CDC25b突变载体的构建及其在小鼠受精卵中的表达

Construction of CDC25b mutation vector and its expression in oosperm of mice

目的构建小鼠细胞分裂周期蛋白25b(CDC25b)突变基因的真核表达载体,并观察其在小鼠G2期受精卵细胞中的表达。方法采用PCR法从野生型CDC25b模版上扩增CDC25b(S15A)和CDC25b(S15D)突变基因,定向插入pcDNA3.1(+)-3flag-C空载体中,由限制性内切酶BamHⅠ和KpnⅠ进行双酶切电泳后测序验证,构建真核表达载体pcDNA3.1(+)-3flag-CDC25b(S15A)和pcDNA3.1(+)-3flag-CDC25b(S15D)。采用脂质体法将其转染至Stbl3感受态细胞,质粒经在感受态细胞内转化、抽提、酶切及测序鉴定正确后,将重组真核表达质粒用显微注射法注射至小鼠G2期受精卵内,采用Western blot法检测重组质粒突变型CDC25b的表达。结果pcDNA3.1(+)-3flag-CDC25b(S15A)和pcDNA3.1(+)-3flag-CDC25b(S15D)真核表达载体经酶切和测序鉴定,酶切结果显示与目的片段大小一致,测序结果显示突变序列正确;将其注射至小鼠G2期受精卵48 h,可检测到细胞内CDC25b蛋白表达增加(P<0.05)。结论成功构建CDC25b(S15A)和CDC25b(S15D)真核表达载体,验证了突变型CDC25b蛋白表达,可为CDC25b对小鼠受精卵发育调节的研究提供实验基础。

Objective To construct the eukaryotic expression vectors of mutant cell division cyclin 25 b(CDC25 b)gene and observe their expressions in oosperm of mice during the G2 stage.Methods CDC25 b(S15 A)and CDC25 b(S15 D)were synthesized and amplified from wild-type CDC-25 b by PCR.The mutant genes were inserted into the empty vector pcDNA3.1(+)-3 flag-C and digested with restriction enzymes BamHⅠand KpnⅠ.After electrophoresis,sequencing and verification,the eukaryotic expression vectors pcDNA3.1(+)-3 flag-CDC25 b(S15 A)and pcDNA3.1(+)-3 flag-CDC25 b(S15 D)were constructed,and then transfected into Stbl3 competent cells by liposome method.After transformation,extraction,digestion and sequencing in competent cells,the eukaryotic expression plasmid was microinjected into oosperm of mice during the G2 stage,and the recombinant plasmids of mutant CDC25 b gene expression were detected by Western blot.Results The eukaryotic expression vectors pcDNA3.1(+)-3 flag-CDC25 b(S15 A)and pcDNA3.1(+)-3 flag-CDC25 b(S15 D)were identified by enzyme digestion and sequencing,which showed that the target fragments were the same size and the mutant sequence was correct.After the cells were injected into oosperm during the G2 stage for 48 hours,the protein expression of CDC25 b was obviously increased(P<0.05).Conclusion The eukaryotic expression vectors of CDC25 b(S15 A)and CDC25 b(S15 D)have been successfully constructed,and the protein expression of mutant CDC25 b is verified,which may provide an experimental foundation for the further study of CDC25 b in the regulation of oosperm development of mice.