人sΔBAFF的高效原核表达及纯化

High Prokaryotic Expression and Purification of Human sΔBAFF

目的克隆TNF家族B细胞激活因子（B cell activating factor to the TNF family,BAFF）胞外区134～285（sBAFF134-285）氨基酸残基段缺失突变体（s△BAFF）的cDNA,并进行表达及纯化.方法以构建的重组质粒pUC19/sBAFF为模板,采用一步反向PCR法,扩增缺失编码sBAFF的142～160位氨基酸的核苷酸序列.经测序证实后,克隆入原核表达载体pQE-80L.经IPTG诱导表达,SDS-PAGE和Western blot检测表达产物,Ni2＋-NTA柱层析纯化目的蛋白.结果经一步反向PCR扩增后得到401 bp的DNA片段,该片段序列与GenBank报道的编码人△BAFF的胞外区（s△BAFF）cDNA序列一致.含s△BAFF的表达载体在大肠杆菌中可表达出相对分子质量为18 000的蛋白质并以包涵体的形式存在,经Ni2＋-NTA柱层析纯化后得到高纯度的目的蛋白.结论已成功地制备出人s△BAFF蛋白,为其功能的研究创造了条件.

Objective To clone the eDNA encoding the mutant（s△BAFF） of human B cell activating factor to the TNF family （BAFF）, with the amino acid residues at sites 142-160 of extraceUular domain（sBAFF142-160） deleted, then express the gene in prokaryotic cells and purify the expressed product. Methods The nucleic acid sequence encoding amino acids 142-160 of sBAFF was deleted by one-step reverse PCR using the constructed recombinant plasmid pUC19/sBAFF （containing the eDNA encoding sBAFF134.245 ） as a template. The amplified eDNA was identified by sequencing and cloned into prokaryotic expression vector pQE-80L for expression under induction of IPTG. The expressed product was analyzed by SDS-PAGE and Western blot, then purified by Ni^2＋-NTA chromatography. Results A eDNA at length of 401 bp was amplified by one-step reverse PCR, and its sequence was consistent with that encoding human sABAFF amino acids reported in GeneBank. Human sABAFF with a relative molecular weight of 18 000 was highly expressed in a form of inclusion body in E. coli and reached a purity of 95.5% after purification by Ni^2＋ -NTA chromatography. Conclusion The successful expression of human sABAFF laid a foundation of further study on its function.