为建立可产业化的大肠杆菌表达的重组新蛭素(neorudin,EH)生产工艺,主要采取从低密度到高密度发酵的优化思路,优化了发酵罐培养基和诱导时间;比较了超声破碎和反复冻融的目的蛋白提取方法;纯化工艺采用离子交换层析2步法进行:SP Sepharose Fast Flow阳离子交换层析和Source 15Q阴离子交换层析;所得产品用免疫印迹法鉴定,经HPLC检测纯度,凝块法检测抗凝比活性;最后对纯化产品进行结构确证,包括高分辨率质谱、C-末端测序、N-末端测序、二硫键分析.结果显示:优化后的发酵工艺确定基于TB培养基的高密度发酵工艺,菌体OD600可达40左右,每升菌体湿质量可达70 g左右,目的蛋白表达量约400 mg/L;菌体反复冻融离心上清经过纯化获得的目的蛋白HPLC纯度均大于97％,纯化收率为26.12％,抗凝比活性为1 024 ATU/mg,用高分辨率质谱检测其分子质量约为7 415.188 0 ku,N-末端、C-末端和二硫键均与理论相符.建立了EH在大肠杆菌中的高密度发酵和纯化工艺.

Objective: To prokaryotic express and purify the fusion protein of human tumor necrosis factor α inhibitory peptide with anti-inflammatory acidic tail in E.coli.Methods: The gene of interest was amplified by overlapping PCR and inserted into the prokaryotic expression vector pET32a with 6×His tag to build recombinant expression plasmid pET32a-T9-ac-9.The recombinant plasmid was transformed into E.coli BL21(DE3),and the expression of fusion protein was induced by IPTG.Ni2+ metal chelating column was utilized for high purification of the target protein.Results: Sequencing and restriction analysis revealed the recombinant plasmid was constructed successfully.The fusion protein was expressed in E.coli,SDS-PAGE showed a clear protein band with a relative molecular weight of 22.917 kD.The expression products were purified by affinity chromatography from ultrasonic supernatant of E.coli.Conclusion: The fusion protein of human tumor necrosis factor α inhibitory peptide with anti-inflammatory acidic tail was successfully expressed and purified,which makes the foundation of further study on its biological function.