Farhangnia Leila, Ghaznavi-Rad Ehsanollah, Mollaee Neda, Abtahi Hamid
Department of Biotechnology, Arak University of Medical Sciences, Arak, IR Iran.
Department of Microbiology and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, IR Iran.
Jundishapur J Microbiol. 2014 May;7(5):e10009. doi: 10.5812/jjm.10009. Epub 2014 May 1.
Staphylococcus aureus is one of the most common causes of nosocomial infections and its resistance to antibiotics is a global concern. Lysostaphin is an antimicrobial agent belonging to a major class of antimicrobial peptides and proteins known as the bacteriocins. It exhibits a high degree of anti-staphylococcal bacteriolytic activity.
In this study, high level of recombinant mature lysostaphin in Escherichia coli was produced by using pET32a expression vector.
The S. simulans gene encoding lysostaphin was extracted, amplified by polymerase chain reaction (PCR), and sub-cloned in prokaryotic expression vector pET32a. E. coli BL21 (DE3) plysS were transformed with pET32a-lys and gene expression was induced by IPTG. The expressed protein was purified by affinity-chromatography using (Ni-NTA) resin.
PCR and sequencing results confirmed the successful cloning of the target gene into the vector. The expression of protein was induced by IPTG and high concentration of the recombinant protein was obtained via the purification process by affinity-chromatography.
Our data showed that the recombinant mature lysostaphin protein produced by pET32a vector in E. coli system was very efficient.
金黄色葡萄球菌是医院感染最常见的病因之一，其对抗生素的耐药性是全球关注的问题。溶葡萄球菌酶是一种抗菌剂，属于一类主要的抗菌肽和蛋白质，即细菌素。它具有高度的抗葡萄球菌溶菌活性。
在本研究中，利用pET32a表达载体在大肠杆菌中高效表达重组成熟溶葡萄球菌酶。
提取编码溶葡萄球菌酶的模仿葡萄球菌基因，通过聚合酶链反应（PCR）扩增，并亚克隆到原核表达载体pET32a中。用pET32a-lys转化大肠杆菌BL21（DE3）plysS，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导基因表达。用镍-亚氨基二乙酸（Ni-NTA）树脂通过亲和层析纯化表达的蛋白。
PCR和测序结果证实目标基因已成功克隆到载体中。IPTG诱导蛋白表达，通过亲和层析纯化过程获得了高浓度的重组蛋白。
我们的数据表明，pET32a载体在大肠杆菌系统中产生的重组成熟溶葡萄球菌酶蛋白非常有效。
