米曲霉中蓝藻抗病毒蛋白-N基因的克隆与表达  

Cloning and expression of cyanovirin-N from Aspergillus oryzae

作　　者：谢露[1,2] 付显令[1,2] 孙萍[1,2] 林俊芳[1,2] 郭丽琼[1,2] 云帆 黎倩怡 

机构地区：[1]华南农业大学食品学院食品生物技术研究所,广东广州510640 [2]广东省微生态制剂工程技术研究中心,广东广州510640 [3]广州市澳键丰泽生物科技有限公司,广东广州510760

出　　处：《生物技术》2016年第2期126-129,157,共5页Biotechnology

基　　金：国家自然基金项目("食用真菌组合表达白藜芦醇的研究";No.31272217);广东省科技计划项目("安全农业投入品新型益生芽孢杆菌素高产菌种选育研究";No.2015A020209121)

摘　　要：[目的]提高蓝藻抗病毒蛋白-N(CVN)的异源表达量,获得大量高纯度可溶性蛋白。[方法]采用RT-PCR从酱油发酵酱醪宏基因组中克隆获得CVN基因cvn-SF,构建了重组表达载体p ET32a-cvn-SF,转化E.coli BL21菌株,获得工程菌株,优化表达条件,通过Ni-NTA凝胶亲和层析对CVN-SF蛋白进行纯化。[结果]工程菌株在温度30℃、添加1 mmol/L的IPTG诱导剂、培养时间12 h的条件下获得最高表达量的可溶性蛋白。Ni-NTA凝胶亲和层析纯化得到了230.8 mg/L的CVN蛋白,占提取总蛋白含量的33.37%。[结论]所得CVN蛋白高于目前报道的CVN在大肠杆菌的最高表达量140 mg/L^([11])。[Objective] A recombinant was constructed to obtain abundant water-soluble CVN protein by the heterologous expression. [Methods] CVN gene was cloned from fermented soybean sauce and constructed into the p ET32 a expression vector.The recombinant protein was expressed in the cytoplasm of E. coli BL21 in a soluble form. Ni-NTA affinity chromatography was used to purify it. [Results]SDS-PAGE assay showed that recombinant CVN was expressed and purified successfully. Meanwhile,the optimal induction conditions were selected. [Conclusion] 230. 8 mg / L purified CVN protein was obtained,which was more than the maximum that had been reported.

关 键 词：蓝藻抗病毒蛋白-N 酱醪 米曲霉 基因克隆 基因表达 

分 类 号：Q786[生物学—分子生物学]