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抗人膀胱癌单链抗体的构建及表达成功为高特异性诊断和治疗膀胱癌带来了新的途径.本研究对其进行表达和复性研究,为其进入中试和临床应用奠定了基础.具体过程和方法如下.采用本室克隆的抗人膀胱癌抗体重轻链可变区基因以及构建的适于单链抗体表达的谷胱甘肽流基转移酶(GST)融合表达载体pROH80,在大肠杆菌XL1-blue中经1mM IPTG,37℃诱导表达抗人膀胱癌单链抗体.SDS-PAGE和Westem印迹分析表明,融合型单链抗体以包涵体形式存在于超声沉淀中,其表达量可达细菌总蛋白量的30%,而空载体表达GST主要以可溶形式存在于超声上清中.降低诱导强度(0.1mM OPTG,28℃)表达并未影响包涵体形式表达.此外,更换其它培养基,如SB2和M9CAA,也未见明显效果.于是,我们对包涵体进行了一系列复性研究.
The construction and expression of anti-human bladder cancer single-chain antibody has brought new methods for the high-specific diagnosis and treatment of bladder cancer. This study has carried out the expression and renaturation research, which laid the foundation for its entry into the pilot and clinical application. Specific procedures and methods are as follows. The anti-human bladder cancer antibody heavy light chain variable region gene cloned in this laboratory and the constructed glutathione flow transferase (GST) fusion expression vector pROH80 suitable for single chain antibody expression, In Escherichia coli XL1-blue, 1 mM IPTG was used to induce the expression of anti-human bladder cancer single-chain antibody at 37°C. SDS-PAGE and Western blot analysis showed that the fusion-type single-chain antibody existed in the form of inclusion bodies in the ultrasonic precipitation, and its expression level It is up to 30% of total bacterial protein, whereas empty vector-expressing GST is mainly present in the sonicated supernatant in a soluble form. Decreased induction intensity (0.1 mM OPTG, 28°C) expression does not affect expression in the form of inclusion bodies. In addition, replacement Other media, such as SB2 and M9CAA, also had no obvious effect. Therefore, we performed a series of renaturation studies on inclusion bodies.