AATF-Che1超家族基因原核表达及其融合蛋白纯化  

Prokaryotic Expression of AATF-Che1 Superfamily Gene and Its Fusion Protein Purification

作　　者：周俊[1] 曹江[1] 孟凡静[1] 冯浩[1] 潘秀英[1] 吴庆运[1] 陈翀[1] 牛铭山[1] 徐开林[1] 

机构地区：[1]江苏省徐州医学院附属医院血液科,221002

出　　处：《国际输血及血液学杂志》2014年第4期337-342,共6页International Journal of Blood Transfusion and Hematology

基　　金：国家自然科学基金项目（81100349）;江苏省“六大人才高峰”资助项目（2011-WS-067）

摘　　要：目的 体外构建抗凋亡转录因子（AATF）-Che1重组蛋白原核表达载体pGEx-4T-1-AATF-Che1,优化pGEX-4T-1-AATF-Che1重组载体原核诱导表达条件,并获得可溶性的纯化谷胱甘肽巯基转移酶（GST）融合蛋白.方法 采用聚合酶链反应（PCR）法从pGEX-4T-1-AATF重组质粒扩增出AATF-Che1基因,将其与原核表达载体pGEX-4T-1双酶切后,回收目的基因片段.通过T4连接酶定向连接AATF-Che1基因和线性化原核表达载体pGEX-4T-1,获得重组蛋白AATF-Che1原核表达质粒pGEX-4T-1-AATF-Che1.将构建正确的表达质粒转化入大肠埃希菌感受态细胞BL21,并采用诱导剂异丙基硫代β-D-半乳糖苷（IPTG）诱导蛋白AATF-Che1的表达.考察不同诱导剂IPTG浓度、诱导时间和诱导温度对重组蛋白表达量及表达方式的影响,从而对诱导条件进行优化.采用谷胱甘肽琼脂糖凝胶珠对可溶形式的GST融合蛋白进行纯化,并采用Western蛋白免疫印记（WB）法鉴定获得的纯化蛋白.结果 原核表达质粒pGEX-4T-1-AATF-Che1的酶切、PCR鉴定结果及测序结果均正确;IPTG诱导蛋白在约65 kD处出现了1条新的蛋白条带,即AATF-Che1与GST的融合蛋白.不同的诱导时间及不同的IPTG浓度条件下,诱导融合蛋白的量均未见明显变化;37℃诱导时,重组蛋白主要以包涵体的形式存在于沉淀中,而16℃诱导时,融合蛋白可溶性地存在于上清液中.最终成功获得AATF-Che1可溶性纯化的融合蛋白,且WB法鉴定其抗原性良好.结论 体外成功构建AATF-Che1基因重组蛋白原核表达载体pGEX-4T-1-AATF-Che1,寻找到优化的重组载体原核诱导表达条件,并获得了大量可溶形式纯化的GST融合蛋白,为进一步研究AATF-Che1超家族基因在肿瘤中的作用奠定了基础.Objective To construct the apoptosis antagonizing transcription factor （AATF）-Che1 recombinant prokaryotic expression vector pGEX-4T-1-AATF-Che1 in vitro,optimize prokaryotic recombinant vector induced expression conditions,and get soluble purified glutathione S-transferase（GST） fusion proteins.Methods The AATF-Che1 gene was amplificated from the pGEX-4T-1-AATF recombinant plasmid by polymerase chain reaction（PCR）.The prokaryotic expression vector pGEX-4T-1 was digested with double enzymes,and recycled purpose fragments.The directional connection of AATF-Che1 gene and linearization prokaryotic expression vector pGEX-4T-1 were through the T4 ligase,to obtain recombinant protein AATF-Che1 prokaryotic expression plasmid pGEX-4T-1-AATF-Che1.Correct plasmid was transformated to escherichia coli cells BL21 （DE3）,and used isopropy1β-D-1-thiogalactopyranoside （IPTG） to induce AATF-Che1 protein expression.Through exploring the influence of the IPTG concentration,induction time and temperature to the expression quantity and form of the recombinant protein,optimized the best induction conditions.Using glutathione sepharose beads purificated soluble GST fusion protein,at last to identificate of the protein by Western blot （WB） mothd.Results The results of enzyme digestion,PCR identification and sequencing were correct,and the prokaryotic expression plasmid was successfully constructed.The electrophoresis showed that a new protein band appear at about 65 kD,which was the fusion protein of AATF-Che1 and GST.The fusion protein was no obvious change with different induction time and IPTG concentration at 37℃ or 16℃.The recombinant protein mainly existed in the form of inclusion body at 37 ℃,while soluble from in the supernatant at 16℃.Soluble purificated GST fusion protein was obtained,and by WB method identified antigenicity of fusion protein was good.Conclusions The prokaryotic expression plasmid was successfully constructed in vitro.The optimized recombinant prokaryotic expression conditions w

关 键 词：AATF-Che1超家族结构域 原核表达 融合蛋白纯化 

分 类 号：R3411[医药卫生—基础医学]