安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
ACTA UNIVERSITY MEDICINALIS ANHUI
2014年
2期
145-148
,共4页
李琳%彭云云%黄成%徐涛%李俊
李琳%彭雲雲%黃成%徐濤%李俊
리림%팽운운%황성%서도%리준
NLRC5%pEGFP-C2%RAW264.7%基因表达%蛋白表达
NLRC5%pEGFP-C2%RAW264.7%基因錶達%蛋白錶達
NLRC5%pEGFP-C2%RAW264.7%기인표체%단백표체
NLRC5%pEGFP-C2%RAW264.7%gene expression%protein expression
目的构建鼠源pEGFP-C2-NLRC5表达质粒,并观察其在 RAW264.7细胞中的表达。方法从小鼠巨噬细胞RAW264.7中获得 NLRC5基因的 cDNA,用 EcoR I和BamH I限制性内切酶双酶切后连接至pEGFP-C2载体上。表达载体经PCR、限制性内切酶酶切和DNA序列分析正确后转染至小鼠巨噬细胞RAW264.7中,荧光显微镜下观察绿色荧光蛋白表达, RT-PCR法鉴定其转录, Western blot法分析其蛋白表达。结果进行双酶切鉴定该质粒可见NLRC5 DNA片段,转染重组质粒后可观察到绿色荧光蛋白的表达, RT-PCR法可检测到NLRC5基因的转录, Western blot法可检测到NLRC5蛋白表达。结论成功构建重组pEGFP-C2-NLRC5表达载体, NLRC5蛋白可与绿色荧光蛋白在RAW264.7细胞中融合表达。
目的構建鼠源pEGFP-C2-NLRC5錶達質粒,併觀察其在 RAW264.7細胞中的錶達。方法從小鼠巨噬細胞RAW264.7中穫得 NLRC5基因的 cDNA,用 EcoR I和BamH I限製性內切酶雙酶切後連接至pEGFP-C2載體上。錶達載體經PCR、限製性內切酶酶切和DNA序列分析正確後轉染至小鼠巨噬細胞RAW264.7中,熒光顯微鏡下觀察綠色熒光蛋白錶達, RT-PCR法鑒定其轉錄, Western blot法分析其蛋白錶達。結果進行雙酶切鑒定該質粒可見NLRC5 DNA片段,轉染重組質粒後可觀察到綠色熒光蛋白的錶達, RT-PCR法可檢測到NLRC5基因的轉錄, Western blot法可檢測到NLRC5蛋白錶達。結論成功構建重組pEGFP-C2-NLRC5錶達載體, NLRC5蛋白可與綠色熒光蛋白在RAW264.7細胞中融閤錶達。
목적구건서원pEGFP-C2-NLRC5표체질립,병관찰기재 RAW264.7세포중적표체。방법종소서거서세포RAW264.7중획득 NLRC5기인적 cDNA,용 EcoR I화BamH I한제성내절매쌍매절후련접지pEGFP-C2재체상。표체재체경PCR、한제성내절매매절화DNA서렬분석정학후전염지소서거서세포RAW264.7중,형광현미경하관찰록색형광단백표체, RT-PCR법감정기전록, Western blot법분석기단백표체。결과진행쌍매절감정해질립가견NLRC5 DNA편단,전염중조질립후가관찰도록색형광단백적표체, RT-PCR법가검측도NLRC5기인적전록, Western blot법가검측도NLRC5단백표체。결론성공구건중조pEGFP-C2-NLRC5표체재체, NLRC5단백가여록색형광단백재RAW264.7세포중융합표체。
Objective To construct the GFP-tagged eukaryotie expression vector of NLRC5 and observe its expres-sion in mouse macrophage RAW264 . 7 . Methods The cDNA of NLRC5 was obtained from mouse macrophage RAW264. 7 cells, amplified by PCR and cut with double enzyme EcoR I and BamHI, then inserted into the eu-karyotic expression vetor pEGFP-C2. The recombinant vector was verified by PCR, restriction enzymes cut and se-quencing identified. Then transfected into mouse macrophage RAW264. 7 cells and the expression of pEGFP-C2-NLRC5 was monitored by fluorescence, PCR and Western blot. Results To deal with recombinant pEGFPC2-NL-RC5 with double digestion, then fragments of NLRC5 could be seen, also GFP could be detected in the transfected RAW264. 7 cells. NLRC5 gene expression could be detected by PCR,and its protein expression was detected by Western blot. Conclusion Eukaryotic expression vector of NLRC5 is successfully constructed, and the fusion ex-pression of NLRC5 protein and GFP can be detected in RAW264 . 7 .