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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 construct chimeric gene of methicillin-resistant Staphylococcus aureus (MRSA) -resistant TG-TPase for prokaryotic expression and purification, and to establish an inhibitor screening system to further utilize its enzymatic activity basis. Methods The gene fragment of PBP2 and the TP fragment of PBP2a were cloned from the MRSA strains screened by the salt plate method by primer design and cloned into the T vector respectively. The positive recombinant was digested / re-ligated to construct the TG-TPase chimera The correct chimeric gene was identified by nucleotide sequencing and subcloned into pET22b to construct expression vector. The recombinant plasmid was transformed into Rosetta (DE3) plysS and induced by IPTG. The expressed protein was analyzed by SDS-PAGE, mass spectrometry and Western blotting Identification. A small amount of fermentative recombinant bacteria, chimeric gene expression products for preliminary purification. Results Two MRSA isolates were screened from 13 clinical isolates of Staphylococcus aureus. The TG fragment of PBP2 and the TP fragment of PBP2a were successfully cloned by PCR. The TG-TPase chimeric Gene and its prokaryotic expression vector and expressed in Escherichia coli, the yield reached 43% of total bacterial protein. Purification analysis of the fusion protein showed that the chimeric protein existed in the form of inclusion bodies and was purified by Ni-NTA affinity column under denaturing conditions with the purity of over 90%. Conclusion The chimeric gene of Staphylococcus aureus TG-TPase and its recombinant expression engineering strain were successfully designed and constructed, which laid the foundation for further screening of anti-drug resistant Staphylococcus aureus with its enzymatic activity.