纤溶酶原饼环区5蛋白重组大肠杆菌高效表达体系的建立

目的　建立纤溶酶原饼环区 5 (Humanplasminogenkringle 5 ,hPK 5 )蛋白重组大肠杆菌高效表达体系 ,为高密度发酵创造条件。方法　观察一级、二级种子的生长状态 ,比较表达hPK 5蛋白的 4种重组工程菌株在相同条件下的表达情况 ,选出首选发酵种子 ;对其培养时间、诱导时间、培养基种类、pH等条件进行优化 ;并用凝胶成像分析系统对SDS PAGE结果进行分析。结果　经过筛选 ,JM10 9 pBV2 2 0 hPK 5 (简称JP5 )是获取hPK 5蛋白的首选工程菌株 ,其最佳表达条件是LB培养基 (pH 7.4、溶解氧充足 )、30℃培养 3h、4 2℃诱导 6h。在此条件下 ,JP5表达目的蛋白占菌体总蛋白的 38%左右。结论　为高密度发酵获取hPK 5蛋白奠定了实验基础

Construction of High Expression System of Human Plasminogen Kringle 5(hPK-5) Protein in E.coli

Objective To construct a high expression system of human plasminogen kringle 5 (hPK-5) protein in E.coli and establish optimal conditions for high density fermentation.Methods Observe the growth of bacterial seeds of classes I and II, compare the expression levels of hPK-5 protein in 4 recombinant strains under the same conditions, select the optimal seeds and further optimize the conditions for fermentation, including the time for culture and induction, medium and pH value. Analyze the bands showed in SDS-PAGE by gel image-forming analysis system.Results JM109/pBV220/hPK-5 (JP5) was selected as the optimal recombinant strain for the expression of hPK-5 protein, and the optimal condition for expression was as follows: inoculate the optimal recombinant strain onto LB medium (pH7.4, with dissolving oxygen supplied well) and incubate at 30℃ for 3h, then induce at 42℃ for 6h. The expression level of JP5 protein under this condition reached about 38% of total somatic protein.Conclusion The study laid an experimental basis of preparing hPK-5 protein by high density fermentation.