摘要
为高效制备可溶性人源N anog蛋白,降低N anog蛋白的使用成本。该研究通过分子克隆技术构建重组大肠杆菌(pET32a-N anog),利用IPTG诱导进行可溶性融合表达。采用镍离子柱亲和层析法纯化产物并将纯化后产物进行肠激酶酶切后得到目的蛋白,Western blot鉴定该蛋白抗体结合的特异性并进一步优化发酵条件。结果表明,原核表达载体pET32a-N anog构建成功,可在大肠杆菌中与硫氧还蛋白融合表达,融合蛋白经肠激酶酶切后得到相对分子质量约为38 kDa的蛋白,Western blot鉴定该蛋白可与N anog抗体特异性结合。在摇瓶培养条件下,得到最优发酵条件是诱导温度37℃、IPTG浓度0.2 mmol/L、甘油为补料碳源;15 L发酵罐上进一步优化溶氧条件及温度控制方式,最终在溶氧设置在30%条件下,37℃(诱导期间30℃维持30 min)发酵12 h后得到最高N anog产量1386.4 mg/L,经蛋白纯化后纯度达到90%,为后续N anog多克隆抗体的制备和基础医学实验研究奠定了基础。
To efficiently prepare soluble human-derived N anog protein and reduce the cost,recombinant expression Escherichia coli(pET32a-N anog)was constructed by molecular cloning technology and soluble fusion expression was induced by isopropyl-β-D-thiogalactopyranoside(IPTG).The product was purified by Ni-NTA affinity chromatography and was digested by enterokinase to obtain the target protein.Western blot was used to identify the specificity of antibody binding to the protein and the fermentation conditions were further optimized.The results showed that the prokaryotic expression vector pET32a-N anog was successfully constructed and could be fused expression with the thioredoxin in E.coli.The fusion protein was digested by enterokinase to obtain a protein with a relative molecular mass of about 38 kDa.The protein could bind to N anog antibodies specifically by Western blot.The optimal fermentation results under shake culture conditions contained induction temperature of 37℃,IPTG concentration 0.2 mmol/L,glycerin as the feed carbon source.The dissolved oxygen and temperature control methods were further optimized in a 15 L fermenter.The highest N anog yield was up to 1386.4 mg/L after 12 hours with the dissolved oxygen set at 30%,37℃(30℃for 30 min during the induction period),and reached 90%purity after protein purification.This study provided the base for the subsequent preparation of N anog polyclonal antibodies and N anog basic medical experimental research.
作者
李正杰
顾正华
石贵阳
李由然
辛瑜
张梁
LI Zhengjie;GU Zhenghua;SHI Guiyang;LI Youran;XIN Yu;ZHANG Liang(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214000,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214000,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2020年第17期15-21,共7页
Food and Fermentation Industries
基金
国家重点研发计划项目:高版本模式微生物底盘细胞(2018YFA0900300)。
关键词
N
anog
原核表达
蛋白质纯化
发酵优化
N anog
prokaryotic expression
protein purification
fermentation optimization
