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目的:克隆、原核表达有酶活性的重组人精胺氧化酶(SMO)蛋白,并制备兔抗人SMO多克隆抗体。方法:采用RT-PCR法从人非小细胞肺腺癌A549细胞株总RNA中克隆人精胺氧化酶cDNA,构建SMO原核表达质粒pET-15b/SMO。将SMO原核表达质粒转化E.coli的BL21(DE3)菌并使用IPTG诱导重组SMO表达。表达的蛋白产物经Ni-NTA亲和层析纯化、透析复性后,化学荧光法测定其酶活性。使用制备性聚丙烯酰胺凝胶电泳分离、纯化重组人SMO蛋白,以此蛋白作为抗原皮内接种日本大耳白兔来制备抗人SMO多克隆抗体。分别以ELISA、Westernblot和免疫细胞化学法检测兔血清中抗体的滴度和抗原特异性。结果:纯化并透析复性后的重组人SMO蛋白具备快速氧化精胺的酶活性。用此重组蛋白制备的抗体具有较高抗体滴度和针对人SMO的特异性。结论:建立了人SMO原核表达、纯化系统,获得了有酶活性的高纯度人SMO蛋白并成功制备了兔抗人SMO的多克隆抗体,为以SMO为靶点的抗肿瘤基础和临床研究提供了有力的研究工具。
OBJECTIVE: To clone and express the prokaryotic recombinant human spermine oxidase (SMO) protein in prokaryotic cells and prepare the polyclonal antibody against rabbit SMO. Methods: Human spermine oxidase cDNA was cloned from total RNA of human non-small cell lung adenocarcinoma A549 cell line by RT-PCR and the prokaryotic expression plasmid pET-15b / SMO was constructed. The SMO prokaryotic expression plasmid was transformed into E. coli BL21 (DE3) bacteria and IPTG was used to induce recombinant SMO expression. The expressed protein product was purified by Ni-NTA affinity chromatography, dialyzed and refolded, and its enzymatic activity was determined by chemical fluorimetry. Recombinant human SMO protein was isolated and purified using preparative polyacrylamide gel electrophoresis, and polyclonal anti-human SMO antibodies were prepared by inoculating Japanese white rabbits with the protein as antigen. The antibody titers and antigenicities of the sera were detected by ELISA, Western blot and immunocytochemistry respectively. RESULTS: Purified and dialyzed renatured recombinant human SMO protein possessed rapid oxidized spermine enzyme activity. Antibodies prepared with this recombinant protein have higher antibody titers and specificity for human SMO. CONCLUSION: The prokaryotic expression and purification system of human SMO was established. High purity human SMO protein with enzyme activity was obtained and polyclonal antibody of rabbit anti-human SMO was successfully prepared. It provides a basis for anti-tumor study with SMO as the target and clinical research A powerful research tool.