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目的 将GM-CSF基因与IL-2基因通过一中间接头G-G-G-S-G-G-S-G连接到一起,并在E.coli中分泌表达具有更高GM-CSF和IL-2生物活性的融合蛋白分子。方法 在引物设计中,通过选择密码子,在Linker的中前部设计了一个BamHI酶切位点,GM-CSF的下游引物和IL-2的上游引物均起始于该位点。然后通过PCR、酶切、连接等一系列分子克隆的方法,构建融合蛋白的克隆及表达载体。结果 经EcoRI/SaII双酶切鉴定,克隆载体pUC19GM-CSF/L/IL-2及表达载体pBVGMCSF/L/IL-2中均插入了正确的外源片段。结论 克隆及表达载体的成功构建,为进一步表达融合蛋白及活性研究打下了基础。
Objective To connect the GM-CSF gene with the IL-2 gene through an intermediate G-G-G-S-G-G-S-G and to secrete the fusion protein with higher GM-CSF and IL-2 activity in E.coli. Methods In the primer design, a BamHI restriction site was designed at the front of Linker through the selection of codons. Both the downstream primer of GM-CSF and the upstream primer of IL-2 began at this site. Then a series of molecular cloning methods, such as PCR, enzyme digestion and ligation, were used to construct the fusion protein cloning and expression vector. Results The recombinant plasmid pUC19GM-CSF / L / IL-2 and its expression vector pBVGMCSF / L / IL-2 were both digested by EcoRI / SaII. Conclusion The successful construction of cloning and expression vector laid the foundation for the further expression of the fusion protein and activity research.