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将在真核细胞表达的pCD-huIL-4质粒,经EcoRV及Bam H1酶切,获人IL-4 cDNA的 EcoRV—Bam H1片段,插入经相应 EcoRV及Bam H1酶切处理的pEX-2ΔH质粒,构成pR-hlL-4质粒。此质粒包含除5’-端9个bp缺失外的编码人成熟1L-4分子的全部bp序列。pR-hlL-4质粒酶切图谱鉴定正确,转化E.colipop 2136菌,表达分子量为60 kd的融合蛋白,它占全部菌体蛋白的30％。表达的融合蛋白无可测出的人IL-4的BCGF或TCGF活性。以融合蛋白免疫小鼠及家兔制成的抗血清,在免疫斑点试验中,与人rIL-4及融合蛋白呈特异反应,表明融合蛋白中具有人IL-4抗原分子。
The pCD-huIL-4 plasmid expressed in eukaryotic cells was digested with EcoRV and BamH1 and the EcoRV-BamHI fragment of human IL-4 cDNA was obtained and inserted into the pEX-2ΔH plasmid digested with EcoRV and BamH1 , Constituting pR-hlL-4 plasmid. This plasmid contains the entire bp sequence encoding human mature 1L-4 molecules except for the 9 bp deletion at the 5’-end. The plasmid pR-hlL-4 was identified by restriction enzyme digestion and transformed into E.colipop 2136. The fusion protein with the molecular weight of 60 kd was expressed, accounting for 30% of all the bacterial proteins. The expressed fusion protein had no detectable BCGF or TCGF activity of human IL-4. Antiserum raised from mice and rabbits immunized with fusion protein showed specific reaction with human rIL-4 and fusion protein in immunoblotting assay, indicating that the fusion protein contains human IL-4 antigen.