人sΔBAFF的高效原核表达及纯化

目的克隆TNF家族B细胞激活因子(BcellactivatingfactortotheTNFfamily,BAFF)胞外区134~285(sBAFF134-285)氨基酸残基段缺失突变体(sΔBAFF)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Westernblot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401bp的DNA片段,该片段序列与GenBank报道的编码人ΔBAFF的胞外区(sΔBAFF)cDNA序列一致。含sΔBAFF的表达载体在大肠杆菌中可表达出相对分子质量为18000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sΔBAFF蛋白,为其功能的研究创造了条件。

High Prokaryotic Expression and Purification of Human sΔBAFF

Objective To clone the cDNA encoding the mutant(sΔBAFF) of human B cell activating factor to the TNF family(BAFF), with the amino acid residues at sites 142-160 of extracellular 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 cDNA encoding sBAFF_ 134-285 ) as a template. The amplified cDNA 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 cDNA at length of 401 bp was amplified by one-step reverse PCR, and its sequence was consistent with that encoding human sΔBAFF amino acids reported in GeneBank. Human sΔBAFF 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 sΔBAFF laid a foundation of further study on its function.