Production of antibody fragments against urokinase by Bacillus brevis and its application

构建了用于生产抗人尿激酶抗体片段（单链（sc）Fv、Fab '和scFab'）的表达-分泌载体，并将其导入短芽孢杆菌中。在载体中使用细胞壁蛋白基因操纵子（cwp）的启动子和来自mwp基因（短B.brevis的主要细胞壁蛋白之一的基因）的翻译起始位点和信号肽编码区。通过PCR从抗尿激酶型纤溶酶原激活物的鼠-人嵌合单克隆抗体的cDNA制备编码Fv和Fab ′片段的轻链和重链的cDNA，并将其插入信号肽编码区的紧下游。为了构建sc片段的基因，通过编码17或24个氨基酸的柔性肽接头的合成寡核苷酸连接编码轻链和重链的cDNA。短B能有效地产生FaB'，导致在培养基中积累100 mg/l。相反，短B.brevis产生的scFv和scFa B '的量在培养基中处于几mg/l的水平。纯化由短B.brevis产生的Fab B '，并证实其具有与亲本抗体相当的抗原结合活性。

Expression-secretion vectors for the production of anti-human urokinase antibody fragments, single chain (sc) Fv, Fab', and scFab', were constructed and inroduced into Bacillus brevis. The promoter of the cell wall protein gene operon (cwp), and the translation start site and the signal pepetide-encoding region from the mwp gene, the gene for one of the major cell wall proteins of B.brevis, were used in the vector. The cDNAs encoding the light and heavy chains of Fv and Fab' fragments were prepared by PCR from the cDNAs for mouse-human chimeric monoclonal antibody against urokinase-type plasminogen activator and inserted immediately downstream of the signal peptide-encoding region. To construct the genes for sc fragments, the cDNAs encoding the light and heavy chains were linked by synthetic oligonucleotides encoding flexible peptide linkers of 17 or 24 amino acids. Fab' was efficiently produced by B.brevis resulting in an accumulation at a level of 100 mg/I in the culture medium. In contrast, the amounts of scFv and scFab' produced by B.brevis were at a level of a few mg/I in the culture medium. Fab' produced by B.brevis was purified and confirmed to have an antigen binding activity comparable to that of the parental antibody.