本文旨在构建抗菌肽parasin Ⅰ大肠杆菌重组表达载体,表达人工重组parasin Ⅰ,并检测其抑菌活性。根据parasin Ⅰ的成熟肽序列和大肠杆菌密码子偏好性,人工合成1段57 bp的基因编码cDNA,通过PCR技术构建大肠杆菌重组表达质粒pET32-Para Ⅰ,并在parasin Ⅰ基因5'-端引入Xa因子酶切位点。重组载体转化大肠杆菌Rosetta(DE3)菌株后,在不同温度(37和20 ℃)条件下对阳性转化子进行异丙基硫代半乳糖苷(IPTG)诱导表达。结果表明:IPTG成功诱导了1个21 ku的融合蛋白表达,重组蛋白占菌体总蛋白质的45%~50%。在低温条件下产生的融合蛋白主要以可溶性形式存在。可溶性重组蛋白经亲和层析纯化后,用Χa因子进行酶切,酶切产物经琼脂孔扩散法抑菌活性检测,结果显示其对金黄色葡萄球菌有一定抑制作用。本试验实现了parasin Ⅰ的重组表达,表达产物经Χa因子酶切后具抑菌活性。
This study was conducted to construct parasin Ⅰ expression vector, produce the recombinant peptide in the E.coli expression system, and analyze the antibacterial activity of the recombinant parasin Ⅰ. According to the amino acid sequence of mature peptide of parasin Ⅰ and codon bias of E. coli, a 57 bp cDNA fragment was synthesized. The recombinant vector pET32-Para Ⅰ in E. coli was constructed by PCR, and a sequence encoding endonuclease site of Xa factor was cloned into the 5'-upstream of the parasin Ⅰ gene. The recombinant vector was delivered into E. coli Rosetta (DE3) cells and isopropyl β-D-thiogalactoside (IPTG) was used to induce recombinant fusion protein under different temperatures (37 and 20 ℃). The results showed that the recombinant fusion protein reached about 45% to 50% of the total protein in E. coli, which had a molecular mass of 21 ku. The fusion protein induced under low temperature (20 ℃) mainly existed in dissolvable form. After purified by affinity chromatography, the recombinant parasin Ⅰ showed potent anti-Staphylococcus aureus activity after digested by Xa fator. An efficient expression system in E. coli for the production of a recombinant parasin Ⅰ with high yield are successfully constructed in this study, and the recombinant protein shows potent anti-bacterial activity after digested by Xa fator.
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