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目的构建HSP65-6×(IA2-P2)-His表达载体并研究其在大肠杆菌中的表达及分离纯化。方法通过酶切酶连的方法将6×(IA2-P2)-His替换pET-28a-HSP65-6×P277中的6×P277,构建好的载体经DNA测序分析构建成功。乳糖诱导表达的蛋白经Ni+柱分离纯化。最后用SDS-PAGE及Western blot鉴定目的蛋白。结果 HSP65-6×(IA2-P2)-His最高表达量占菌体总蛋白的65%,超声破碎后蛋白以可溶性的形式存在于裂解上清中。Ni+柱纯化后蛋白纯度可达80%电泳纯以上。最后West-ern blot鉴定目的蛋白具有抗原性和His标签。结论重组载体pET-28a-HSP65-6×(IA2-P2)-His成功转入BL21中,以可溶性的形式高效表达,该工作为进一步验证融合蛋白的生物学活性奠定了一定的基础。
Objective To construct HSP65-6 × (IA2-P2) -His expression vector and study its expression in Escherichia coli and to isolate and purify it. Methods 6 × P277 in pET-28a-HSP65-6 × P277 was replaced by 6 × (IA2-P2) -His by restriction enzyme digestion. The constructed vector was successfully constructed by DNA sequencing. The lactose-induced protein was purified by Ni + column. Finally, the target protein was identified by SDS-PAGE and Western blot. Results The highest expression level of HSP65-6 × (IA2-P2) -His accounted for 65% of the total bacterial proteins. The protein was soluble in lysate supernatant after sonicated. Purity of Ni + column protein purity of up to 80% electrophoresis pure above. Finally West-ern blot identification of the target protein with antigenicity and His tag. Conclusion The recombinant vector pET-28a-HSP65-6 × (IA2-P2) -His was successfully transformed into BL21 and expressed in soluble form. This work laid a foundation for further verifying the biological activity of the fusion protein.