重组降血压肽表达系统的构建及表达多肽的鉴定

Construction of the Expression system for Angiotensin-Converting Enzyme Inhibitor Peptide and Identification of the Expressed Peptide

通过对降血压肽结构和功能的分析,设计合成了一条小肽VPVLPK。通过测定其对ACE酶的抑制活性,发现此肽具有很好的降血压活性IC50为4.2μmol/L。根据大肠杆菌偏爱密码子人工合成此肽的6拷贝片段,将其克隆至表达载体pET-15b并转化到宿主菌E.coli BL21(DE3)中。筛选阳性克隆,通过双酶切、PCR及测序表明,本研究已成功构建出重组降血压肽质粒pET-15b-ACEIP的表达系统。IPTG诱导表达后的菌体通过Tricne-SDS-PAGE小分子多肽电泳检测在5.8～7.8kDa之间出现目的蛋白条带,这与理论蛋白分子量6.2kDa相符,且目的蛋白主要存在于超声破碎的上清液中。上清液用胰蛋白酶酶切后,通过HPLC鉴定证实为目的多肽,且多肽的含量达130mg/L。

According to the relationship between the function and the structure of Angiotensin-Converting Enzyme Inhibitor Peptide （ACEIP） , an angiotensin-eonverting enzyme inhibitor peptide （VPVLPK） was designed and synthesized. Results of the bioactivity measurement of the inhibitor to ACE enzyme showed that this pepfide effectively decreased blood pressure IC50 to 4.21amol/L. A six-copy fusion peptide was synthesized according to the code choice of Escheria. coli and then the corresponding coding gene was cloned into pET-15b. The fusion expression vector of pET-15b-ACEIP was finally tmnsfonned into E. coli BL21 （DE3）. The results of double enzyme-cleavage, PCR and the sequence analysis all indicated that the recombinant plasmid pET-15b-ACEIP was successfully constmcted. After induced by IPTG the expressed fusion protein was identified by Tricne-SDS-PAGE, which mainly existed in the supematant after sonication. The supematant was digested by trypsin and identified by HPLC, showing that the expressed pepetide can reach the level of 130mg/L.