摘要
目的 :克隆人细胞因子IL 2 1全长cDNA及其在大肠杆菌中表达 ,并进一步检测其表达产物的功能。方法 :抽取外周血 ,分离淋巴细胞 ;抗CD3抗体刺激后 ,提取总RNA ,通过RT PCR获得两个部分重叠的IL 2 1cDNA片段 (分别为 5′端 2 4 2bp ,3′端 4 2 5bp) ,重组PCR扩增出IL 2 1全长cDNA。DNA测序正确后 ,将成熟IL 2 1cDNA的编码框序列构建到原核表达载体pET2 8a(+)中。卡那霉素平板筛选及PCR鉴定 ,挑出阳性克隆进行扩增、转化大肠杆菌进行IPTG诱导表达。SDS PAGE、Westernblotting鉴定 ,亲和层析分离纯化得到Mr 为 186 0 0的带有组氨酸标签重组人IL 2 1融合蛋白。透析法重折叠复性 ,MTT法测定其对T细胞增殖活性。结果 :成功获得人IL 2 1全长cDNA ,并以包涵体形式表达于大肠杆菌中。重折叠后的rhIL 2 1融合蛋白具有与抗CD3抗体共刺激T细胞增殖作用。结论 :获得具有生物学活性的rhIL 2 1细胞因子 ,为进一步研究其功能奠定基础。
AIM: To clone full length cDNA of humam interleukin-21 (IL-21) and express it in E.coli. METHODS: Total RNA was isolated from peripheral lymphocyte stimulated with anti-CD3 antibody. 5′ and 3′ terminal fragments of IL-21 gene (242 bp and 425 bp fragments respectively) were amplified using RT-PCR. The full length IL-21 cDNA was amplified by recombination-PCR from the products of RT-PCR .The expression plasimd pET28a(+)-IL21 was constructed by inserting IL-21 cDNA into pET28a(+)and then was transformed into BL21(DE3). Expression of hIL-21 was induced by IPTG at 37℃ for 5 h. The target protein was purified through Ni 2+-chelating Sepharose Fast Flow. Purified rhIL-21 was refolded by using dialysis method. And the bioactivity was detected by MTT on costimulating the T cell proliferation with anti-CD3. RESULTS: IL-21 was cloned and expressed in E.coli successfully. SDS-PAGE analysis showed the IL-21 was expressed in the form of insoluble inclusion body. The refolded rhIL-21 could stimulate the proliferation of mature human T-cells in the presence of anti-CD3. CONCLUSION: The rhIL-21 with bioactivity was obtained, which lays the foundation for study of its function.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期406-409,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
安徽省科技计划项目基金资助 (No .0 4 0 1 1 0 1 0 )
