【摘要】 目的 :筛选出能高效表达且可溶性好的NGAL(neutrophilgelatinase associatedlipocalin)融合表达载体。方法 :将NGALcDNA全长片段分别定向克隆到表达载体的BamHI +EcoRI位点 ,构建NGAL基因的 4种融合表达载体 ,然后分别转化大肠杆菌并进行IPTG诱导小样表达 ,监测各NGAL融合蛋白在不同诱导时间内的表达量及其可溶性等。选择pHis NGAL和pDsbA2 0 NGAL进行IPTG诱导大样表达 ,通过SDS PAGE比较表达产物的量与可溶性。结果 :成功构建出 4种NGAL融合表达载体。经IPTG诱导小样表达后 ,4种载体的表达水平及其可溶性明显不同 ,DsbA2 0 NGAL、His NGAL和Trx NGAL融合蛋白表达量分别占菌体总蛋白的 33 3%、32 3%、2 8 7% ,而它们的可溶性部分分别占各自总NGAL融合蛋白的96 8%、95 3%和 6 3 0 %。对pDsbA2 0 NGAL和pHis NGAL进行IPTG诱导大样表达后发现 ,DsbA2 0 NGAL融合蛋白的表达量与可溶性较好。结论 :pDsbA2 0 NGAL表达载体是能高效表达且可溶性好的NGAL融合蛋白表达载体。更多还原

【Abstract】 Objective To screen the high efficiency NGAL fusion expression vector with good solubility.Methods Full length cDNA of NGAL was directly cloned into the BamHI and EcoRI sites of four expression vectors respectively,and identification was done by digestion.Then they all were transformed into E.coli and induced to express with IPTG.Expression production and solubility of four expressed NGAL fusion proteins were detected in different induced time via.After that a large scale expression of pHis NGAL and pDsbA2 0 NGAL were carried out.Analysis of productivity and solubility were done via SDS PAGE.Results Four NGAL expression vectors were constructed successfully And their expression levels and solubility are different distinctly:The expression productivity of DsbA2 0 NGAL,His NGAL,Trx NGAL fusion proteins is 33 3 %,32 3%,28 7% of their total proteins respectively and their soluble fractions are 96 79%,95 34%,62 95% of their total NGAL fusion proteins respectively.Large scale expression of pHis NGAL and pDsbA2 0 NGAL suggest that productivity and solubility of DsbA2 0 NGAL are good.Conclusions pDsbA2 0 NGAL expression vector is the satisfing vector that can express NGAL fusion protein efficiently with good solubility.更多还原