| 摘要: |
| 【目的】克隆和表达鸡β-防御素3(Gal-3)基因，并对其抑菌活性进行研究，为寻找替代抗生素的抗菌肽提供试验依据。【方法】利用 RT-PCR 方法，从鸡心脏中分离并克隆了鸡Gal-3基因，将其与分子伴侣基因SUMO相融合，再将该融合基因与原核表达载体pET-20b连接后转化至BL21（DE3）宿主细胞中，经IPTG 诱导后，对表达产物进行SDS-PAGE和Western blot分析；最后，对Gal-3融合蛋白进行纯化，并对表达产物进行体外抑菌试验。【结果】通过RT-PCR扩增获得了约240 bp的鸡Gal-3基因，经IPTG诱导获得约26 ku的蛋白。IPTG在终浓度为0.6 mmol/L，33 ℃诱导4 h，诱导时菌体A600为0.6，蛋白表达量最高，且多数以可溶形式存在，33 ℃上清中的蛋白含量比37 ℃上清高。纯化的蛋白对大肠埃希菌和金黄色葡萄球菌均有明显的抑菌活性。【结论】成功克隆并表达了鸡Gal-3基因，其原核表达产物对大肠埃希菌和金黄色葡萄球菌具有一定的抑菌活性 |
| 关键词: 防御素 Gal-3基因 原核表达 抑菌活性 |
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| 基金项目:吉林省科技发展计划项目（20090244）；吉林省教育厅“十一五”科学技术研究项目（2009297） |
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| Cloning,expression and antibacterial activity of chicken Gal-3 gene |
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| Abstract: |
| 【Objective】The study aimed to clone and express the chicken Gal-3 gene,purify the recombinant Gal-3 protein and analyze its antimicrobial activity.It contributed to further investigation on biological function of defensing and exploitation of new antibiotics.【Method】The chicken beta-defensin Gal-3 gene was amplified from cDNA of chicken heart using reverse transcription-polymerase chain reaction (RT-PCR),and fused with SUMO(small ubiquitin-related modifier).Then the fused gene was inserted into pET-20b vector and expressed in E.coli BL21.Recombinant Gal-3 protein was analyzed by SDS-PAGE and Western blot after IPTG induction.At last,obtained protein was purified and the antibacterial activity was assayed by agar hole diffusion-inhibition zone method.【Result】Complete Gal-3 cDNA coding sequence was successfully obtained by PCR,the full lengh of Gal-3 gene was 240 bp.The optimal parameters of IPTG induction,at which highest expression was observed,were 33 ℃ for culture temperature,0.6 mmol/L for joining IPTG final concentration,and 0.6 for recombinant strain growth density A600 after being induced for 4 hours.Soluble Gal-3 protein at 33 ℃ was more than that at 37 ℃,and most of them were in soluble form.Recombinant protein with relative molecular mass of 26 ku was produced and purified.Antibacterial activity outcomes demonstrated that the recombinant Gal-3 protein was able to suppress the growth of E.coli and Staphyloccocus.【Conclusion】The chicken Gal-3 gene from chicken heart was successfully cloned and expressed in E.coli and Staphyloccocus.The purified recombinant protein showed antimicrobial activity for E.coli and Staphyloccocus. |
| Key words: defensin Gal-3 gene prokaryotic expression antimicrobial activity |
