CAJ | 학술논문

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.
목적 체외구건항조망전록인자(AATF)-Che1중조단백원핵표체재체pGEx-4T-1-AATF-Che1,우화pGEX-4T-1-AATF-Che1중조재체원핵유도표체조건,병획득가용성적순화곡광감태구기전이매(GST)융합단백.방법 채용취합매련반응(PCR)법종pGEX-4T-1-AATF중조질립확증출AATF-Che1기인,장기여원핵표체재체pGEX-4T-1쌍매절후,회수목적기인편단.통과T4련접매정향련접AATF-Che1기인화선성화원핵표체재체pGEX-4T-1,획득중조단백AATF-Che1원핵표체질립pGEX-4T-1-AATF-Che1.장구건정학적표체질립전화입대장애희균감수태세포BL21,병채용유도제이병기류대β-D-반유당감(IPTG)유도단백AATF-Che1적표체.고찰불동유도제IPTG농도、유도시간화유도온도대중조단백표체량급표체방식적영향,종이대유도조건진행우화.채용곡광감태경지당응효주대가용형식적GST융합단백진행순화,병채용Western단백면역인기(WB)법감정획득적순화단백.결과 원핵표체질립pGEX-4T-1-AATF-Che1적매절、PCR감정결과급측서결과균정학;IPTG유도단백재약65 kD처출현료1조신적단백조대,즉AATF-Che1여GST적융합단백.불동적유도시간급불동적IPTG농도조건하,유도융합단백적량균미견명현변화;37℃유도시,중조단백주요이포함체적형식존재우침정중,이16℃유도시,융합단백가용성지존재우상청액중.최종성공획득AATF-Che1가용성순화적융합단백,차WB법감정기항원성량호.결론 체외성공구건AATF-Che1기인중조단백원핵표체재체pGEX-4T-1-AATF-Che1,심조도우화적중조재체원핵유도표체조건,병획득료대량가용형식순화적GST융합단백,위진일보연구AATF-Che1초가족기인재종류중적작용전정료기출.
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 were found and purificated GST fusion protein was obtained.It maybe lay the foundation for the further study on the role of AATF-Che1 in tumor.