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目的:构建单链抗体scFv3的原核表达载体pET28a-scFv3并诱导表达,纯化并检测表达产物的免疫抗原性。方法:用PCR扩增抗肾小球基底膜(GBM)抗体单链抗体scFv3基因,构建pGEM-scFv3重组质粒并测序。将重组基因亚克隆于原核表达载体pET28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达,亲和层析法纯化,用间接ELISA检测纯化的单链抗体scFv3的免疫抗原性。结果:酶切和测序证实得到的scFv3基因序列正确。间接ELISA检测表明,纯化获得的scFv3能与抗GBM抗体结合。结论:成功构建了pET28a-scFv3原核表达系统并获得纯化的scFv3蛋白。表达产物具有良好的抗原性,为进一步研究该单链抗体在抗GBM抗体病中的治疗作用奠定了基础。
OBJECTIVE: To construct the prokaryotic expression vector pET28a-scFv3 of scFv3 and induce its expression, purification and detection of the immunogenicity of the expressed product. Methods: scFv3 gene of anti-glomerular basement membrane (GBM) antibody was amplified by PCR, and the recombinant plasmid pGEM-scFv3 was constructed and sequenced. The recombinant gene was subcloned into prokaryotic expression vector pET28a (+), transformed into E. coli BL21 (DE3), induced by IPTG and purified by affinity chromatography. The immunogenicity of scFv3 was detected by indirect ELISA. Results: The scFv3 gene sequence confirmed by restriction analysis and sequencing was correct. Indirect ELISA showed that purified scFv3 could bind to anti-GBM antibody. Conclusion: The prokaryotic expression system pET28a-scFv3 was successfully constructed and the purified scFv3 protein was obtained. The expression product has good antigenicity, which lays the foundation for further study on the therapeutic effect of the single chain antibody in anti-GBM antibody.