CAJ | 학술논문

利用PCR、DNA重组、原核与真核生物表达等技术从新疆短命植物东方旱麦草(Eremopyrum orientale)基因组中扩增出Rubisco大亚基基因rbcL(GenBank登录号为FJ346562),并构建了原核与真核表达载体pGEX4T-1-rbcL和pcDNA3-rbcL,随后分别在原核宿主BL21(DE3) 和小鼠中进行了表达,并利用真核表达载体和纯化蛋白制备的抗体,对表达产物的特异性进行了Western 印迹检测.结果表明:东方旱麦草的rbcL基因可读区包含1431 bp,与旱麦草rbcL基因的同源性达99.86%,推测编码476个氨基酸,分子量56 kD.该基因在BL21中以融合蛋白GST-RBCL形式表达并存在于包含体中,融合蛋白大小约为82 kD.RT-PCR检测到该基因在小鼠肝脏中的表达.利用真核表达载体与纯化融合蛋白进行联合免疫制备的RBCL抗体可与RBCL特异性地结合,这为短命植物东方旱麦草基因后续的免疫定位检测奠定了基础.
이용PCR、DNA중조、원핵여진핵생물표체등기술종신강단명식물동방한맥초(Eremopyrum orientale)기인조중확증출Rubisco대아기기인rbcL(GenBank등록호위FJ346562),병구건료원핵여진핵표체재체pGEX4T-1-rbcL화pcDNA3-rbcL,수후분별재원핵숙주BL21(DE3) 화소서중진행료표체,병이용진핵표체재체화순화단백제비적항체,대표체산물적특이성진행료Western 인적검측.결과표명:동방한맥초적rbcL기인가독구포함1431 bp,여한맥초rbcL기인적동원성체99.86%,추측편마476개안기산,분자량56 kD.해기인재BL21중이융합단백GST-RBCL형식표체병존재우포함체중,융합단백대소약위82 kD.RT-PCR검측도해기인재소서간장중적표체.이용진핵표체재체여순화융합단백진행연합면역제비적RBCL항체가여RBCL특이성지결합,저위단명식물동방한맥초기인후속적면역정위검측전정료기출.
The rbcL (large subunit of ribulose-1,5-bisphosphosphate carboxylase/oxygenase) gene of ephemeral plant Eremopyrum orientale L.was amplified from the genomic DNA and the correct gene sequence was registered in GenBank (Accession No.FJ346562 ).The rbcL gene was cloned into prokaryotic expression vector pGEX4T-1(harbored with gene encoding GST and will form a fusion protein with foreign gene) and eukaryotic expression vector pcDNA3,and then expressed in the BL21 (DE3) and mouse,respectively.The expressed RBCL protein was detected by Western blot.The results showed that the ORF of rbcL included 1431 nucleotides and encoded 476 putative amino acids.It shares 99.86% homology with that of Eremopyrum triticeum.The fusion protein GST-RBCL with the size of 82 kD was expressed in inclusion body in BL21 (DE3);the rbcL gene was shown being transcribed in the mouse liver by RT-PCR analysis.Western blot results showed that the purified RBCL was able to specifically bind to its antibody.These preliminary results would provide evidence for further immunohistochemistry assay in E.orientale.