摘要
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。
AIM： To prepare recombinant human spermine oxidase （SMO） and polyclonal antibody against human SMO by gene recombination techniques. METHODS： Human SMO cDNA was amplified from total RNA of A549 cells through reverse transcription PCR. The cDNA was then cloned into pET-15b to construct SMO prokaryotic expression vector. After transforming, the vector was induced to express recombinant SMO by IPTG in E. coil BL21 （ DE3 ）. Recombinant SMO was purified by Ni-NTA resin under denaturing condition and then was dialyzed to renature. The enzyme activity of recombinant SMO was analyzed by chemical fluorescent method. SMO polyclonal antibody was prepared by using recombinant human SMO protein purified by polyacrylamide gel electrophoresis as antigen to inoculate rabbit intradermally. The titer and specificity of anti-sera were determined by ELISA, Western blot and Immune Cell Chem- istry. RESULTS： Purified and dialyzed recombinant human SMO has the specificicity of oxidizing the spermine. The polyclonal antibody has high titer and specificity against human SMO. CONCLUSION： This research established a method for prokaryotic expression, purification and polyclonal antibody preparation of human SMO. The method lays a foundation for the future functional research of SMO.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2009年第10期920-923,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30772590)
关键词
精胺氧化酶
原核表达
抗体制备
spermine oxidase
prokaryotic expression
antibody preparation
