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目的利用原核表达系统融合表达人胰岛素样生长因子-1(human insulin-like growth factor-1,hIGF-1),并进行纯化及鉴定。方法根据大肠埃希菌密码子偏好性对hIGF-1天然基因序列进行同义突变,并人工合成,PCR扩增后,克隆至pET48b(+)载体,构建重组原核表达质粒,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的融合蛋白Trx A-hIGF-1经Ni2+亲和层析进行纯化,纯化产物经肠激酶酶切后,进行Western blot鉴定。结果重组表达质粒pET48b-Trx A-hIGF-1经菌落PCR及测序证实构建正确;表达融合蛋白相对分子质量约为26 000,表达量约为菌体总蛋白的40%,主要以可溶形式存在于菌体裂解上清中,可溶性蛋白占总目的蛋白的比例不低于90%,纯化后纯度不低于90%,蛋白浓度为0.5 mg/ml;纯化的Trx A-hIGF-1融合蛋白可被肠激酶切割为相对分子质量约为8 000的hIGF-1蛋白和18 000的Trx A蛋白,hIGF-1蛋白可与兔抗hIGF-1多克隆抗体特异性结合。结论成功表达了hIGF-1融合蛋白,为其生物学活性的研究及规模化生产奠定了基础。
Objective To express human insulin-like growth factor-1 (hIGF-1) by using prokaryotic expression system and to purify and identify it. Methods According to the codon preference of Escherichia coli, the hIGF-1 natural gene sequence was synonymous mutated and synthesized by PCR. After amplification by PCR, it was cloned into pET48b (+) vector to construct recombinant prokaryotic expression plasmid, which was transformed into Escherichia coli BL21 (DE3), IPTG induced expression. The expressed fusion protein Trx A-hIGF-1 was purified by Ni2 + affinity chromatography. The purified product was digested with enterokinase and identified by Western blot. Results The recombinant plasmid pET48b-Trx A-hIGF-1 was constructed correctly by colony PCR and sequencing. The relative molecular mass of the expressed fusion protein was about 26 000 and the expression level was about 40% of the total bacterial proteins, mainly in soluble form In the cell lysate supernatant, the ratio of the soluble protein to the total protein was not less than 90%, the purity of the purified protein was not less than 90% and the protein concentration was 0.5 mg / ml. The purified Trx A-hIGF-1 fusion protein The enterokinase cleaves hIGF-1 protein with a molecular weight of about 8000 and TrxA protein with 18000, and the hIGF-1 protein can specifically bind with the rabbit anti-hIGF-1 polyclonal antibody. Conclusion The hIGF-1 fusion protein was successfully expressed, which laid the foundation for its biological activity and large-scale production.