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目的克隆人血红蛋白δ(delta)链蛋白基因,并在大肠杆菌中进行表达,经纯化与鉴定,获得δ链蛋白,为建立β-地中海贫血的免疫学筛查方法提供特异性抗原。方法从K562细胞中提取总RNA,采用RT-PCR技术获得目的片段并将其克隆到pET-32a及pET43.1a载体中,经PCR、酶切及测序验证,以IPTG诱导其在大肠杆菌中表达。包涵体经变性、复性后以Ni2+亲和层析柱纯化,可溶性蛋白直接以Ni2+亲和层析柱纯化。采用Western Blot和ELISA分析鉴定表达产物。结果成功构建两种融合表达载体pET32-Hbδ及pET43.1-Hbδ,分别为包涵体表达和可溶性表达,纯化产物经Western blotting和ELISA鉴定均为δ链蛋白。结论克隆表达并获得了纯化的人血红蛋白δ链,为后续的抗体制备及地中海贫血的免疫筛选方法的建立提供了抗原。
Objective To clone the human hemoglobin delta (delta) chain protein gene and express it in E.coli. After purification and identification, delta-chain protein was obtained, which provided specific antigen for immunological screening of beta-thalassemia. Methods Total RNA was extracted from K562 cells. The target fragment was obtained by RT-PCR and cloned into pET-32a and pET43.1a vector. The recombinant plasmid was induced by IPTG in E. coli . The inclusion bodies were denatured and renatured and purified by Ni2 + affinity chromatography. The soluble protein was directly purified by Ni2 + affinity chromatography. Western Blot and ELISA analysis were used to identify the expression product. Results Two fusion expression vectors, pET32-Hbδ and pET43.1-Hbδ, were successfully constructed and expressed in inclusion bodies respectively. The purified products were all identified as δ-chain proteins by Western blotting and ELISA. Conclusion Cloning and expression of purified human hemoglobin δ chain provided the antigen for the subsequent antibody preparation and establishment of immune screening method for thalassemia.