CAJ | 학술논문

采用PCR方法从重组质柱pTG19-T-M扩增得到缺失N端疏水区序列的基因片段tM.将tM与原核表达载体pET-32a进行连接并转化,重组质粒经鉴定并测序.结果表明,目的基因在大肠杆菌BL21细胞中成功地表达了醉繁殖与呼吸病毒重组蛋白pET-tM.表达量为31%.表达产物经SDS-PAGE分析,得到分子量约为29 kD的重组蛋白且以包涵体形式存在.8 mol·L~(-1)尿素变性溶解包涵体,再用稀释与透析相结合的方法对重组蛋白进行复性.复性蛋白经Western-blot检测,结果证明复性蛋白可被PRRSV阳性血清识别用.
채용PCR방법종중조질주pTG19-T-M확증득도결실N단소수구서렬적기인편단tM.장tM여원핵표체재체pET-32a진행련접병전화,중조질립경감정병측서.결과표명,목적기인재대장간균BL21세포중성공지표체료취번식여호흡병독중조단백pET-tM.표체량위31%.표체산물경SDS-PAGE분석,득도분자량약위29 kD적중조단백차이포함체형식존재.8 mol·L~(-1)뇨소변성용해포함체,재용희석여투석상결합적방법대중조단백진행복성.복성단백경Western-blot검측,결과증명복성단백가피PRRSV양성혈청식별용.
The gene segments tM deleting hydrophobic region sequence were successfully amplified from recombinant plasmids pTG19-T-M by PCR. The fragment was inserted into the prokaryotic expression vector pET-32a to construct the recombinant plasmid pET-tM,and expressed in E. Coli BL21. The result indicated that the fusion protein pET-tM was successfully expressed. SDS-PAGE analysis demonstrated the molecular weight of reorganization protein existing in cytorrhyctes type was 29 kD and amounted to 31% of the mass of bacterial proteins in cvtorrhvctes. The renaturation of reorganization protein was carried on by dilution and the dialysis after treatment with the 8 mol ·L~(-1) urea. The renaturation reorganization protein was analyzed by Western-blot,the result demonstrated that the renaturation reorganization protein was recognized by the PRRSV masculine blood serum.