利用PCR技术构建GM-CSF/Fc_(γ2)^-融合蛋白真核表达载体

CONSTRUCTION AND EXPRESSION OF THE VECTOR OF hGM-CSF/Fc_(γ2)^- FUSION PROTEIN

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-。

Objective To eliminate the C1q binding site of the Fc fragment of GM-CSF/Fc γ2 - fusion protein and construct the eukaryotic expression vector of GM-CSF/Fc γ2 - fusion protein. Methods Replace the c1q binding motif Glu318, Lys320, Lys322 of fusion protein GM-CSF/Fc γ2 - with Ala residues by PCR oligonucieotide site-directed mutagenesis . Then, the mutated GM-CSF/ Fc γ2 - was cloned into eukaryotic expression plasmid pRc/CMV2 by the strategy of T-A cloning, and the result was confirmed by restriction enzyme cutting and sequence analysis. Results fusion protein GM-CSF/Fc γ2 - was mutated successfully, and the vector of the eukaryotic expression pRc/CMV2/GM-CSF/Fc γ2 - was constructed. Conclusion The methde of PCR olionucleotide site-directed mutagenesis is feasible.