人γ干扰素的高效表达

Study on the High Expression of Human Interferon-γ

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。经酶切鉴定和核苷酸序列测定证实重组质粒pET32a(+)-hIFN-γ含有hIFN-γ基因且阅读框架正确,以IPTG诱导hIFN-γ基因表达的优化条件是:培养基pH7.0,培养温度37℃,IPTG浓度0.8 mmol/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%。从而实现了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。

The recombinant plasmid pET32a（＋）-hIFN-γ was identified with restriction endonucleases digestion and the expressional level of differently conditional hIFN-γ protein was detected by SDS-PAGE.The expression products were identified and purified by Western blot and affinity chromatography.The expression conditions were optimized.By identification of endonucleases digestion and sequence analysis,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 0.8 mmol/L when the recombinant strain growth density OD600 achieved 0.8,and induction time 5 h.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.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.