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目的 克隆人B淋巴细胞刺激因子(B-lymphocyte stimulator,BLyS)胞外区cDNA,并行高效表达、纯化及功能鉴定。方法 提取 HL-60细胞总RNA,经RT-PCR扩增编码人 BLyS胞外区 78-285氨基酸 cDNA,行序列测定后,构建高效原核表达载体pQE-80L,经 IPTG诱导表达及 Ni-NTA层析纯化,SDS-PAGE和 Wester blot检测活性。结果 RT-PCR扩增得到627bp的DNA片段,序列分析与CenBank中报道的编码BLyS 78-285的cDNA序列一致,并在大肠杆菌中得到高效表达,纯化后纯度可达95％,并能刺激B淋巴细胞增殖。结论 成功克隆人 BLyS胞外区cD-NA,并获得了高效表达,纯化产物具有生物学活性,为进一步研究奠定了基础。
Objective To clone the cDNA of extracellular domain of human B lymphocyte stimulator (BLyS) and express in vitro efficiently. Methods The total RNA of HL-60 cells was extracted and the cDNA of 78-285 amino acids encoding the extracellular domain of human BLyS was amplified by RT-PCR. After sequencing, the recombinant plasmid pQE-80L was constructed and induced by IPTG. Purification, SDS-PAGE and Wester blot were used to detect the activity. Results A 627bp DNA fragment was amplified by RT-PCR. The sequence analysis was consistent with the cDNA sequence of BLyS 78-285 reported in CenBank and was highly expressed in E. coli. The purity of purified DNA was 95% Lymphocyte proliferation. Conclusion The cD-NA of human extracellular domain of BLyS was successfully cloned and highly expressed. The purified product has the biological activity, which lays the foundation for further research.