CAJ | 학술논문

目的:构建富含脯氨酸精氨酸的39位氨基酸抗菌肽(Proline-arginine rich 39-amino acid peptide,PR-39)的真核表达载体,并检测它在293 T细胞中的表达情况.方法:以含有PR-39核心编码区(Core ceding regions,CDS)序列的质粒pBluescript ⅡSK-PR-39为模板,PCR扩增PR-39 CDS序列并测序.扩增片段插入真核表达质粒pGC-FU中,构建包含PR-39基因的重组表达质粒pGC-FU-PR-39.阳性克隆用限制性内切酶酶切及测序鉴定后用脂质体2000转染293 T细胞,荧光显像观察其转染效率,Western blot检测目的蛋白表达情况.结果:成功扩增出PR-39基因,通过酶切测序鉴定证明成功构建了包含PR-39的真核表达载体pGC-FU-PR-39,转化293 T细胞24 h后观察到荧光,Western blot检测到目的蛋白.结论:本实验成功构建PR-39真核表达载体,并能在293 T细胞中表达PR-39-GFP融合蛋白,这为进一步研究PR-39的作用奠定了基础.
목적:구건부함포안산정안산적39위안기산항균태(Proline-arginine rich 39-amino acid peptide,PR-39)적진핵표체재체,병검측타재293 T세포중적표체정황.방법:이함유PR-39핵심편마구(Core ceding regions,CDS)서렬적질립pBluescript ⅡSK-PR-39위모판,PCR확증PR-39 CDS서렬병측서.확증편단삽입진핵표체질립pGC-FU중,구건포함PR-39기인적중조표체질립pGC-FU-PR-39.양성극륭용한제성내절매매절급측서감정후용지질체2000전염293 T세포,형광현상관찰기전염효솔,Western blot검측목적단백표체정황.결과:성공확증출PR-39기인,통과매절측서감정증명성공구건료포함PR-39적진핵표체재체pGC-FU-PR-39,전화293 T세포24 h후관찰도형광,Western blot검측도목적단백.결론:본실험성공구건PR-39진핵표체재체,병능재293 T세포중표체PR-39-GFP융합단백,저위진일보연구PR-39적작용전정료기출.
Objective:To construct eukaryotic plasmid expressing PR-39 and test whether the plasmid can be expressed in the 293 T cell.Methods:The core coding regions (CDS) sequence of PR-39 was amplified by PCR from pBluescript Ⅱ SK-PR-39.Then the PR-39 gene was inserted into the eukaryotic expression plasmid pGC-FU. The resultant recombinant plasmid was confirmed by restriction enzyme cutting and sequencing, and then was designated as pGC-FU-PR-39. The recombinant plasmid pGC-FU-PR-39 was transfected into 293 T cell by Lipofectamine 2000. Transfected efficiency was observed by fluorographic unit. Western blot was used to certify whether the purpose protein was correctly expressed in 293 T cell. Results:The CDS sequence of PR-39 was correctly amplified. The recombinant plasmid was successfully constructed and could be correctly expressed in 293 T cell. Conclusion :We successfully constructed PR-39 eukaryotic expression plasmid and observed the expression of PR-39-GFP fusion protein. It lays a foundation for further studies of PR-39.