抗柑桔溃疡病菌可溶性单链抗体的表达及鉴定

前期采用核糖体展示技术筛选了抗柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,Xac)高亲和力单链抗体(Single chain variable fragment,scFv)GX13、GX44和GX95,为高效表达具有生物活性的单链抗体,本研究将前期筛选的高亲和力单链抗体基因转入大肠杆菌HB2151中进行可溶性表达,点印迹和Western印迹检测可溶性单链抗体的表达水平,亲和层析纯化表达的单链抗体,酶联免疫吸附法(ELISA)检测纯化单链抗体的抗原结合活性.结果显示,3株抗柑桔溃疡病菌可溶性单链抗体在大肠杆菌HB2151中均获得成功表达,表达产物分子量(Mr)约为32.0×103,主要集中于细菌周质腔中.ELISA检测纯化的单链抗体都具备抗原结合活性,可进一步应用于柑桔溃疡病菌的免疫诊断和病害防治.图4参14

Expression and Identification of Soluble Single Chain Variable Fragments Against Xanthomonas axonopodis pv. Citri

The single chain variable fragments (scFvs) GX13, GX44 and GX95 against Xanthomonas axonopodis pv. citri (Xac) had been selected by ribosome display in previous research. In order to express the scFvs with bio-activity efficiently, the genes of the selected scFvs were transformed into Escherichia coli HB2151 for expressing resoluble antibodies. The expression levels of scFvs were detected by dot blotting and Western blotting, and the expressed scFvs were purified by affinity chromatography, and then the activity of the purified scFvs was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the three scFvs specific for Xac were successfully expressed in E. coli HB2151. The molecular weight (M_r) of expressed products was about 32.0×10~3 and the products were mainly located in the periplasm. All the purified scFvs had the antigen-binding activity by ELISA and could be further used for immunoassay and disease control for citrus bacterial canker disease. Fig 4, Ref 14