利用同尾酶技术构建pET15bPEP1CAT重组质粒
同尾酶,,],同尾酶;过氧化氢酶;TA克隆;DNA重组;PEP-1肽,1材料和方法,2结果,3讨论,[参考文献]
[摘 要] 目的: 利用同尾酶技术构建pET15b-PEP-1-CAT重组质粒，为制备PEP-1-CAT融合蛋白进而为防治心肌缺血再灌注损伤奠定基础。方法:设计并合成两端分别带SalⅠ和BglⅡ酶切位点的CAT引物，用PCR方法以pZeoSV2(+)-CAT质粒为模板，特异性扩增CAT 全长cDNA。将扩增的CAT cDNA 与dATP 反应,利用Taq DNA聚合酶具有的非模板依赖性末端转移酶活性在CAT cDNA的3′末端加"A"并纯化，然后应用TA克隆策略将纯化后的CAT cDNA插入至中间载体pGEM-T Easy Vector中，经PCR、限制性酶切分析鉴定重组子pGEM-T-CAT。人工合成编码PEP-1的双链寡核苷酸，两端分别引入NdeⅠ和XhoⅠ酶切位点，将其插入pET15b中得到重组质粒pET15b-PEP-1，然后酶切鉴定。pGEM-T-CAT和pET15b-PEP-1分别经SalⅠ-BglⅡ和XhoⅠ-BamHⅠ双酶切，利用同尾酶(SalⅠ与XhoⅠ，BglⅡ与BamHⅠ)能酶切产生相同黏性末端的特性，将回收得到的CATcDNA片段与pET15b-PEP-1相连，构建出重组质粒pET15b-PEP-1-CAT，经PCR及酶切鉴定，并通过核苷酸序列测序证实。 结果: 获得pET15b-PEP-1-CAT重组子，经测序分析证实，克隆入pET15b的PEP-1序列与设计合成的PEP-1序列一致，CAT序列与GeneBank中登录号为“AY028632”的CAT cDNA序列一致。 结论: pET15b-PEP-1-CAT的成功构建为产生融合蛋白His tag-PEP -1-CAT进而为防治心肌缺血再灌注损伤奠定了基础。[关键词] 同尾酶;过氧化氢酶;TA克隆;DNA重组;PEP-1肽
Construction of the Recombinant pET15b- PEP-1-CAT plasmid with Isocaudamer Technique
YAO Ling-ling, WANG Jia-ning*, HUANG Yong-zhang, et al.
(Institute of Clincal Medicine, Renmin Hospital, Yunyang Medical College, Shiyan, Hubei province 442000, China)
Abstract:Objective To construct the recombinant plasmid pET15b- PEP-1-CAT with isocaudamer technique and provide a vector expressing PEP-1-CAT fusion protein for the prevention and therapy of myocardial ischemia-reprefusion injury.Method Using pfu DNA polymerase,the full-length human catalase cDNA was specifically amplified via PCR from pZeoSV2(+)-CAT plasmid. The PCR product was added"A"by using the template-independent terminal transferase activity of Taq DNA polymerase. The purified products of CAT cDNA whose 3' end was added "A" were ligated with pGEM-T Easy vector ,and were transformed into DH5α. The correct recombinant was identified by PCR and SalⅠ-BglⅡ digestion and named after pGEM-T-CAT.Two oligoncleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide.The PEP-1 peptide sequence was directly ligated into NdeI-XhoI-digested pET15b.The recombinant plasmid was identified by double-enzyme digested and named after pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by XhoI-BamHI and SalI-BglⅡ,respectively. The purified linear fragment of pET15b-PEP-1 and the purified CAT cDNA fragment were ligated by using two pairs of isocaudamers which have different recognition sequences but produce compatible cohesive ends. The plasmid with the expected insert was selected using a XhoI restriction analysis and then analysed by sequencing. Results The recombinant plasmid was confirmed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT by sequencing were consistent with designed PEP-1 peptide and the human catalase cDNA sequence of GeneBank"AY028632", respectively. The pET15b-PEP-1-CAT of prokaryotic expression plasmid was constructed successfully. Conclusion Construction of pET15b-PEP-1-CAT recombinant plasmid provides a basis for production of His tag-PEP-1-CAT fusion protein to prevent and treat myocardial ischemia reperfusion injury. ......
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