MRSA耐药相关TG-TPase嵌合基因的原核表达与纯化

Prokaryotic expression and purification of methicillin-resistant Staphylococcus aureus TP-TGase domain

目的设计、构建耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcus aureus,MRSA)耐药相关TG-TPase嵌合基因,进行原核表达、纯化探索,为进一步利用其酶学活性建立抑制剂筛选系统奠定基础。方法通过引物设计,从盐平板法筛选的MRSA菌株中分别克隆PBP2的TG基因片段和PBP2a的TP片段,分别克隆入T载体,对阳性重组子进行酶切/再连接,构建TG-TPase嵌合基因,经核苷酸测序鉴定正确的嵌合基因再亚克隆到pET22b,构建表达载体,转化Rosetta(DE3)plysS,用IPTG进行诱导表达,并对表达的蛋白进行SDS-PAGE、质谱和Western blotting鉴定。小量发酵重组菌,对嵌合基因表达产物进行初步纯化。结果从13株临床分离的金葡菌中筛选出2株高耐药性MRSA菌株,用PCR法从中成功地克隆到青霉素结合蛋白PBP2的TG片段和PBP2a的TP片段,构建了TG-TPase嵌合基因及其原核表达载体,并在大肠埃希菌中表达,产量达菌体总蛋白的43%。融合蛋白纯化分析表明,嵌合蛋白以包涵体形式存在,在变性条件下经Ni-NTA亲和柱纯化,纯度达90%以上。结论成功设计并构建了耐药性金葡菌TG-TPase嵌合基因及其重组表达工程菌,为进一步利用其酶学活性进行抗耐药性金葡菌抑制剂的筛选奠定基础。

Objective To design and express a chimeric gene fused by the transglycosylase （TGase） domain and transpeptidase （TPase） domain of methicillin - resistant Staphylococcus aureus （MRSA）, and facilitate the subsequent screening of inhibitors against MRSA based on the TGase and/or TPase activities. Methods Two fragments encoding PBP2a TPase and PBP2 TGase of MRSA were amplified by PCR with pair of primers, and then cloned into pMD18-T vector. The TP-TGase chimeric gene was merged into pET22b by proper restriction enzyme digestion and DNA ligation. The recombinants of interest were screened out by restriction enzyme analysis and direct DNA sequencing, and then transformed into Rosetta （DE3） plysS. Fusion proteins expressed by engineering bacteria harboring the chimeric gene were verified by SDS-PAGE, mass spectrum, and Western blotting analysis. Results Two out of thirteen were isolated as MRSA strains by salt-plate assay. The genomic DNA of one MRSA strain served as DNA template and PBP2a TPase and PBP2 TGase coding sequences were amplified successfully by PCR respectively. The chimeric gene were constructed by merging the TPase and TGase fragment into pET22b, which designated as pET22b- PBP2-2a. The engineered bacteria bearing pET22b-PBP2-2a could express the fusion protein of interest induced by IPTG. The expressed protein was verified by SDS-PAGE, mass spectrum, and Western blotting analysis, and it can be purified to 90% homogeneity by one-step Ni-NTA affinity chromatography. Conclusion The TP-TGase chimeric gene and the engineering bacteria pET-22b-PBP2-2a/Rosetta are successful constructed, which may lay a great foundation for later screening of inhibitors against MRSA.