人γ干扰素的高效表达

Study on the High Expression of Interferon-γ

关键词:  大肠杆菌，hIFN-γ基因，基因表达，优化

Key words:  Escherichia coli,h IFN-γ gene,Gene expression,optimization

摘要:

对已构建的重组表达菌株BL21(DE3)(pET32a( )-hIFN-γ)进行鉴定和表达工艺研究。采用限制性核酸内切酶酶切鉴定含hIFN-γ基因的重组质粒， SDS-PAGE检测不同条件下hIFN-γ基因的表达情况，用Western blot和亲和层析对表达产物进行鉴定和纯化，并对表达条件进行优化。酶切鉴定证实重组质粒pET32a( )-hIFN-γ含有hIFN-γ基因且阅读框架正确，以IPTG为诱导剂诱导hIFN-γ基因表达的优化条件是：培养基pH 7.0，培养温度37℃，IPTG浓度0.8mmol/L，菌体生长密度OD600达到0.8时加入IPTG，诱导时间5h，此时hIFN-γ蛋白表达量为52%。实现了hIFN-γ基因的高效表达并优化了其表达工艺，从而为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 identify the recombinant expression strain BL21(DE3) (pET32a( )-hIFN-γ) and study the expression technique of the hIFN-γ. Methods: To identify the recombinant plasmid pET32a( )-hIFN-γ with restriction endonucleases digestion and detect expressional level of differently conditional hIFN-γ protein by SDS-PAGE. The expression products were identified and purified by Western blot and affinity chromatography. The expression conditions were optimized. Results:By identification of endonucleases digestion, the recombinant expression plasmid pET32a( )-hIFN-γ contained hIFN-γ gene having positive reading frame. The expression optimization result indicated that the hIFN-γ gene expression optimized condition with IPTG induction is culture medium pH 7.0,culture temperature 37℃, joining IPTG to final concentration 0.8mmol/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the hIFN-γ protein were about 52% of total cellular protein with IPTG induction by SDS-PAGE and gel system analysis. Conclusion:The hIFN-γ protein is expressed high by recombinant strain BL21(DE3)( pET32a( )-hIFN-γ). The expression technology was optimized. This provided experimental data to research the biology activity and productive technology of hIFN-γ.