耐甲氧西林金黄色葡萄球菌CHIPS基因的克隆、序列分析及原核表达  

Amplification,cloning,and expression of methicillin resistant staphylococcus aureus CHIPS gene

作　　者：容莉莉[1] 杨镒宇[1] 关锐梨[1] 关小珊[1] 邓秋连[1] 谢永强[1] 周珍文[1] 

机构地区：[1]广州医学院附属广州市妇女儿童医疗中心,510120

出　　处：《广州医药》2013年第4期1-4,共4页Guangzhou Medical Journal

摘　　要：目的对耐甲氧西林金黄色葡萄球菌(MRSA)趋化抑制蛋白(CHIPS)进行扩增、克隆、序列分析、原核表达及纯化,为后续的疫苗研究奠定基础。方法根据GenBank中趋化抑制蛋白(CHIPS)的序列,设计一对特异性引物,以耐甲氧西林金黄色葡萄球菌DNA为模板PCR扩增CHIPS基因,纯化DNA进行EcoRI、XhoI双酶切鉴定及测序鉴定,并将CHIPS亚克隆入表达载体PET-28α,转化感受态大肠杆菌BL21,对质粒进行双酶切鉴定及基因序列分析,用IPTG诱导表达重组蛋白,His标签单克隆抗体进行免疫印迹验证重组蛋白的表达。结果以金黄色葡萄球菌临床儿童分离株基因组为模板,成功扩增CHIPS基因,基因大小为450bp;重组PET-28a(+)-CHIPS双酶切鉴定可见目的片段,测序结果显示CHIPS在正确读框中,序列比对分析显示其与相关报道核苷酸序列一致性达99.98%。经IPTG诱导后,pET-28a(+)-CHIPS/BL21在相应分子量(17kDa)可见融合蛋白在上清表达,免疫印迹检测到目的蛋白。结论成功从儿童分离株MRSA中构建了CHIPS的原核表达系统,并成功在大肠杆菌BL21中融合表达,为后续的疫苗研究奠定基础。Objective To amplify chemotaxis inhibitory protein which excreted by methicillin resistant staphylococcus aureus, conduct cloning, sequence analysis , prokaryotic expression and purification, laid foundation for further research for the follow-up study of the vaccine. Methods A pair of specific primer was designed according to chemotaxis inhibitory protein （CHIPS） gene in the GenBank, using Staphylococcus aureus DNA as template, CHIPS gene was PCR by specific primer, the PCR product was transformed to E. coli DH5a, after digested by BamH I, Xhol I the production was ligated with pET-28a （ ＋ ） which digested with same enzyme, the recombinant plasmid was transformed into E. coli BL21. Extrated recombinant plasmid was identified by double enzyme digestion and sequence analysis, the sequence results were compared with gene sequence in the GenBank. And recombinant fusion protein was induced by IPTG, west-blotting with HRP Conjugated Anti His-Tag Mouse Mono- clonal Antibody was used to identify the fusion protein. Results CHIPS gene was successfully amplified from Staphylococcus aureus, it was 450bp, recombinant plasmid was identified by double enzyme digestion. DNA sequence showed that CHIPS was in the correct open reading frame, sequence comparison analysis showed its nucleotide sequence identities were 99. 8% compared with CHIPS sequence of Staphylococcus aureus in GenBank. Recombinant fusion protein expression product can be seen at about 17kDa, and the fusion protein has been identified by west-blotting. Conclusion The CHIPS gene of Methicillin Resistant Staphylococcus aureus from a clinical child was successfully cloned and prokaryotic expression, which laid a good foundation for the follow-up study of the vaccine.

关 键 词：MRSA CHIPS 克隆 原核表达 

分 类 号：R378[医药卫生—病原生物学]