摘要
目的克隆人B淋巴细胞刺激因子 (BLyS)胞外区 12 8 2 85氨基酸cDNA ,并行高效表达、纯化及功能鉴定。方法提取HL 6 0细胞总RNA ,经RT PCR扩增编码人BLyS胞外区 12 8 2 85氨基酸的cDNA ,行序列测定后 ,构建于高效原核表达载体pQE 80L ,经IPTG诱导表达 ,Ni NTA色谱纯化 ,进而行免疫学活性检测。结果RT PCR扩增得到了 4 77bp的DNA片段 ,序列分析与GenBank中报道的编码BLyS 12 8 2 85的cDNA序列一致 ,并在大肠杆菌中获得了高效表达 ,Ni NTA色谱纯化后 ,活性检测发现它能刺激小鼠脾淋巴细胞增殖。结论成功克隆了人BLyS胞外区cDNA ,并获得了高效表达及纯化 。
PurposeTo clone the cDNA encoding the extracellular region of human B-lymphocyte stimulator (BLyS), to highly express and purify and to functionally analyze.MethodsThe total RNA was extracted from HL-60 cells and the cDNA encoding human BLyS 128-285 amino acids were amplified by using RT-PCR. After being sequenced, the cDNA was ligated into the prokaryotic expression vector pQE-80L and induced by IPTG to express the corresponding soluble BLyS. The protein was purified by Ni-NTA chromatography, and its immunological activity was assayed.Results477bp cDNA amplified by RT-PCR was consistent with the sequence encoding human BLyS128-285 amino acids reported in GenBank. The soluble BLyS was highly expressed. The protein could strongly stimulate proliferation of lymphocyte of mouse.ConclusionThe cDNA encoding the extracellular region of human BLyS was successfully cloned, the relevant protein was highly expressed and purified, and the purified product possesses biological activity.
出处
《中国生化药物杂志》
CAS
CSCD
2004年第1期1-5,共5页
Chinese Journal of Biochemical Pharmaceutics
基金
国家自然科学基金资助项目 (No .3 0 2 712 2 8)
关键词
B淋巴细胞刺激因子
CDNA克隆
原核表达
纯化
B-lymphocyte stimulator
cDNA cloning
prokaryotic expression
protein purification
immunological function
