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目的构建和表达日本血吸虫单克隆抗独特型抗体NP30单特异性双链抗体,初步鉴定表达产物的活性。方法用overlap PCR法扩增双链抗体基因VH-GGGGS-VL,将双链抗体基因重组入原核表达载体pBAD/gⅢ。表达质粒转化E.coli TOP10F′,左旋阿拉伯糖诱导表达。对表达产物进行分离纯化,ELISA检测纯化蛋白与血吸虫病人血清抗体的结合活性。结果测序证实双链抗体基因正确,构建了双链抗体的原核表达系统,双链抗体在细菌超声上清和沉淀内均有表达,分子量约为34kD。纯化产物经ELISA鉴定,结果表明NP30单特异性双链抗体可与血吸虫病人血清抗体特异性结合。结论构建和表达的日本血吸虫单克隆抗独特型抗体NP30单特异性双链抗体具有与亲本单抗相同的结合活性。
Objective To construct and express the monospecific anti-idiotypic antibody NP30 monospecific diabody of Schistosoma japonicum and to identify the activity of the expressed product. Methods The double-stranded antibody gene VH-GGGGS-VL was amplified by overlap PCR, and the double-stranded antibody gene was recombined into prokaryotic expression vector pBAD / gⅢ. The expression plasmid was transformed into E.coli TOP10F ’and induced by L-arabinose. The expressed product was isolated and purified, and the binding activity of the purified protein to the serum antibody of schistosoma patients was detected by ELISA. Results The correct double-stranded antibody gene was confirmed by sequencing. The prokaryotic expression system of the double-stranded antibody was constructed. The double-stranded antibody was expressed in both bacterial supernatant and sediment. The molecular weight was about 34kD. The purified product was identified by ELISA, the results showed that NP30 monospecific diabodies with schistosomiasis serum antibody specific binding. CONCLUSION: The constructed and expressed monovalent anti-idiotypic antibody NP30 monospecific diabody of Schistosoma japonicum has the same binding activity as the parental mAb.