人心肌型脂肪酸结合蛋白(H-FABP)在大肠杆菌中的表达和纯化

Cloning, Expression and Purification of Human Heart Type Fatty Acid Binding Protein in E.coli

目的获得重组人心肌型脂肪酸结合蛋白,为进一步开发应用奠定基础。方法根据已报道的心肌型脂肪酸结合蛋白(H-FABP)基因序列,采用RT-PCR的方法获得心肌型脂肪酸结合蛋白基因。将所得的PCR产物插入原核表达载体pQE30中,得重组质粒(pQE-H-FABP)并转化大肠杆菌(E.coli)M15,通过IPTG诱导表达出带有六个组氨酸的融合蛋白。经镍固定金属亲和层析纯化。结果序列分析表明:H-FABP基因成熟肽编码区含有396bp,编码132个氨基酸;与GenBank(NM004012)中已报道的H-FABP分离物的核苷酸序列有99·9%同源性。经IPTG诱导表达,SDS-PAGE电泳和免疫印迹分析显示,表达的融合蛋白占菌体蛋白总量的27%,分子质量约为15kDa并与商品化的H-FABP单抗(Clone6B6)呈特异性反应。经Ni+2-NTAagarose纯化获得SDS-PAGE电泳下单一条带。结论在大肠杆菌中获得了人心肌型脂肪酸结合蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础。

Objective To obtain recombinant human heart type fatty acid binding protein(H-FABP) by prokaryotic expression for clinical use in diagnosis. Methods The encoding sequence for mature peptide of human H-FABP was amplified with RT-PCR and inserted into pQE30 vector to establish the prokaryotic expressing system coupled with 6His. The competent cells of host strain of M15 were transformed by the recombinant plasmid(pQE-H-FABP). Expression of the target protein was induced with IPTG and assayed by SDS-PAGE and immunoblot after sequencing. The recombinant products were purified by Ni^+-NTA agarose column. Results The cloned fragment of human H-FABP was 99.9% consistent with that in genbank(NM004012), which was deduced to express 132 Aa mature peptide of human H-FABP correctly. The Expressed fusion-protein was 15 kD in SDS-PAGE as expected, was recognized by a commercial McAb(Clone 6B6) and was 27% in the total quatiy of germ proteins. Conclusion The homologous recombinant human H-FABP is obtained after Ni^+-affinity chromatograph, which may benefit for preparation of specific antibodies and diagnosis for some related diseases.