抗耐药性金黄色葡萄球菌嵌合药靶的设计与构建

Design and construction of a chimeric drug target of methicillin-resistant St-aphylococcus aureus

目的设计耐甲氧西林金黄色葡萄球菌(MRSA)耐药相关TG-TPase嵌合基因,进行分子建模及三维结构观察,并构建了原核表达质粒,进行嵌合基因的表达、纯化,为进一步利用其酶学活性建立抑制分子筛选系统奠定基础。方法运用Bioedit和DNAStar软件对MRSA耐药性相关转糖基酶(TGase)和转肽酶(TPase)活性片段序列和活性区结构特点及其活性发挥进行分析,设计TG-TPase嵌合药靶,并通过引物设计从MRSA菌株中克隆PBP2的TG基因片段和PBP2a的TP基因片段,进一步用引物延伸法、酶切连接等分子生物学操作构建TG-TPase嵌合基因并亚克隆至原核表达质粒pET30b中,转化Rosetta(DE3)plysS大肠埃希菌,用IPTG进行诱导表达,并小量发酵重组菌,对嵌合基因表达产物进行初步纯化和Western blotting鉴定。结果设计的TG-TPase嵌合药靶包括PBP2分子的TGase活性区、绞链区的N-端,PBP2a分子绞链区C-端及完整的TPase结构域,融合基因为1 935 bp,编码645个氨基酸,等电点为7.10,相对分子质量72 000。用PCR法从MRSA菌株中成功克隆了青霉素结合蛋白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质粒,并在大肠埃希菌中表达出药靶蛋白,表达结果与预期设计相符合。融合蛋白纯化分析表明,融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、构建了耐药性金葡菌TG-TPase嵌合基因及其重组表达工程菌,并对融合蛋白进行了初步纯化,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。

This paper is aimed to design the drug-resistance related TG-TPase chimeric gene derived from methicillin-resistant Staphylococcus aureus（MRSA） by the methods of molecular modeling and molecule three-dimensional structure analysis.TG-TPase chimeric gene was constructed and expressed in a prokaryotic system,and then the expression products were purified.The multiple sequences alignments of MRSA transglycosylase（TGase） and transpeptidase（TPase） active domain were performed by Bioedit and DNAStar software and a TG-TPase chimeric gene was designed based on the analysis of TPase and TGase structural features and activity performance.Two fragments encoding MRSA PBP2a TPase and PBP2 TGase domains were respectively amplified by PCR.Then the TG-TPase chimeric gene was constructed by the method of primer extension and cloned into pMD18-T vector,then subcloned into pET30b.The recombinants of interest were screened out by restriction enzyme analysis and direct DNA sequencing,then transformed into E.coli strain Rosetta（DE3） plysS.The fusion proteins expressed by the engineering bacteria harboring the chimeric gene were analyzed by SDS-PAGE.The designed TG-TPase gene consists of 1 935 bp,including the TGase domain and N-terminal hinge region of PBP2 plus the C-terminal hinge region and TPase domain of PBP2a.The fusion protein encoded by the designed chimeric gene has an isoelectric point（pI） of 7.10 and a molecular weight of 72 000,and can be purified to 90 % homogeneity by one-step Ni-NTA affinity chromatography.In this study,a TP-TGase chimeric gene was designed and the engineered bacteria carrying the chimeric gene are successful constructed,which may lay a foundation for further screening of inhibitors against MRSA.