重组人γ-干扰素表达菌株的构建及纯化

The Construction and Purification of the expression stain of Recombinant Human Interferon-γ

关键词:  大肠杆菌，hIFN-γ基因，反转录聚合酶链反应，基因表达，纯化

Key words:  Escherichia coli,h IFN-γ gene,RT-PCR,Gene expression,Purification

摘要:

目的：构建重组人γ-干扰素表达菌株并对表达产物进行纯化。方法：从健康人外周血分离单核细胞,并提取总RNA , 利用RT - PCR 扩增出hIFN-γ基因。构建了重组表达菌株BL21(DE3)(pET32a( )-hIFN-γ)。结果：经酶切鉴定和序列测定证实，构建的重组质粒pET32a( )-hIFN-γ含有hIFN-γ基因，且基因序列和阅读框架正确。经Western blot鉴定证实为hIFN-γ，并实现了在大肠杆菌中的高效表达。表达产物占菌体总蛋白的52%。经Ni2 -NTA 亲和层析纯化,纯度达90%以上。结论：已构建了重组人γ-干扰素表达菌株，获得了纯度较高的目的蛋白，为hIFN-γ的生物活性和生产工艺研究提供了可靠的试验数据。

Abstract:

AIM: To construct the recombinant expression strain BL21(DE3) (pET32a( )-hIFN-γ) and purified the expressed product. Methods:Monocytes were obtained from healthy volunteers and total RNA was prepared from these cells. Human interferon-γ(hIFN-γ) gene was amplified with RT-PCR. The recombinant plasmid pET32a( )-hIFN-γ containing hIFN-γ gene was obtained and transformed into Escherichia coli BL21(DE3). Results:The recombinant plasmid pET32a( )-hIFN-γ contained the gene hIFN-γ which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The result of Western blot showed that the expression protein was correct and expressed high in Escherichia coli. The expression level of the hIFN-γ protein were about 52% of total cellular protein. The expression product was purified by Ni-NTA affinity chromatography and the purity of hIFN-γ is above 90%. Conclusion: The recombinant expression strain BL21(DE3) (pET32a( )-hIFN-γ) had been constructed and the highly purified hIFN-γ was obtained. This provided experimental data to research the biology activity and productive technique of hIFN-γ.