CAJ | 학술논문

目的克隆人角蛋白19(CK19)基因,原核表达重组蛋白并纯化,为建立一种新的非小细胞肺癌诊断方法奠定基础.方法从宫颈癌HeLa细胞株中提取总RNA,经RT-PCR合成cDNA,设计特异性的引物,用PCR的方法扩增目的片段,构建pET-32a-CK19和pGEX-4T1-CK19表达载体,在BL21大肠杆菌中表达CK19重组蛋白纯化,并进行SDS-PAGE及Western blot鉴定.结果经PCR扩增后得到一条420 bp的DNA片段,构建载体后DNA测序,结果与预期序列一致.初步结果鉴定表明,表达和纯化的融合蛋白,与预期结果一致.结论成功克隆CK19基因,并获得高纯度的CK19融合蛋白,为进一步制备体外诊断试剂打下基础.
목적 극륭인각단백19(CK19)기인,원핵표체중조단백병순화,위건립일충신적비소세포폐암진단방법전정기출.방법 종궁경암HeLa세포주중제취총RNA,경RT-PCR합성cDNA,설계특이성적인물,용PCR적방법확증목적편단,구건pET-32a-CK19화pGEX-4T1-CK19표체재체,재BL21대장간균중표체CK19중조단백순화,병진행SDS-PAGE급Western blot감정.결과 경PCR확증후득도일조420 bp적DNA편단,구건재체후DNA측서,결과여예기서렬일치.초보결과감정표명,표체화순화적융합단백,여예기결과일치.결론 성공극륭CK19기인,병획득고순도적CK19융합단백,위진일보제비체외진단시제타하기출.
Objective To clone human keratin 19 (CK19)gene and carry out prokaryotic expression and purification of CK19 protein,in order to establish an early diagnostic method of non-small cell lung cancer.Methods Human CK19 gene was cloned through RT-PCR and PCR techniques from total RNA of a HeLa cell strain.The recombinant plasmids PET-32a-CK19 and PGEX-4T1-CK19 were constructed and transformed into E.coli BL21,and CK19 was expressed,purified and identified by SDS-PAGE and Western blotting.Results The 420 bp DNA fragment was confirmed as CK19 gene by DNA sequencing.After expression and purification of the cloned gene,the protein was identified as CK19 by SDS-PAGE and Western blotting.Conclusions The gene encoding CK19 is successfully cloned,expressed and purified.The study lays a foundation for further development of diagnostic reagent of non-small cell lung cancer.