CAJ | 학술논문

目的构建含有人沉默调节蛋白6(hSIRT6)基因的重组原核表达载体,获得高效表达hSIRT6蛋白的基因工程菌,以及较高产量的SIRT6蛋白.方法取205 ng HEK293细胞总RNA逆转录后合成的cDNA为模板,聚合酶链反应(PCR)扩增hSIRT6的编码序列,并克隆至原核表达载体pGEX-4T-3中,构建重组的质粒pGEX-4T-3/SIRT6;将重组质粒pGEX-4T-3/SIRT6转化感受态细菌BL21( DE3),在含100 mg/L氨苄西林的LB平板37℃培养过夜,以pGEX-4T-3空载体作为对照,在相同条件下转化并异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达谷胱甘肽巯基转移酶(GST)蛋白.产物经SDS-PAGE电泳及Western blot鉴定.结果 PCR产物大小及双酶切鉴定证明所克隆的基因是hSIRT6,DNA测序进一步证实与GeneBank序列完全一致.获得高表达及纯化的SIRT6-GST融合蛋白,该可溶蛋白的相对分子质量为72×103,经Western blot鉴定证实为目的蛋白.结论成功构建了基因重组体pGEX-4T-3/hSIRT6,并制备出可溶性SIRT6-GST融合蛋白.
목적 구건함유인침묵조절단백6(hSIRT6)기인적중조원핵표체재체,획득고효표체hSIRT6단백적기인공정균,이급교고산량적SIRT6단백.방법 취205 ng HEK293세포총RNA역전록후합성적cDNA위모판,취합매련반응(PCR)확증hSIRT6적편마서렬,병극륭지원핵표체재체pGEX-4T-3중,구건중조적질립pGEX-4T-3/SIRT6;장중조질립pGEX-4T-3/SIRT6전화감수태세균BL21( DE3),재함100 mg/L안변서림적LB평판37℃배양과야,이pGEX-4T-3공재체작위대조,재상동조건하전화병이병기-β-D-류대반유당감(IPTG)유도표체곡광감태구기전이매(GST)단백.산물경SDS-PAGE전영급Western blot감정.결과 PCR산물대소급쌍매절감정증명소극륭적기인시hSIRT6,DNA측서진일보증실여GeneBank서렬완전일치.획득고표체급순화적SIRT6-GST융합단백,해가용단백적상대분자질량위72×103,경Western blot감정증실위목적단백.결론 성공구건료기인중조체pGEX-4T-3/hSIRT6,병제비출가용성SIRT6-GST융합단백.
Objective To construct a recombinant prokaryotic expression vector and obtain gene engineering bacteria which can efficiently express human sirtuin6.Methods 205 ng SIRT6 gene obtained from HEK293 cells by using reverse transcription-polymerase chain reaction (RT-PCR) was cloned into vector pGEX-4T-3 to generate the recombinant plasmid named as pGEX-4T-3/SIRT6.The recombinant plasmid was transformed into E.coli BL21 under the 37 ℃ cultivation overnight on the 100 mg/L ampicillin plate.After inducing expression with isopropyl-β-D-thiogalactopyranoside (IPTG),the SIRT6-glutathione S-transferases (GST) fusion protein was purified by Glutathione Sepharose,and then analyzed by SDS PAGE and Western blotting.Results The cloned gene was determined as human SIRT6 via detection.Highly expressed and purified SIRT6-GST fusion protein was obtained.The specificity of fusion protein was verified by SDS PAGE and Western blotting.The relative molecular weight of target protein was 72 × 103.Conclusion The recombinant plasmid pGEX-4T-3/SIRT6 was successfully constructed.SIRT6-GST fusion protein was successfully expressed and purified.