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CAT cDNA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H is tag-PEP-1-CAT进而为防治心肌缺血再灌注损伤奠定了基础。
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 ar, plified 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 DHSa. The correct recombinant was identified by PCR and Sal Ⅰ - BglⅡ digestion and named after pGEM - T - CAT. Two oligoncleotides were synthesized and annealed to generate a double - stranded olgonucleotide 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 isocaudameL~ 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 recom- binant＂ plasn,id provides a basis for production of His tag - PEP - 1 - CAT fusion protein to prevent and treat myocardial ischemia rcperfusion injury.
出处
《郧阳医学院学报》
2006年第1期1-5,F0002,共6页
Journal of Yunyang Medical College
