li, Xiaoe;Shen, Chuanlai
目前，可溶性多肽主要组织相容性复合体(PMHC)的制备耗时且成本高，限制了其在监测抗原特异性T细胞方面的广泛应用。多肽-β2M-MHC-I类重链的单链三聚体(SCT)作为一种替代策略被开发出来，但由于多个限制性内切酶与质粒载体的限制性内切酶位点不相容，其基因融合在许多情况下受到阻碍。为了克服这一限制，本研究采用重叠延伸聚合酶链式反应和一步克隆的方法。构建了OVA(257-264)肽-(GS(4))(3)-β2M-(GS(4))(4)-H-2K(B)重链的SCT基因，通过重叠延伸聚合酶链式反应和一步克隆的方法将其插入到pET28a中，不需要限制性内切酶。在大肠杆菌中表达SCT蛋白，纯化后复性。得到的H-2K(B)/OVA(257-264)复合体显示出正确的结构构象和与OVA(257-264)特异性T细胞受体结合的能力。重叠延伸聚合酶链式反应和一步法克隆可确保构建与随机表位相关的单链MHC I类分子，并有助于制备可溶的pMHC多聚体。
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-beta 2m-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 OVA(257-264) peptide-(GS(4))(3)-beta 2m-(GS(4))(4)-H-2K(b) 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-2K(b)/OVA(257-264) complex showed the correct structural conformation and capability to bind with OVA(257-264)-specific T-cell 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.