CAJ | 학술논문

目的构建三叶肽(TFF)原核重组质粒pET32a-TFF3,实现TFF3融合蛋白在大肠杆菌中的高效表达并鉴定表达产物.方法用Trlzol试剂提取人结肠组织mRNA,逆转录后经PCR扩增得到不含信号肽的TFF3编码序列,插入原核表达载体pET32a,构建pET32a-TFF3重组质粒,转化大肠杆菌BL-21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,用SDS-PAGE和Western blot检测表达蛋白.结果酶切和测序证实pET32a-TFF3重组质粒构建成功.SDS-PAGE分析表明在IPTG浓度为1 mmol/L时,诱导6 h后蛋白表达量最大.Western blot分析表明,TFF3融合蛋白相对分子质量约为24×10~3,其能与兔源TFF3抗体特异性结合.结论成功构建了pET32a-TFF3重组质粒,实现了该基因在大肠杆菌中的高效表达,为后续深入研究TFF3奠定了基础.
목적 구건삼협태(TFF)원핵중조질립pET32a-TFF3,실현TFF3융합단백재대장간균중적고효표체병감정표체산물.방법 용Trlzol시제제취인결장조직mRNA,역전록후경PCR확증득도불함신호태적TFF3편마서렬,삽입원핵표체재체pET32a,구건pET32a-TFF3중조질립,전화대장간균BL-21(DE3),이병기-β-D-류대반유당감(IPTG)유도중조단백표체,용SDS-PAGE화Western blot검측표체단백.결과 매절화측서증실pET32a-TFF3중조질립구건성공.SDS-PAGE분석표명재IPTG농도위1 mmol/L시,유도6 h후단백표체량최대.Western blot분석표명,TFF3융합단백상대분자질량약위24×10~3,기능여토원TFF3항체특이성결합.결론 성공구건료pET32a-TFF3중조질립,실현료해기인재대장간균중적고효표체,위후속심입연구TFF3전정료기출.
Objective To construct recombinant human TFF3 prokaryotic expressing plasmid,express it in E.coli and identify the expressed protein.Methods The cDNA for mature peptide of TFF3 was amplified by RT-PCR from RNA of human colon tissue and inserted into the MCS of the prokaryotic expressing plasmid pET32a(+).Then TFF3 was expressed as a fusion protein by IPTG induction.The recombinant protein was determined by SDS-PAGE and Western blot with a rabbit anti-TFF3 polyclonal antibody.Results Sequencing result indicated that the obtained TFF3 fragment was inserted into plasmid pET32a(+)successfully and the sequence was correct.The expression level of the fusion protein was highest after 6h induction with 1 mmol/L IPTG.The result of Western blot demonstrated that the relative molecular mass of recombinant protein was about 24×10~3 and the protein had good antigenicity and specificity.Conclusion The expression plasmid pET32a-TFF3 was consctructed and expressed successfully.This study will provide a substantial basis for further study of human TFF3.