摘要
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 EcoRV-BamH1 fragment of human interleukin 4(huIL-4) cDNA cleaved from pCD-huIL-4plasmid was inserted into EcoRV/BamH1 treated pEX2ΔH plasmid via ligation, which resulted in theconstruction of pR-hIL-4 plasmid. The validity of the construction was conformed by the restrictionmap of the pR-hIL-4 plasmid, and by the expression of novel fusion protein of 60 Kd in host E.coli transformed with pR-hIL-4. The constructed pR-hIL-4 plasmid contains all cDNA sequence butnine bp at the 5' terminal coding for mature human IL-4 molecule. The β-galactosidase-human IL-4fusion protein did not have human IL-4 biological activity, indicating the critical role of -NH2terminal three amino acids of IL-4 in the exhibition of IL-4 activity, which are lacked in the IL-4fusion protein. The antiserum has been prepared by immunization of mice and rabbits with fusionproteins. The antisera immunoprecipitated with fusion proteins and specifically bound to purifiedhuman IL-4 with high titer in the dot--blotting assay. It indicates that the fusion protein containshuman IL-4 moiety. The antiserum was unable to block human IL-4 biological activity. Because ofgood immunogenicity and high yield of fusion protein. which accounting for 30% of total bactarialproteins, as well as its specific binding with human rIL-4, the potencial application of this fusionprotein is to develop immunoassay for the measurement of human IL-4.
出处
《北京医科大学学报》
CSCD
1990年第4期241-245,共5页
Journal of Peking University(Health Sciences)
关键词
白细胞介素
大肠杆菌
融合蛋白
Human interleukin 4
cDNA
Plasmid
Fusion protein antiserum
