重组降血压肽ACEIP的表达及活性鉴定

Expression and Activity Identification of Recombinant Antihypertensive Peptide(Angiotensin I-Converting Enzyme Inhibitory Peptide)

选择降血压肽WQVLPNAVPAK,人工合成大肠杆菌密码子优化后六串联体血管紧张素转换酶抑制肽(angiotensin I-converting enzyme inhibitory peptide,ACEIP)基因片段ACEIP,将其克隆至表达载体pET30a后构建重组质粒pET30a-ACEIP。该重组质粒经限制性内切酶Nde I和HindIII双酶切鉴定、菌落聚合酶链式反应鉴定后,将其化转至大肠杆菌BL21(DE3)感受态细胞中,构建了表达工程菌大肠杆菌BL21(DE3)/pET30a-ACEIP。工程菌经终浓度为0.8 mmol/L异丙基-β-D-硫代半乳糖苷导后,Tricine-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示在分子质量约为8.7 kDa处出现一条明显条带。经Ni^(2+)亲和层析、肠激酶酶切后纯化得到串联多肽ACEIP。其中融合蛋白的表达量为61.30 mg/L,串联多肽ACEIP的表达量为57.84 mg/L。串联多肽经胰蛋白酶酶解后的IC_(50)值为6.39 mg/m L,ACE抑制活性高于单体肽WQVLPNAVPAK的抑制活性(IC_(50)为12.34 mg/mL)。

In this study,the antihypertensive peptide WQVLPNAVPAK was selected for the synthesis of the gene encoding the hexameric peptide angiotensin I-converting enzyme inhibitory peptide(ACEIP)based on the codon preference of Escherichia coli.The synthetic gene was cloned into the expression vector pET30a to construct recombinant plasmid pET30a-ACEIP.The recombinant plasmid was identified by double digestion with restriction endonucleases NdeI and HindIII and polymerase chain reaction(PCR),and transformed into competent E.coli BL21(DE3)cells to construct the expression system E.coli BL21(DE3)/pET30a-ACEIP,which was induced with isopropyl-β-D-thiogalactopyranoside(IPTG)at a final concentration of 0.8 mmol/L.A clear band of 8.7 kDa was seen on Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The hexameric peptide ACEIP was purified by Ni^(2+)affinity chromatography and the expression of the fusion protein was 61.30 mg/L.The expression of ACEIP was 57.84 mg/L.The half-maximal inhibitory concentration(IC_(50))of the hexameric peptide was 6.39 mg/mL after trypsin digestion,whose ACE inhibitory activity was higher than that of the monomeric peptide WQVLPNAVPAK(IC_(50) of 12.34 mg/mL).