【摘要】 利用RT-PCR技术,从稳定分泌抗人Lipocalin型前列腺素D合酶(Lipocalin-typeprostaglandinDsynthase,L-PGDS)单克隆抗体的鼠杂交瘤细胞中获得抗体可变区基因;通过一柔性连接短肽[(Gly)4Ser]3,SOEPCR法将此重、轻链可变区拼接成完整的抗人L-PGDS单链抗体基因,并装入表达载体pET-28a(+),在BL21宿主菌中进行表达。经SDS-PAGE检测显示,在低剂量0.02mmol/LIPTG诱导和较低温度25℃或28℃条件下培养,重组菌表达了一定量的可溶性单链抗体。后经Ni-NTA亲和层析柱纯化,得率为2mg/100ml,纯度达92%;双夹心ELISA和免疫荧光竞争抑制实验证实,纯化后的单链抗体具有较好的亲和力和特异性。更多还原

【Abstract】 The variable region genes of Lipocalin-type prostaglandin D synthase(L-PGDS) were cloned from hybridoma cells by RT-PCR, and then assembled to a whole ScFv gene by a linker [(Gly)4Ser]3. After connecting ScFv to the expression vector pET-28a(+), it was expressed in E.coli BL21. As a result, the recombined cells BL21 induced by 0.02 mmol/L and cultivated under 25℃ or 28℃ could express soluble ScFv protein. Then the soluble protein was purified by Ni-NTA affinity chromatography, and its concentration was found to be 2 mg/100 ml. By ELISA and immunofluorescence competitive test, the purified ScFv was proved to have antigen binding activity and specificity to L-PGDS.更多还原