CAJ | 학술논문

目的构建含NLRC5蛋白功能区的cDNA序列的真核表达载体 pEGFP-C2-NLRC5,并检测其在肾成纤维细胞(COS-7)中的表达。方法采用RT-PCR法从人肝星状细胞(LX-2)中获得 NLRC5蛋白功能区的 cDNA 序列片段,用EcoR玉和BamH 玉双切酶将片段和载体pEGFP-C2双酶切后,酶切产物加入T4连接酶16益连接过夜,构建真核表达载体pEGFP-C2-NLRC5。将构建成功的pEGFP-C2-NLRC5重组质粒经PCR,限制性内切酶酶切和测序鉴定后用阳离子脂质体LipofectamineTM2000将其转染至COS-7细胞,荧光显微镜下观察绿色荧光蛋白表达, Western blot法鉴定融合蛋白表达。结果阳性克隆经双酶切法鉴定可见NLRC5基因片段。转染重组质粒后可观察到绿色荧光蛋白的表达,而且主要分布在细胞质中,Western blot法也可检测到在74 ku处有一明显条带,其大小符合EGFP-NLRC5表达的蛋白( NLRC5蛋白大小约为47 ku,EGFP蛋白大小约为27 ku),表明融合蛋白可在哺乳动物细胞中成功表达。结论成功构建pEG-FP-C2-NLRC5质粒, NLRC5蛋白可在 COS-7细胞中成功表达。
목적구건함NLRC5단백공능구적cDNA서렬적진핵표체재체 pEGFP-C2-NLRC5,병검측기재신성섬유세포(COS-7)중적표체。방법채용RT-PCR법종인간성상세포(LX-2)중획득 NLRC5단백공능구적 cDNA 서렬편단,용EcoR옥화BamH 옥쌍절매장편단화재체pEGFP-C2쌍매절후,매절산물가입T4련접매16익련접과야,구건진핵표체재체pEGFP-C2-NLRC5。장구건성공적pEGFP-C2-NLRC5중조질립경PCR,한제성내절매매절화측서감정후용양리자지질체LipofectamineTM2000장기전염지COS-7세포,형광현미경하관찰록색형광단백표체, Western blot법감정융합단백표체。결과양성극륭경쌍매절법감정가견NLRC5기인편단。전염중조질립후가관찰도록색형광단백적표체,이차주요분포재세포질중,Western blot법야가검측도재74 ku처유일명현조대,기대소부합EGFP-NLRC5표체적단백( NLRC5단백대소약위47 ku,EGFP단백대소약위27 ku),표명융합단백가재포유동물세포중성공표체。결론성공구건pEG-FP-C2-NLRC5질립, NLRC5단백가재 COS-7세포중성공표체。
Objective To construct pEGFP-C2-NLRC5 of expression plasmid, and observe its expression in COS-7 cells. Methods Get the cDNA sequence of NLRC5 protein from the LX-2 by RT-PCR. Then NLRC5 cDNA and the vector pEGFP-C2 were digested with restriction enzymes EcoR Iand BamH I, and the digested productions were connected by T4 enzyme at 16 ℃, and then the eukaryotic vector of pEGFP-C2-NLRC5 was constructed. The recombinant vector was identified by the double digestion with restriction enzymes EcoRIand BamHIand DNA sequencing. After the analysis, pEGFP-C2-NLRC5 was transfected into renal fibroblasts COS-7 cells by Lipo-fectamineTM2000, and the expression of pEGFP-C2-NLRC5 was monitored by fluorescence and confocal microscope and Western blot. Results The NLRC5 fragment was contained in the positive recombination by identification of restriction enzymes. After transfectting recombinant plasmid, green fluorescent protein could be observed, and they were mainly distributed in the cytoplasm. A stripe of 74 ku protein could be detected by Western blot, whose size was accord with EGFP-NLRC5 of protein expression ( NLRC5 protein:47 ku, EGFP protein:27 ku) . Therefore, fusion protein could be successfully expressed in mammalian cell. Conclusion The eukaryotic expression vector of pEGFP-C2-NLRC5 is constructed successfully,and the fusion expression of NLRC5 protein and GFP can be detec-ted in COS-7 .