【摘要】 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gremlin]基因的真核表达载体。方法:采用PCR技术获得全长大鼠gremlinl基因(gremlin])和无信号肽的gremlin]基因(gremlinl-DS),以全长gremlinl为模板运用重叠延伸PCR获得无核定位系统的gremlin]基因(gremlinl-DN),继而在gremlinl-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gremlinl基因(gremlinl-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gremlin]基因的表达。结果:构建的真核表达载体pEFl/Myc-His C-gremlin]、pEFl/Myc-His C-gremlinl-DS、pEF1/Myc-His C_gremlinl-DN和pEF1/Myc-His C-gremlin]-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-gremlinl中的gremlinl片段与Gene Bank中的gremlin]序列(NM-019282)完全吻合;pEF1/Myc-His c_gremlinl-DS中的gremlinl基因缺失信号肽序列;pEFl/Myc-His C-gremlinl-DN中的gremlinl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 Cgremlinl-DS-DN中的gremlinl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-His C-gremlinl、pEF1/Myc-His C-gremlinl-DS、pEF1/Myc-His C-gremlinl-DN、pEF1/Myc-His C-gremlinl-DS-DN的真核表达载体。更多还原

【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 gremlinl.Methods:We got the gremlinl gene(gremlinl)and the gremlinl gene without the signal peptide(gremlinl-DS) from the plasmid pcDNA3.1(+)gremlinl by PCR.We cloned the gremlinl gene without NLS(gremlinl-DN) on the basis of the gremlinl as the template using gene splicing by overlap extension PCR(SOE PCR) and then cloned the gremlinl gene neither signal sequence nor NLS(gremlin1-DS-DN) on the basis of the gremlinl-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 pEFl/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 pEFl/Myc-His C-gremlin1,the pEFl/Myc-His C-gremlinl-DS,the pEFl/Myc-His C-gremlin1-DN and the pEFl/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 gremlinl in the pEFl/Myc-His C-gremlin1 was same as the sequence of the NM-019282 in the GeneBank,that the one in the pEFl/Myc-His C-gremlinl-DS lost the signal peptide,that the one in the pEFl/Myc-His C-gremlin1-DN mutated four sites,and that the one in the pEFl/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 pEFl/Myc-His C-gremlin1,the pEFl/Myc-His C-gremlin1-DS,the pEFl/Myc-His C-gremlin1-DN and the pEFl/Myc-His C-gremlin1-DS-DN.更多还原