建鲤组织蛋白酶L 的原核表达、纯化鉴定及多克隆抗体的制备

对原核表达的重组建鲤组织蛋白酶L(Cathepsin L,CAT L)蛋白进行尿素洗涤和Ni-NTA亲和层析纯化,该目的蛋白经300 mmol/L咪唑洗脱为单一峰,SDS-PAGE结合TSK-GEL G2000SWxl凝胶过滤高效液相色谱分析表明重组CAT L获得了高度纯化,分子量约28 k D,纯度超过95%。Z-Phe-Arg-MCA底物测活法显示该重组CAT L表现为半胱氨酸蛋白酶活性,能与其内源抑制因子Cystatin以1︰1的摩尔比结合,具有生物学活性。以纯化的重组CAT L蛋白免疫Balb/C小鼠获得抗血清,经ELISA法检测获得的CAT L抗血清效价高于1︰512000;Western blotting鉴定结果表明该抗体具有良好的特异性,能够识别原核表达的重组CAT L蛋白。免疫组织化学分析结果表明,该抗体还能识别建鲤小肠、肝胰脏、脾、背肌和心肌组织表达的内源性CAT L蛋白。因此可利用该抗体从蛋白水平检测CAT L在鱼类不同组织中的表达和分布情况。

Prokaryotic expression, purification, characterization, and polyclonalantibody preparation of Jian carp (Cyprinus carpio var. jian) CathepsinL

) expressed inprokaryotic cells was washed by a gradient of urea concentrations and then purified by Ni-NTA agarose affinitychromatography. The target protein appeared as a single peak when eluted by 300 mmol/L imidazole in affinitychromatography. SDS-PAGE analysis and gel-filtration HPLC on a TSK-GEL G2000SWxl column revealed thatrecombinant CAT L was highly purified, and the molecular weight was about 28 kD with purity greater than 95%.The activity assay with Z-Phe-Arg-MCA as a substrate indicated that the recombinant CAT L could combine withits endogenesis inhibitor of Cystatin at a 11 ratio, and thus took on the biological activity of cysteine protease.Balb/C mice were immunized by the purified protein to obtain antiserum, and ELISA showed that the titer of CATL antiserum was higher than 1512000. Western blotting showed that the CAT L polyclonal antibody was highlyspecific for recognizing recombinant CAT L protein expressed in prokaryotic cells. Immunohistochemistry analysisindicated that this antibody also recognized endogenous CAT L protein expressed in the hepatopancreas, muscle,small intestine, heart, and spleen of Jian carp. Based on these results, the polyclonal antibody obtained in thisstudy could be used to detect CAT L expression and distribution in different tissues of fish based on protein level.