摘要本研究从转基因玉米基因组上成功克隆出潮霉素磷酸转移酶基因(hpt)，通过BamHⅠ和Hind III 双酶切作用，将hpt基因与pET30a进行粘性末端连接成功构建pET30a-hpt质粒，并转化到大肠杆菌BL21中进行蛋白表达。在异丙基硫代-β-D-半乳糖苷（IPTG）诱导下，pET30a-hpt融合表达载体在大肠杆菌BL21(DE3) 菌株中以可溶性蛋白的形式高效表达了潮霉素磷酸转移酶（HPT），并通过金属鏊和层析纯化了目的蛋白。用肠激酶切除了融合蛋白的融合部分之后再一次通过金属鏊和层析，经过透析后获得了HPT纯品,并且使用纯化的HPT蛋白免疫兔子制备了特异性强的多克隆抗体。
Abstract：Hpt gene was cloned from genetically modified plants’ genome and digested by BamHⅠ和Hind III. pET30a vector was digested by the same enzymes and ligated to hpt gene to construct plasmid pET30a-hpt. HPT protein was expressed by E.coli BL21 and purified firstly by ammonium sulfate, then by Ni+-NTA column. Hygromycin B phosphotransferase (HPT) was highly expressed in Escherichia coli BL21(DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) and most products existed in soluble form. After cleavage of His•Tag fusion protein by enterokinase, intact、biologically active HPT was released from the fusion protein and was purified to homogeneity with Ni2+ affinity chromatography. Polyclonal antibodies against were raised in three rabbits and the purified polyclonal anti-RCT antiserum appears a very high degree of sensitivity and specificity.
