人血红素加氧酶-1的原核表达、纯化及其中和抗体的制备

目的确定人血红素加氧酶-1（human heme oxygenase-1，hHO-1）在大肠埃希菌中的表达条件与纯化方法，利用纯化的蛋白制备具有中和活性的hHO—1多克隆抗体。方法将hHO-1原核表达质粒pMW172／hHO-1转人大肠埃希菌菌株BL21，通过改变摇床转速、诱导剂IPTG浓度和培养时间确定hHO-1蛋白的最佳可溶性表达条件；利用超声破碎、高速离心、分级盐析、分子筛层析等方法纯化hHO-1蛋白，建立体外HO-1活性测定方法检测hHO-1蛋白的活性；利用纯化的hHO-1活性蛋白作为抗原免疫新西兰兔，制备多克隆抗体；利用ELISA方法和Western印迹技术分别测定抗体的效价和特异性，通过HO-1活性测定检测抗体的中和活性。结果确定hHO-1最佳可溶性表达条件为：37℃、200r／min培养3h后，0．1 mmoL／LIPTG诱导培养4h。超声破碎菌体，上清经30％～40％盐析纯化及分子筛层析纯化，获得活性hHO-1蛋白，收得率为30．3％，纯化倍数为2．83倍，纯度为90％。制备的抗hHO-1的兔血清效价达到10^6，并能中和掉46％hHO-1的催化活性。结论为hHO-1蛋白的表达和纯化以及多克隆抗体制备确立了可行的技术方案；获得了高纯度活性hHO-1蛋白及hHO-1多克隆抗体，为HO-1功能、结构研究，以及相关疾病研究奠定了基础。

Expression and Purification of Human Heme Oxygenase-1 in E. coli BL21 and Preparation of Its Neutralizing Antibody

Objective To define optimal expression condition in E. coli and purification methods of human HO-1 （hHO-1）, and to prepare neutralizing antibody using the purified protein. Methods pMW172/hHO-1 plasmid coding hHO-1 was transformed into E. coli BL21. Ex- pression condition was optimized with different concentration of IPTG, rotation speed and time of shaking, hHO-1 protein purification of was performed by ultrasonication, high-speed centrifugation, salt fractionation and sieve chromatography, hHO-1 activity was examined by an established HO-1 in vitro activity assay. Antibodies against hHO-1 were prepared by immunizing New-Zealand rabbits with purified hHO-1. The titer and specificity of hHO-1 antibodies were determined by ELISA and Western Blot respectively, and the neutralizing activity of hHO-1 antibodies was determined by HO-1 activity assay. Results The optimal expression condition of hHO-1 was determined as 3 h cul- ture of E. coli BL21 at 200 r/min, 37℃ and subsequent d h induction by 0. 1 mmol/L IPTG. The purified HO-1 was obtained by 35 %-55 % salt fractionation and chromatography, with final purity of 90%, purification fold of 2. 83 and yield rate of 30. 3%. The polyclonal antibody specific to h HO-1 showed neutralizing activity, with high titer up to 10^6. Conclusions We established feasible experimental procedures for expression and purification of hHO-1 as well as the preparation of the polyclonal antibody specific to hHO-1. Our purified hHO-1 protein and specific hHO-1 polyclonal antibody are provided for study of HO-1 function and structure and HO-1 related diseases.