解剖学杂志2013，Vol.36Issue(4)：747-750,836,5.DOI:10.3969/j.issn.1001-1633.2013.04.011
gremlin1基因的诱变及其真核表达载体的构建
Mutation of rat gremlin1 gene and construction of eukaryotic expression vectors
摘要
Abstract
Objective:To construct the eukaryotic expression vectors of the full length,the non-signal sequence,the nonnuclear localization signal sequences (NLS) and both non-signal sequence and non-NLS rat gremlin1.Methods:We got the gremlin1 gene(gremlin1)and the gremlin1 gene without the signal peptide (gremlin1-DS) from the plasmid pcDNA3.1 (+)-gremlin1 by PCR.We cloned the gremlin1 gene without NLS (gremlinl-DN) on the basis of the gremlin1 as the template using gene splicing by overlap extension PCR (SOE PCR) and then cloned the gremlin1 gene neither signal sequence nor NLS (gremlin1-DS-DN) on the basis of the gremlin1-DN continuously.We identified the plasmids using the restriction and the DNA sequencing methods after we had cloned them into the eukaryotic expression vectors of the pEF1/Myc-His C.We also evaluated the expression of the plasmids through the immunofluorescence and Western blotting methods when they had been transfected into the line cos-7 respectively.Results:The identified results through methods of the PCR and the restriction showed that the flags of the pEF1/Myc-His C-gremlin1,the pEF1/Myc-His C-gremlin1-DS,the pEF1/Myc-His C-gremlin1-DN and the pEF1/Myc-His C-gremlin1-DS-DN were almost consistent with the results of the anticipation.The results using DNA sequence method also showed that the sequence of the gremlin1 in the pEF1/Myc-His C-gremlinl was same as the sequence of the NM-019282 in the GeneBank,that the one in the pEF1/Myc-His C-gremlin1-DS lost the signal peptide,that the one in the pEF1/Myc-His C-gremlin1-DN mutated four sites,and that the one in the pEF1/Myc-His C-gremlin1-DS-DN not only lost the signal peptide,but also mutated four sites.Both the immunofluorescence and Western blotting showed that the four plasmids expressed the corresponding proteins when they had been transfected into the line cos-7.Conclusion:We have successfully constructed the eukaryotic expression vectors of the pEF1/Myc-His C-gremlin1,the pEF1/Myc-His C-gremlin1-DS,the pEF1/Myc-His C-gremlin1-DN and the pEF1/Myc-His C-gremlin1-DS-DN.关键词
gremlin1/诱变/真核表达Key words
gremlin1 / mutation/ eukaryotic expression引用本文复制引用
万林燕,张巧娟,彭虎,夏鑫,柳长柏,吴江锋,张艳琼..gremlin1基因的诱变及其真核表达载体的构建[J].解剖学杂志,2013,36(4):747-750,836,5.基金项目
国家自然科学基金(81200307/H0317) （81200307/H0317）
湖北省卫生厅青年科技人才项目(QUX2010-28) （QUX2010-28）
三峡大学"求索"大学生创新基金(三峡大学[2012]10号) （三峡大学[2012]10号）
湖北省卫生厅科研指导性项目(JX6C-26) （JX6C-26）
