重组人BAFF_(134～285)的克隆及在大肠杆菌中的高效表达

Gene Cloning and High Expression of Recombinant Human BAFF_(134-285) in E.coli

目的克隆人TNF家族的B细胞激活因子(BcellactivatingfactortotheTNFfamily,BAFF)胞外区cDNA,并进行高效表达和纯化。方法提取人新鲜扁桃体组织总RNA,经RTPCR扩增编码人BAFF胞外区134～285氨基酸残基cDNA,经序列测定后,克隆至pQE80L载体中,并转化大肠杆菌DH5α,经IPTG诱导表达及Ni2+NTA柱层析纯化目的蛋白,最后经SDSPAGE和Westernblot检测。结果RTPCR扩增得到了459bp的cDNA片段,序列分析与GenBank中报道的编码人BAFF134～285的cDNA序列一致,SDSPAGE及Westernblot证实表达蛋白确实为6×HisBAFF134～285融合蛋白,并存在于包涵体中。结论利用大肠杆菌可高效表达rhBAFF134285,为进一步研究其生物学活性奠定了基础。

Objective To clone the cDNA encoding extracellular domain of B cell activating factor to human TNF family,BAFF) and highly express and purify recombinant BAFF_~134-285 protein.Methods The total RNA was extracted from fresh human tonsil tissue,and the cDNA encoding amino acids 134-285 at extracellar domain of human BAFF was amplified by RT-PCR,identified by sequencing and inserted into prokaryotic expression vector pQE-80L.The constructed recombinant plasmid was transformed to E.coli DH5α for expression under induction of IPTG.The expressed protein was purified by Ni^2+ -NTA column chromatography and identified by SDS-PAGE and Western blot.Results A cDNA fragment,at length of 459 bp,was amplified by RT-PCR,and its sequence was consistent with that of cDNA encoding human BAFF_~134-285 reported in GenBank.SDS-PAGE and Western blot proved that the expressed product was 6×His-BAFF_~134-285 fusion protein existing in inclusion body.Conclusion rhBAFF_~134-285 was highly expressed in E.coli.It laid a foundation of further study on the biological activity of rhBAFF_~134-285 .