特异性结合革兰氏阴性菌外膜蛋白C的单链抗体核糖体展示文库的构建

Constmction of single chain antibody fragmenl library binding specially with G-outer membrane protein C for ribosome display

以纯化的重组大肠杆菌外膜蛋白Omp C免疫BALB/c小鼠,提取骨髓浆细胞总RNA,逆转录获得c DNA,作为模板通过PCR扩增获得重链和轻链可变区序列。由(Gly4Ser)3 linker连接两片段,获得单链抗体可变区片段基因库。重叠延伸PCR添加T7启动子、核糖体结合位点、gⅢtether和两端茎环结构等元件,成功构建对外膜蛋白C具有特异性亲和力的单链抗体可变区的核糖体展示文库,为特异性强、亲和力高的单链抗体可变区筛选提供了技术和材料基础。构建能与革兰氏阴性菌外膜蛋白C特异性结合的鼠源单链抗体核糖体展示文库,是奠定构建饲料革兰氏阴性腐败菌分子预警和靶向抗菌剂的靶向定位区的高亲和力单链抗体可变区片段的重要基础,对建立有害革兰氏阴性菌分子检测方法和靶向抗革兰氏阴性菌菌剂设计方法具有借鉴作用。

In order to develop a new and efficient feed gram-negative pathogens detection agent or guiding domain for the designing of bactericidal antibody drug conjugate,a single chain antibody fragment （scFv）library against outer membrane protein C （OmpC）for ribosome display was constructed in this study.Firstly,the BALB/c mice were immunized with OmpC.The total RNA was extracted from the bone marrow plasma cells, and followed by being reverse transcribed to cDNA.Then,the heavy （VL）and light （VH）chain variable regions were amplified by PCR using the above cDNA as tem- plates,respectively.Both VL and VH regions were combined by （Gly4Ser）3 linker by overlapping PCR.The promoter T7, Shine Dalgarno（SD）,gⅢ tether and loop regions were fused into one fragment of scFv by the overlapping PCR.Finally,the scFv library against OmpC was constructed successfully.The construction of ribosome library provided a reliable technical basis for the screening of high specificity and high affinity binding antibody fragment.