耐甲氧西林金黄色葡萄球菌IsdB活性片段的克隆、表达及抗原免疫保护性初步研究

目的探讨耐甲氧西林金黄色葡萄球菌(MRSA)抗原IsdB活性片段(IsdB2)免疫保护作用。方法利用生物信息学技术预测分析出IsdB活性片段(IsdB2),PCR扩增编码IsdB2的基因片段,亚克隆至GST标签融合表达的原核表达载体pGEX-6P-2中,将载体转化入大肠杆菌XL-1 blue,通过IPTG诱导表达IsdB2/GST融合蛋白,利用GST亲和层析初步纯化获取IsdB2蛋白。用IsdB2蛋白抗原辅以氢氧化铝佐剂对小鼠进行免疫实验,统计小鼠存活率对IsdB2抗原的免疫性进行初步研究。结果重组质粒经过BamHⅠ和NotⅠ双酶切鉴定、核酸序列测定和IPTG诱导表达IsdB2/GST及酶切获取IsdB2蛋白的SDS-PAGE分析表明,IsdB2蛋白相对分子质量大小约72 000,GST标签相对分子质量大小约26 000,与预期相符合。用IsdB2蛋白对小鼠进行3次疫苗免疫实验,IsdB2对小鼠的保护率分别为84.6%、50%和60%。结论成功构建重组表达载体pGEX-6P-2-IsdB2,利用大肠杆菌表达系统、GST亲和层析和酶切方法获得IsdB2蛋白抗原,通过3次动物疫苗免疫实验结果表明IsdB2具有免疫保护性,为研制新型有效的MRSA疫苗奠定实验基础。

Cloning,expression,and antigen immune protection of IsdB active fragment protein from Methicillin-resistant Staphylococcus aureus

In this study,we aimed to express IsdB active fragment protein and evaluate its immunoprotection.Firstly,we used the bioinformatics to predict active domain of IsdB and select active fragment IsdB2.The sequence coding IsdB2 was amplified by PCR and subcloned into pGEX-6p-2 vector.After the target gene was sequenced,the plasmid was transformed into E.coil XL-1 and was induced with IPTG to express fusion protein Isdb2/GST.Then,the expressed fusion protein Isdb2/GST was purified with affinity chromatography method and separated by enzyme digestion.SDS-PAGE analysis showed the expected molecular mass was about 72 000 and the purity reached 95%.The immune protection of Isdb2 protein was evaluated in three animal trials,in which the protection rates were 84.6%,50%,and 60%,respectively.All the results suggest that the Isdb2,emulsified in an alum adjuvant,can provide strong immune protection against S.aureus in mice,therefore could be a vaccine candidate against infection induced by S.aureus.