胱抑素C原核表达载体的构建、表达及蛋白纯化

摘要

目的：构建胱抑素C(cystatin C，Cys-C)原核表达载体并表达、纯化、鉴定目的蛋白。方法根据GeneBank报道的Cys-C基因序列，采用RT-PCR技术从人胚肾细胞系HEK293中获得Cys-C的cDNA序列，经PCR、酶切及测序确认最终获得重组载体pET-30a(+)-CysC，转化大肠埃希菌Rosetta，通过异丙基-β-D-硫代吡喃半乳糖苷(isopropyl-β-D-thiogalac-topyranoside，IPTG)诱导表达、经镍柱亲和层析法纯化获得Cys-C融合蛋白。结果序列分析表明，人Cys-C基因成熟肽编码区编码122个氨基酸；与GeneBank(NM-000099.2)中已报道的序列有100%的同源性。经IPTG诱导表达， SDS-PAGE电泳和ELISA分析显示，表达的融合蛋白占菌体蛋白总量的20%，重组蛋白相对分子质量约为20000，经Ni2+-NTA agarose纯化获得SDS-PAGE电泳下单一条带并与商品化的人Cys-C单抗呈特异性反应。结论在大肠埃希菌中获得了Cys-C的表达，并经镍柱亲和层析得到较纯的胱抑素。%Objective To construct, express and purify the prokaryotic expression vector cystatin C (Cys-C). Methods The cDNA sequences of Cys-C gene in human embryonic kidney cell line HEK293 were detected from GeneBank by RT-PCR. The recombinant vector pET-30a (+)-CysC, identified by restriction enzyme restriction and sequencing, was transformed into E. coli. The Cys-C fusion protein was induced by IPTG and purified by Ni-chelating affinity chromatography. Results Sequence analysis showed 122 amino acids in peptide-encoded human Cys-C, which is consistent with the reported sequences in GeneBank (NM-000099.2). The expression of Cys-C gene was induced by IPTG. SDS-PAGE and ELISA showed that the expressed fusion protein accounted for 20%of the total bacterial protein. The molecular weight of recombinant protein was 20 000. The Ni2+-NTA agarose-purified SDS-PAGE bind could specifically react with the commercially-available human Cys-C monoclonal antibody. Conclusion Cys-C can be expressed in E.coli and purified by Ni-chelating affinity chromatography.