一种抗菌肽和aFGF融合蛋白的构建和表达

利用PCR技术扩增出带有凝血酶Xa因子切割位点的天蚕素蜂毒素杂合肽和aFGF的融合基因，插入大肠杆菌表达载体pET-3c中,构建出表达质粒pET-aF-CM，并转化至大肠杆菌BL21（DE3）中,氨苄青霉素抗性筛选重组转化子。IPTG诱导4h后，以包涵体形式表达的融合蛋白约占菌体总蛋白的17%。将包涵体溶解后透析复性，并利用肝素亲和层析纯化，得到电泳纯的融合蛋白。Western blot分析表明，该蛋白能与aFGF抗体产生免疫反应。MTT法检测显示，融合蛋白具有促3T3Bal/b细胞分裂活性，其比活为1.471×106IU/mg。利用凝血酶Xa因子裂解融合蛋白，可以获得抗菌肽和含凝血酶Xa因子裂解序列的aFGF蛋白。分子筛回收含杂合抗菌肽，抑菌活性检测表明其对大肠杆菌K12D31具有明显抑菌活性。微量稀释法检测结果表明，回收的抗菌肽对大肠杆菌DH5α、大肠杆菌K12D31、沙门氏菌、金黄色葡萄球菌、枯草芽孢杆菌和绿脓杆菌的MIC分别达6.25μg/ml、10μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml和5μg/ml。

Construction and expression of a fusion protein composed of cecropina-melittin and haFGF

A new type of fusion protein was composed of cecropin A melittin peptide CA(1 7)M(5 12)NH2 and human acidic fibroblast growth factor ( haFGF ). The DNA fragment coding the fusion protein was cloned into the vector pET 3c and constructed the recombinant expression vector pET aF CM.The recombinant plasmid was transformed into E.coli BL21(DE3). After induced with IPTG for 4h, the expression of the fusion protein was detected by 12% SDS PAGE and Western blot. The fusion protein was about 17% of total cellular protein and expressed as inclusion bodies. The fusion protein, which purified by affinity chromatography, was cleaved by Xa factor. The cecropin A melittin was purified by gel filtration. The mitogenic activity of the fusion protein detected by MTT was about 1.471×106 IU/mg. The cecropin A melittin peptide had the antibacterial activities against several bacteria including E.coli，Salmonella, staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa etc.