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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。
The prokaryotic recombinant protein of Cathepsin L (CAT L) was purified by urea and Ni-NTA affinity chromatography. The target protein was eluted as a single peak by 300 mmol / L imidazole. SDS-PAGE TSK-GEL G2000SWxl gel filtration high performance liquid chromatography analysis showed that the recombinant CAT L was highly purified, with a molecular weight of about 28 kD and a purity of over 95%. The Z-Phe-Arg-MCA substrate assay showed that the recombinant CAT L exhibited cysteine protease activity and was able to bind with its endogenous inhibitory factor Cystatin at a molar ratio of 1: 1 and was biologically active. The antiserum was obtained by immunizing Balb / C mice with purified recombinant CAT L protein. The titer of antiserum against CAT L was higher than 1︰512000 by ELISA assay. The result of Western blotting showed that the antiserum had good specificity and could recognize Prokaryotic expression of recombinant CAT L protein. The results of immunohistochemistry showed that this antibody could recognize the endogenous CAT L protein expressed in the intestine, hepatopancreas, spleen, back muscle and myocardium of Jian carp. Therefore, this antibody can be used to detect the expression and distribution of CAT L in different tissues of fish from the protein level.