拟穴青蟹抗菌肽hyastatin基因原核表达条件的优化  

Optimization of Prokaryotic Expression of Antibacterial Peptide hyastatin Gene in Scylla paramamosain

作　　者：彭银辉[1,2] 蔡小辉[1,2,3,4] 熊向英[1,2] 刘旭佳[1,2] 黄国强[1,2] 

机构地区：[1]广西海洋生物技术重点实验室,北海536000 [2]广西海洋研究所,北海536000 [3]广东海洋大学海洋学院,湛江524088 [4]广东省水产经济动物病原生物学及流行病学重点实验室,湛江524088

出　　处：《生物技术通报》2015年第7期138-142,共5页Biotechnology Bulletin

基　　金：广西自然科学基金资助项目(桂科自2010GXNSFA013079);广西科技攻关资助项目(桂科攻1114012-11)

摘　　要：旨在优化拟穴青蟹抗菌肽hyastatin基因原核表达条件。克隆拟穴青蟹抗菌肽hyastatin基因成熟肽片段,与p ET-28a表达载体连接后,转入到大肠杆菌BL21(DE3)中,通过优化条件进行诱导表达。结果显示,在异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.2 mmol/L、37℃条件下培养4 h后表达量最大,分子大小与预期值相符。融合蛋白主要以包涵体形式高效表达,通过His Trap HP柱子使其得到进一步纯化。Western blot分析表明,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合。说明通过优化表达条件,获得拟穴青蟹抗菌肽hyastatin基因原核表达表达产物。This study aims to optimize the prokaryotic expression of hyastatin gene in Scylla paramamosain. The fragment of cloned mature peptide in S. paramamosain hyastatin was ligated with the expression vector pET-28a. This recombinant was transformed into Escherichia coli BL21 （ DE3 ） , and the gene was expressed by inducing under the optimal conditions. The expression of this protein was the highest under the 37℃ and in 0.2 mmol/L of IPTG for 4 hours. The molecular weight of the expressed product was identical to that of expected protein by SDS-PAGE analysis. The fusion protein was efficiently expressed as inclusion bodies, and further purified by HisTrap HP column. The result of Western blot showed that the fusion protein could be bound specifically with mouse anti-His-tag Mab. In conclusion, the prokaryotic expression product of hyastatin gene in S. paramamosain may be obtained by optimizing expression conditions.

关 键 词：拟穴青蟹 hyastatin基因 原核表达 条件优化 

分 类 号：Q78[生物学—分子生物学]