重组P128蛋白的表达纯化及其对抗牛源葡萄球菌活性研究

Fusion expression,purification and anti-bacterial activity of recombinant P128 protein against bovine mastitis-associated staphylococci

为了制备重组P128蛋白用于葡萄球菌性奶牛乳房炎治疗。将引入烟草蚀纹病毒蛋白酶(TEVp)识别位点的P128编码序列插入类弹性蛋白多肽(ELP)融合表达载体,转化大肠杆菌后用IPTG诱导融合蛋白表达,用温度敏感相变循环纯化,用TEVp活性包涵体切割融合标签,用最小抑菌浓度(MIC)和最小杀菌浓度(MBC)试验测定重组P128对奶牛乳房炎葡萄球菌抗菌活性。结果显示,融合蛋白以可溶性蛋白表达,两次相变循环纯化后的纯度达90%,产量为400 mg/mL; TEVp活性包涵体切割融合标签的效率接近100%,回收的重组P128纯度达98%,对牛源葡萄球菌的MIC和MBC分别为0. 47μg/mL和1. 88μg/mL;除个别多重耐药菌株外,重组P128蛋白对多数金黄色葡萄球菌和凝固酶阴性葡萄球菌的溶(杀)作用很强。这些研究结果表明,重组P128可用于葡萄球菌性奶牛乳房炎治疗。

To prepare recombinant P128 protein for treating staphylococcal bovine mastitis.The P128-coding sequence with tobacco etch virus protease(TEVp)recognition site introduced was inserted into elastin-like polypeptide(ELP)fusion expression vector and the recombinant vector was transformed into E.coli.The expression of fusion protein was induced with IPTG and purified by temperature-sensitive inverse transition cycling(ITC).After cleavage with TEVp active inclusion bodies and removal of the fusion tag,the anti-staphylococcal activity of the recombinant P128 was analyzed by minimum inhibitory concentration(MIC)and minimum bacteriocidal concentration(MBC)assays.The fusion protein was expressed as soluble protein and purified to 90%purity after two rounds of ITC with a yield of 400mg/L bacterial culture.Almost 100%of the fusion protein was cleaved by TEVp and the purity of recovered P128 was up to 98%.The MIC and MBC of P128 against mastitis-causing Staphylococci were 0.47μg/mL and 1.88μg/ml,respectively.Except for few multi-resistant isolates,the P128 protein had strong anti-staphylococcal activity,including Staphyloccocus aureus and coagulase-negative Staphylococci.These data suggest that recombinant P128 could be used to treat staphylococcal bovine mastitis.