摘要
目的:构建真核表达载体GFP-hArgBP2并检测在骨肉瘤细胞内的表达及定位。方法:以pcDNA3.1-hArgBP2为模板,利用PCR扩增hArgBP2基因cDNA全长,并将其克隆至真核表达载体pEGFP-C1中。进一步将构建的重组质粒进行酶切和测序鉴定,并转染到骨肉瘤细胞MG-63中,提取细胞蛋白进行Western blot检测。同时利用共聚焦激光扫描显微镜观察GFP-hArgBP2在MG-63细胞中的定位,免疫沉淀的方法纯化hArgBP2蛋白。结果:hArgBP2基因cDNA全长成功构建到真核表达载体pEGFP-C1中,Western blot检测到了GFP-hArgBP2融合蛋白表达,相对分子质量(Mr)约为97 000。GFP-hArgBP2在骨肉瘤细胞MG-63中主要定位于细胞质和核周,并成功纯化hArgBP2蛋白。结论:成功地构建了GFP-hArgBP2真核表达质粒,同时鉴定了GFP-hArgBP2融合蛋白的表达,并纯化hArgBP2蛋白。GFP-hArgBP2蛋白主要定位在细胞质和核周。
AIM： To construct an eukaryotic expression vector GFP-hArgBP2 and identify the expression and localization of hArgBP2 in osteosarcoma MG-63 cells.METHODS： Using pcDNA3.1-hArgBP2 as a template,we obtained human ArgBP2 coding sequence by polymerase chain reaction（PCR） amplification and cloned it into the eukaryotic expression vector.The insert was identified by restriction enzyme digestion and DNA sequencing.GFP-hArgBP2 was transfected into osteosarcoma MG-63 cells and examined by Western blot.The localization of GFP-hArgBP2 in MG-63 cells was also observed with laser scanning confocal microscopy.hArgBP2 protein was purified by immunoprecipitation assay.RESULTS： hArgBP2 was successfully constructed into the expressing vector pEGFP-C1.The length of the fragment identified by restriction enzyme digestion was 1 935 bp.The expression of GFP-hArgBP2 fusion protein with a molecular weight of 97 000Da was detected by Western blot and pulled down by GFP antibody,and its localization was in the cytoplasm and perinucleus in MG-63 cells.CONCLUSION： The recombinant plasmid of hArgBP2 gene was successfully constructed.The expression of GFP-hArgBP2 fusion protein was identified and pulled down by GFP antibody.GFP-hArgBP2 was mainly localized in the cytoplasm and perinucleus of MG-63 cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第3期237-239,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30800415
81070688)
