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

目的：分别构建全长、无信号肽、无核定位系统和既无信号肽也无核定位系统的大鼠gremlin1基因的真核表达载体.方法：采用PCR技术获得全长大鼠gremlin1基因（gremlin1）和无信号肽的gremlin1基因（gremlin1-DS）,以全长gremlin1为模板运用重叠延伸PCR获得无核定位系统的gremlin1基因（gremlin1-DN）,继而在gremlin1-DN的基础上去掉信号肽获得既无信号肽也无核定位系统的gremlin1基因（gremlin1-DS-DN）.分别将4组重组质粒转染到非洲绿猴肾细胞株cos7中,免疫荧光和蛋白免疫印迹检测gremlin1基因的表达.结果：构建的真核表达载体pEF1/Myc-HisC-gremlin1、pEF1/Myc-His C-gremlin1-DS、pEF1/Myc-His C-gremlin1-DN和pEF1/Myc-His C-gremlin1-DS-DN经PCR与酶切鉴定显示目的片段大小与预期结果一致；经DNA测序显示pEF1/Myc-His C-gremlin1中的gremlin1片段与Gene Bank中的gremlin1序列（NM-019282）完全吻合；pEF1/Myc-His C-gremlin1-DS中的gremlin1基因缺失信号肽序列；pEF1/Myc-His C-gremlin1-DN中的gremlin1片段实现了4个定点突变：433-435位的AAG（赖氨酸）、487-489位的CCA（脯氨酸）、490-492位的CCC（脯氨酸）、496-498位的AAG（赖氨酸）均突变为TTC（苯丙氨酸）；pEF1/Myc-His Cgremlin1-DS-DN中的gremlin1基因既缺失信号肽序列又实现了4个定点突变.将它们分别转染到cos7细胞后,免疫荧光和蛋白免疫印迹检测显示,4者均能在细胞内成功表达.结论：成功构建了pEF1/Myc-HisC-gremlin1、pEF1/Myc-HisC-gremlin1-DS、pEF1/Myc-His C-gremlin1-DN、pEF1/Myc-His C-gremlin1-DS-DN的真核表达载体.

Abstract Objective： To construct the eukaryotic expression vectors of the full length, the non-signal sequence, the non- nuclear localization signal sequences （NLS） and both non-signal sequence and non-NLS rat gremlird. Methods： We got the gremlird gene（gremlinl ）and the gremlin1 gene without the signal peptide （gremllnJ-DS） from the plasmid pcDNA3.1 （＋）- gremlird by PCR. We cloned the gremlin1 gene without NLS （gremlinl-DN） on the basis of the grernlird as the template using gene splicing by overlap extension PCR （SOE PCR） and then cloned the gremlird gene neither signal sequence nor NLS （gremlird-DS-DN） on the basis of the gremlird-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-gremlird, the pEF1/Myc-His C-gremlinl-DS, the pEF1/Myc-His C-gremlinl-DN and the pEF1/Myc-His C-gremlird-DS-DN were almost consistent with the results of the anticipation. The results using DNA sequence method also showed that the sequence of the grernlinl 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-gremlinl-DS lost the signal peptide, that the one in the pEF1/Myc-His C-grernlinl-DN mutated four sites, and that the one in the pEF1/Myc-His C-gremlird-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, thepEF1/Myc-His C-gremlinl-DS, the pEF1/Myc-His C-gremlinl-DN and the pEF1/Myc-His C-gremlinl-DS-DN.