| 摘要: |
| 【目的】克隆果蝇抗菌肽Andropin基因，以期利用原核表达系统生产高活性抗菌肽。【方法】首先化学合成了目的基因的2个片段和1对引物，然后利用重叠区互补PCR法得到抗菌肽Andropin的全基因序列。构建了融合表达载体pET32a-Anp，并转入感受态细胞OrigammiB进行原核融合表达。重组菌株诱导表达后，用His-Tag Purification kit亲和层析柱纯化融合蛋白，纯化的蛋白用肠激酶切割并进行活性分析。【结果】在诱导温度37 ℃、1 mmol/L IPTG 诱导培养4 h 的条件下，可获得高效表达的融合蛋白；融合蛋白的表达量占总蛋白的30%，分子质量约为28 ku，可溶性达70%以上；抗菌活性试验表明，表达产物对大肠杆菌和金黄色葡萄球菌均有抑制作用。【结论】重组工程菌表达得到Andropin的融合抗菌肽，且其具有抗菌生物学活性。 |
| 关键词: 果蝇 抗菌肽 基因融合表达 大肠杆菌 抗菌活性 |
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| Prokaryotic expression and activity analysis of drosophila Andropin gene |
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| Abstract: |
| 【Objective】In this study,the andropin gene of drosophila was cloned into Escherichia coli to produce high activity antibacterial peptide by prokaryotic expression system.【Method】Two sequences and a pair of primers of the Drosophila Andropin gene were designed and synthesized by chemical method and the whole Andropin sequence was obtained by complementary overlay area PCR method.A recombination fusion expressing vector pET32a－Anp was constructed and transformed into an E.coli host named OrigammiB.The expressed recombinant protein was purified by His-tag Purification kit,digested by enterokinase,and then its antibacterial activity was tested by agar diffusion assay.【Result】The result showed that the Andropin was expressed as a fusion protein in a 70% soluble form with a theoretical molecular weight of 28 ku.Following induction under the condition of 1 mmol/L IPTG for 4 h at 37 ℃,the high－level expression of the fusion protein can be achieved and the amount of the fusion protein reached 30% of the total proteins.The activity assay demonstrated that the peptide had active ability against the E.coli and Staphylococcus aureaus.【Conclusion】The recombinant engineered bacteria expressed the Andropin fusion peptide and activity assay demonstrated that the peptide had antibacterial biological activity. |
| Key words: drosophila antibacterial peptide gene fusion expression E.coli antibacterial activity |
