食管癌相关基因NGAL四种融合表达载体的构建及其蛋白表达产物的比较分析  

Construction of Four Esophageal Cancer-related NGAL Fusion Expression Vectors and Comparison Analysis of Their Expression Production

作　　者：王朝阳[1] 许丽艳[1] 荣举[2] 韩溟[1] 吉坤美 沈忠英[1] 李恩民[2] 

机构地区：[1]汕头大学医学院肿瘤病理研究室,广东汕头515031 [2]汕头大学医学院生物化学与分子生物学教研室,广东汕头515031 [3]深圳市三九医药研究院,广东深圳518028

出　　处：《肿瘤防治杂志》2003年第10期1019-1023,共5页China Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金 (No .3 990 0 0 69和No .3 0 170 42 8);广东省自然科学基金 (No .0 10 43 1);广东省高校自然科学研究基金 (No .2 0 0 0 3 3 );广东省医学科研基金 (No .A2 0 0 14 19);汕头大学研究与发展基金 (No .L0 0 0 4)

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。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.

关 键 词：基因 重组融合蛋白类 生物合成 大肠杆菌 基因表达 

分 类 号：R735.1[医药卫生—肿瘤]