论文部分内容阅读
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。
OBJECTIVE: To design a chimeric gene of methicillin-resistant Staphylococcus aureus (MRSA) -resistant TG-TPase for molecular modeling and three-dimensional structure observation and to construct a prokaryotic expression plasmid for expression and purification of chimeric genes. Using its enzymatic activity to establish an inhibitory molecular screening system lays the foundation. Methods Bioactiveit and DNAStar software were used to analyze the structure and activity of MRSA-resistant TGase and TPase active fragments. The design of TG-TPase chimeric target, The TG gene fragment of PBP2 and the TP gene fragment of PBP2a were cloned from the MRSA strain by primer design. The chimeric gene of TG-TPase was further constructed by molecular biological manipulation with primer extension and restriction enzyme digestion, and subcloned into the prokaryotic expression plasmid pET30b The recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) plysS, induced with IPTG, and then fermented in a small amount. The chimeric gene was purified and identified by Western blotting. Results The designed TG-TPase chimeric targets include the TGase activity region of PBP2, the N-terminus of hinge region, the C-terminus of hinge region of PBP2a and the complete TPase domain. The fusion gene was 1 935 bp, encoding 645 Amino acids, isoelectric point of 7.10, the relative molecular mass of 72 000. The TG fragment of PBP2 and the TP fragment of PBP2a were successfully cloned from MRSA by PCR. The chimeric gene of TG-TPase and its prokaryotic expression plasmid were constructed and the target protein was expressed in Escherichia coli. The results are in line with the expected design. Purification analysis of the fusion protein showed that the fusion protein existed in the form of inclusion bodies and was purified by Ni-NTA affinity column with the purity of over 90% under denaturing conditions. Conclusion The chimeric gene of Staphylococcus aureus TG-TPase was successfully designed and constructed, and the recombinant protein was successfully purified. In order to further utilize its enzymatic activity against Staphylococcus aureus Agent screening laid the foundation.