【摘要】 胰蛋白酶原 2是急性胰腺炎相关的敏感而特异的标志物,并且与多种肿瘤转移的过程密切相关.对胰蛋白酶原 2基因5’端4个氨基酸的密码子碱基进行简并突变,并在其3’端连接6个组氨酸编码序列构建含重组质粒pBVTg2的大肠杆菌工程菌─pBVTg2/DH5α.重组大肠杆菌经温度诱导后获得了高效表达,重组蛋白占总菌体蛋白的20%,并以包涵体形式存在.包涵体经尿素缓冲液裂解后,通过Ni NTA亲和树脂一步分离纯化.纯化蛋白经SDS PAGE分析及利用anti histidinemAb进行Westernblotting分析,以及蛋白N末端测序分析,结果表明与目的蛋白特征相符合.纯化的重组人胰蛋白酶原 2经复性后能与天然人胰蛋白酶原 2的单抗anti hTrypsinogen2mAb特异结合,复性后的蛋白在肠激酶作用后测得具有74BAEEU/mg的胰蛋白酶比活力,为进行胰蛋白酶原 2单克隆抗体的制备及其检测和应用奠定了基础.更多还原

【Abstract】 Recent research indicates that the human trypsinogen-2(Tg2) is a sensitive and specific diagnostic biomarker of severe acute pancreatitis(AP) and local expression of trypsinogen is found to be associated with tumor metastasis. The Tg2 cDNA was obtained from human pancreas cDNA library by PCR and cloned into the expression vector pBV220 with a 6×histidine tag at the C terminus to construct the recombinant plasmid pBVTg2. Then the plasmid pBVTg2 was transformed into E.coli DH5α. Upon temperature induction, about 20% expression level of trypsinogen-2 was achieved. After dissolved in 8 mol/L urea lysis buffer, the recombinant protein expressed as inclusion body was purified by Ni-NTA chelating resin, according to the high affinity of 6×histidine with Ni. The final product displayed a single band with a corresponding molecular weight 32 ku in SDS-PAGE. Western blotting showed that the anti-histidine mAb and the anti Tg2 mAb could bind with the purified Tg2 specifically. N-terminal amino acid sequencing indicated that the N-terminus of purified Tg2 possessed the same amino acid sequence as the natural one. And the specific activity of purified Tg2 through renaturation and activation by Enterokinase was 74 BAEE Units/mg. It shows that the purified Tg2 not only can be used to prepare the monoclonal antibody which can be used for clinical diagnosis, but also has potential application value to the study of its biological activities in the metastasis of tumor cells.更多还原