中国白兔白介素-10基因的克隆、表达及其抗体的制备

Expression and Cloning of IL-10 Gene in Chinese White Rabbits and Preparation of Antibodies against Recombinant IL-10

目的克隆并表达中国白兔IL-10基因,制备抗IL-10多克隆抗体。方法运用RT-PCR对从ConA诱导后的中国白兔外周血单核细胞(PMBCr)总RNA中扩增IL-10基因,克隆后进行测序和遗传进化分析,同时将其亚克隆pET28a中,在大肠杆菌中诱导表达,并用纯化的表达产物制备多克隆抗体。结果该基因全长537 bp,编码178个氨基酸。与欧洲兔IL-10基因同源性很高,但与鼠、鸡、河豚和斑马鱼等不同物种IL-10基因差异较大。经IPTG诱导后,重组菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为23.4×103的重组蛋白。Western blot分析表明,中国白兔IL-10基因已经表达。重组表达的蛋白量可占菌体蛋白的15.2%。结论成功实现了中国白兔IL-10的原核表达,制备了小鼠抗兔IL-10的多克隆抗体。

Objective To study the expression and cloning of IL-10 gene in Chinese white rabbits and prepare the polyclonal antibody against recombinant IL-10. Methods The Chinese white rabbit IL-10 gene was amplified by RT-PCR method from the PMBCr stimulated by ConA, and then cloned, sequenced and analysed. The IL-10 gene was subeloned into prokaryotic expressing vector pET28a for expression in E.coli. Results The complete length of white rabbit IL-10 gene was 537 bp, encoding 178 amino acids. The IL-10 of Chinese white rabbit shared higher identities with that of European rabbit, but differed significantly from IL-10 genes of mouse, chicken and zebrafish. After the induction of IPTG, the expressed recombinmlt IL-10 protein, with a molecular weight of 23.4 kDa, was successfully detected by SDS-PAGE and confirmed by Western blot assay, which amounted to 15.2% in the total protein of the induced bacteria. Conclusion IL-10 has been successfully expressed in E. coli and mouse anti-rabbit IL-10 polyclonal antibody has been prepared by immunization with the purified recombinant IL-10 protein.