重组人血红蛋白δ链的制备与鉴定

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Ni^2＋亲和层析柱纯化，可溶性蛋白直接以Ni^2＋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。

Preparation and Identification of Recombinant Human Hemoglobin 8 Chain

Objective To prepare recombinant human hemoglobin δ chain for the establishment of HbA2 immunoassay in the screening of β thalassemia. Methods Hbδ gene was cloned from K562 cells and amplified by RT-PCR. Two expression vectors were constructed. The fusion proteins were expressed in E. coli and identified by Western blotting and ELISA. Results Two expression vectors, namely pET32-Hbδ and pET43.1-Hbδ, were constructed and expressed in inclusion and soluble phase. The fusion protein was purified and was confirmed by Western blot and ELISA as Hbδ. Conclusion Two recombinant human hemoglobin 8 chains were obtained. The recombinant proteins may be used as immunogens for immunoassay of HbA2 in the screening of βthalassemia.