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目的 对融合基因GM -CSF Fcγ2 进行定点突变 ,去除其补体结合位点 ,构建GM -CSF Fcγ2-融合蛋白真核表达载体。方法 通过设计突变引物 ,利用PCR定点突变技术扩增融合基因GM -CSF Fcγ2-。并通过T -A克隆策略 ,把突变后的融合基因GM -CSF Fcγ2-重组到真核表达载体pRc CMV2中 ,构建真核表达质粒pRc CMV2 GM -CSF Fcγ2-并经过酶切和测序确证。结果 成功对融合基因GM -CSF Fcγ2-进行了突变 ,构建了真核表达载体pRc CMV2 GM -CSF Fcγ2-。结论 利用该PCR方法进行基因定点突变可行
Objective To perform site-directed mutagenesis of fusion gene GM-CSF Fcγ2 and remove its complement binding site to construct a GM-CSF Fcγ2-fusion protein eukaryotic expression vector. Methods By designing mutant primers, the fusion gene GM-CSF Fcγ2- was amplified by site-directed mutagenesis using PCR. Through the T-A cloning strategy, the mutated fusion gene GM-CSF Fcγ2-was recombined into the eukaryotic expression vector pRc CMV2 to construct the eukaryotic expression plasmid pRc CMV2 GM-CSF Fcγ2- and confirmed by restriction enzyme digestion and sequencing. Results The fusion gene GM-CSF Fcγ2- was successfully mutated and the eukaryotic expression vector pRc CMV2 GM-CSF Fcγ2- was constructed. Conclusion The use of this PCR method for genetic site-directed mutagenesis is feasible