人α防御素融合表达、纯化及生物活性检测

Fusion expression,purification and bioactivity detection of human defensinα (HDα)

目的:获得大量重组人α防御素(HDα)并检测其活性,为其深入研究提供必要的材料。方法:体外化学合成得到带有羟氨裂解位点编码序列的HDα基因片段,克隆入pBV220-IL-4表达载体构建重组表达载体pBV220-IL-4-HDα。测序正确后将重组质粒转入大肠杆菌DH5α中进行温度诱导表达。经羟氨裂解去除IL-4后,对所得蛋白进行纯化、复性,以SDS-PAGE和生物学活性检测鉴定其特性。结果:经温度诱导后,融合蛋白主要以包涵体的形式表达,表达产物约占菌体总蛋白的20%;羟氨裂解切除IL-4后,所得目的蛋白的纯度约为99.8%。抑菌活性试验和克隆形成试验显示,所得HDα对细菌生长有明显的抑制作用。结论:成功地构建了HDα基因的重组表达质粒,获得稳定表达的工程菌,并建立了复性与纯化技术,为进一步对其进行功能研究与应用奠定了基础。

AIM： To obtain recombinant human defensin α（HDα） and detect its biological activity, so as to facilitate further research. METHODS： The HDα gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4- HDα, The constructed vector which was confirmed to be correct by sequencing was transformed into E. coli HD5α and the IL-4-HDα fusion protein was expressed under temperature induction, After fusion protein was cleaved to re- move IL-4 by hydroxylamine, purification and renaturation was performed, HDα＇s characteristics were identified by SDS-PAGE and bioactivity detection, RESULTS： After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body, After cleaving by hydroxylamine, the purity of obtained HDα was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDα could obviously inhibit the growth of bacteria. CONCLUSION： The recombinant expression plasmid for HDα gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDα.