人防御素HNP-1在大肠杆菌中的融合表达及可溶性研究

Construction and soluble expression of fusion vector of human neutrophil peptide 1(HNP-1)

根据大肠杆菌密码子偏爱性用PCR方法分别获得人防御素HNP-1基因序列,利用SOE技术和不含信号肽的DsbA基因片段拼接后将产物克隆到质粒pET-11b中构建人防御素HNP-1与分子伴侣DsbA的融合表达载体,转化E.coliJM109,提取阳性克隆进行测序后抽取质粒转化表达宿主E.coliBL21(DE3).工程菌经IPTG诱导后可在胞内以可溶形式大量表达DsbA-HNP-1蛋白.重组蛋白经亲和层析法纯化后达到电泳纯,经体外复性,在抗菌试验中表现出对短小杆菌的抗菌活性.

In this study, the DNA fragments of human neutrophil peptide 1（HNP-1） was synthesized according to the condon preference of E. coli and was linked with DsbA without signal peptide by means of SOE. Then, the product of SOE was inserted into vecter （pET11b） by the digesting of two restriction enzyme BarnH Ⅰ and Nde Ⅰ After the recombinant plasmid （pETllb- DsbA-HNP-1） was transformed into E. coli JM109, the interested plasmid was extracted and transformed into E. coli BL21（DE3） . The results indicated that the genetically engineered bacteria expressed a large quantity of intracellular and soluble DsbA-HNP-1 protein when it was inducted by IPTG, and the reconbinant HNP-1 fusion proteins possessed an antimicrobial activity to Bacillus pumilus after purified by Ni^2＋-Chelating chromatography and refoding in vitro.