论文部分内容阅读
目的筛选可与A549细胞相互作用的抗旋毛虫7TR单链抗体。方法 PCR法克隆旋毛虫7TR基因,连接至表达载体p ET-32a,转化E.coli BL21(DE3),IPTG诱导后,将表达的7TR蛋白进行复性及亲合层析纯化;以纯化后的旋毛虫7TR蛋白为抗原,筛选Tomlinson(I+J)人源噬菌体单链抗体库,筛选并纯化阳性抗体,免疫荧光试验检测抗体与A549细胞的结合活性。结果重组表达质粒p ET-32a-7TR经双酶切及测序鉴定证明构建正确;表达的旋毛虫7TR蛋白相对分子质量约50 000。共获得2株稳定分泌抗旋毛虫7TR蛋白单链抗体的E.coli HB2151菌株,命名为4C7和8G5;8G5可与重组7TR蛋白发生特异性结合,具有良好的反应原性,也可与A549细胞发生特异性结合。结论筛选出了与A549细胞具有结合活性的抗7TR单链抗体,为后续的临床应用奠定了基础。
Objective To screen anti-Trichinella 7TR single chain antibody that interacts with A549 cells. Methods The Trichinella spiralis 7TR gene was cloned by PCR and ligated into the expression vector p ET-32a and transformed into E. coli BL21 (DE3). After IPTG induction, the expressed 7TR protein was renatured and purified by affinity chromatography. The Trichinella spiralis 7TR protein was used as antigen to screen out the Tomlinson (I + J) human phage scFv library, and the positive antibody was screened and purified. The binding activity of the antibody to A549 cells was detected by immunofluorescence assay. Results The recombinant plasmid p ET-32a-7TR was confirmed by double enzyme digestion and sequencing. The relative molecular mass of Trichinella spiralis 7TR protein was about 50,000. A total of 2 strains of E.coli HB2151 stably secreting anti-Trichinella 7TR single chain antibody were obtained and named as 4C7 and 8G5. 8G5 could specifically bind with recombinant 7TR protein and had good reactivity with A549 cells Specific binding occurs. Conclusion The anti-7TR single-chain antibody with the binding activity to A549 cells was screened, which laid the foundation for the subsequent clinical application.