摘要
目的 :构建人细胞因子IL 2 1表达载体并在Hela细胞中稳定表达 ,分析rhIL 2 1体外促T细胞增殖作用。方法 :以已构建的pMD IL2 1为模板 ,PCR扩增成熟IL 2 1cDNA的编码框序列并构建到真核表达载体pCDNA3 1/hismyc B中。挑出阳性克隆进行扩增 ,脂质体转染入Hela细胞中并以G4 18加压筛选。有限稀释法、RT PCR及Westernblot筛选出稳定表达的细胞株。培养上清液经金属离子螯合层析分离纯化后 ,SDS PAGE、Westernblot鉴定。MTT法测定其与抗CD3单克隆抗体共刺激T细胞增殖活性。结果 :SDS PAGE、Westernblot显示Mr为 180 0 0的带有myc重组人IL 2 1融合蛋白 (rhIL 2 1) ;并成功地获得hIL 2 1稳定表达细胞株。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。
Objective:To construct Interleukin 21 (IL 21) expression plasimd pCDNA3.1 IL21 and express it in Hela cell and analysis its acitivity of costimilating T cell proliferation with anti CD3 monoclonal antibody in vitro.Methods:The CDs of IL 21 was amplified by PCR using the pMD IL21 as templet.The expression plasimd pCDNA3.1 IL21 was constructed by inserting the sequence of coding region of the IL 21 into pCDNA3.1/Hismyc B containing CMV promoter and transfected into Hela cells.The stability expression stain was screened by the condition media containing 400 mg/L G418 and cloned through the limited dilution method.The target protein was purified through Ni 2+ chelating Sepharose Fast Flow.The bioactivity was confirmed by MTT method on costimulating the T cell proliferation with anti CD3.SDS PAGE and Western blot analysis the rhIL 21 expressed.Results:hIL 21 was expressed in Hela cell successfully.SDS PAGE analysis showed the IL 21 fusion protein with Mr 18 000 or so was expressed in supernatant of the cells.The rhIL 21 has significant proliferation effect on mature human T cells in the presence of anti CD3 monoantibody.Conclusion:The rhIL 21 with bioactivity has been obtained,which may help researcher study its new function and effects. [
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2005年第1期22-24,共3页
Chinese Journal of Immunology
基金
安徽省科技计划项目基金资助 (0 40 110 10 )
关键词
白细胞介素21
克隆表达
T细胞增殖
Interleukin-21(IL-21)
Cloning and expression
T cells proliferation
