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目的探讨抗红细胞H抗原单链抗体(scFv)中可变区重链(VH)和轻链(VL)顺序对该单链抗体活性的影响。方法采用PCR方法从质粒pMD-18T-2E8scFv(VH-linker-VL型)中克隆出VL和VH可变区基因,通过重叠引物延伸法将这2种基因连接,并引入连接肽(linker)编码序列,构建VL-linker-VH型的单链抗体基因,将VL-linker-VH和原有的VH-linker-VL2种形式的单链抗体基因分别插入到原核表达载体pET-his中,转化BL21(DE3)plysS细胞,异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,表达产物用镍亲和层析柱纯化;免疫荧光法、竞争ELISA和低离子聚凝胺法检测纯化后scFv活性。结果酶切及测序表明VL-linker-VH型单链抗体表达载体构建成功,SDS-PAGE和Western blot分析表明VL-linker-VH和VH-linker-VL2型抗人红细胞scFv均在BL21菌中获得高效表达,重组蛋白的相对分子质量(Mr)为27kD,表达产物以包涵体形式存在,分别占菌体总蛋白的26.8%和27.6%,纯化后获得了高纯度的scFv;免疫荧光、竞争ELISA和低离子聚凝胺试验分析证明2种单链抗体均可特异结合红细胞表面H抗原,VH-linker-VL结合活性高于VL-linker-VH。结论成功制备了VL-linker-VH型2E8scFv;VH-linker-VL型2E8scFv抗原的纯合活性高于VL-linker-VH型2E8scFv。
Objective To investigate the effect of variable region heavy chain (VH) and light chain (VL) sequences on the activity of this single chain antibody in anti-erythrocytic H-antigen single chain antibody (scFv). Methods The VL and VH variable region genes were cloned from plasmid pMD-18T-2E8scFv (VH-linker-VL) by PCR. The two genes were ligated by overlap primer extension and introduced with linker coding VL-linker-VH and single-chain VH-linker-VL2 single-chain antibody genes were inserted into the prokaryotic expression vector pET-his and transformed into BL21 (DE3) plysS cells induced by isopropylthio-β-D-galactoside (IPTG), and the expressed products were purified by nickel affinity chromatography. Immunofluorescence, competitive ELISA and low ion polybrene assay Purified scFv activity. Results The digestion and sequencing of VL-linker-VH scFv showed that VL-linker-VH scFv was successfully constructed in BL21 by SDS-PAGE and Western blot analysis Highly expressed recombinant protein had a molecular weight of 27 kD and expressed as inclusion bodies, accounting for 26.8% and 27.6% of the total bacterial proteins, respectively. High purity scFv was obtained after purification. Immunofluorescence and competition ELISA And low ion polyamine test analysis showed that both single chain antibodies can specifically bind to the red blood cell surface antigen H, VH-linker-VL binding activity than VL-linker-VH. Conclusion The VL-linker-VH 2E8scFv was successfully prepared. The homozygous activity of VH-linker-VL 2E8scFv antigen was higher than that of VL-linker-VH 2E8scFv.