摘要
[目的]探索一种简便可行的白细胞介素-10(IL-10)基因的克隆方法。[方法]以人Burkitt’s淋巴瘤细胞系Raji细胞为材料,提取培养的Raji细胞中的总RNA。根据GenBank中人IL-10基因序列设计1对特异性引物,用RT-PCR方法扩增人IL-10基因的编码cDNA。回收目的片段,将其与pMD18-T载体连接并转化到大肠杆菌感受态细胞DH5α,构建该基因的重组质粒。重组质粒经PCR和双酶切鉴定后进行测序。[结果]以骨髓细胞组织总RNA为模板进行RT-PCR,可从Raji细胞中获得了预期的约550bp特异性条带。成功构建了IL-10基因的重组质粒,PCR扩增和双酶切的鉴定结果说明该插入片段可能为IL-10cDNA。从获得的阳性克隆中挑选1个菌落来分析重组质粒的插入DNA片段序列。序列分析结果证实其与报道的IL-10基因的序列一致。[结论]该研究为IL-10的重组表达及其生物学活性分析奠定了基础。
[Objective] The aim of the research was to discuss a simple and feasible method of cloning human interleukin-10（IL-10） gene.[Method] With Raji cells of human Burkitt＇s lymphoma cell line as materials,total RNA was extracted from the cultured Raji cells.A pair of specific primers was designed according to the sequence of human IL-10 gene and the coded cDNA of human IL-10 gene was amplified by the method of RT-PCR.After being reclaimed,the target fragments were connected with pMD18-T vector and transformed into competent cell DH5α of Escherichia coli to construct the recombinant plasmid of this gene.The recombinant plasmid was identified by PCR and double-enzyme digestion and sequenced.[Result] With total RNA of bone marrow cell tissue as template,RT-PCR was conducted and about 550 bp specific band was obtained from Raji cells.The recombinant plasmid of IL-10 gene was successfully constructed.The identification results of PCR amplification and double-enzyme digestion indicated that this inserted fragment wa
s IL-10 cDNA possibly.One colony was selected from the obtained positive clones to analyze the inserted DNA fragment sequence of the recombinant plasmid.The results of the sequence analysis proved that it was accordant with the sequence of the reported IL-10 gene.[Conclusion] This research laid the foundation for the recombinant expression of IL-10 and the analysis of its biological activity.
出处
《安徽农业科学》
CAS
北大核心
2007年第36期11764-11765,11781,共3页
Journal of Anhui Agricultural Sciences
基金
河南省教育厅自然基金项目(2007230003)
河南省科技攻关项目(052403011)
