摘要
目的构建pET15b-PEP-1-CAT原核表达质粒,并表达、纯化得到融合蛋白PEP-1-CAT,为防治与氧化应激有关的疾病奠定基础。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CATcDNA与dATP反应,利用TaqDNA聚合酶具有的非模板依赖性末端转移酶活性在CATcDNA的3'末端加“A”并纯化,然后应用TA克隆策略将纯化后的CATcDNA插入至中间载体pGEM-TEasyVector中得到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。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。将鉴定正确的pET15b-PEP-1-CAT重组质粒转化BL21(DE3),用IPTG诱导目的蛋白表达。然后利用重组蛋白N端的组氨酸“标签”(His-tag)对其进行金属螯合亲和层析纯化。结果测序分析证实,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CATcDNA序列一致。SDS-PAGE和Westernblot结果表明,pET15b-PEP-1-CAT在E.coli中获得可溶性高表达,经Ni2+-NTA-树脂柱亲和层析纯化得到了PAGE纯的融合蛋白PEP-1-CAT,并在体外检测到其具有特异的过氧化氢酶活性,活性值为77.15U/g。结论成功构建了原核表达载体pET15b-PEP-1-CAT,并经表达、纯化得到可以天然活性形式穿透细胞的重组过氧化氢酶蛋白,从而为防治心肌缺血再灌注损伤等与氧化应激有关的疾病奠定了基础。
Objective To construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-l-CAT. Methods Using pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2 （＋）-CAT plasmid, and the PCR product was added with ＂A＂ using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3＇ end was ligated with pGEM-T Easy vector and transformed into DH5α. The correct recombinant was identified by PCR and Sol Ⅰ /Bgl Ⅱ digestion and named as pGEM-T-CAT. Two oligoncleotides were synthesized and annealed to gencrate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho 1-digcsted pET15b.The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1, pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and So/I/Bgl Ⅱ, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E.coli BL21 （DE3） which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni~＋-NTA-resin column. The fusion protein PEP-l-CAT was produced and confirmed by specific enzyme activity in vitro. Results Sequence analysis showed that the PEP-1 and the human CAT eDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase eDNA sequence in GenBank （accession No. AY028632）, respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-I-CAT fusion protein with specific activity of 77.15 U/g. Conclusion The prokaryotic expression plasmid pET 15b-PEP- 1 -CAT has be
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2006年第9期1319-1325,共7页
Journal of Southern Medical University
