摘要
为进一步研究刺参甘露糖结合凝集素(AJ-MBL)的功能及提高刺参自身免疫力,对AJ-MBL基因的CDS区进行原核表达、纯化并初步鉴定其生物学活性。采用RT-PCR法扩增AJ-MBL基因编码区,经酶切鉴定、序列分析后,将其CDS区498 bp片段克隆至原核表达载体pET28a中,得到重组质粒pET-AJ-MBL。重组质粒转化大肠杆菌BL21(DE3)感受态细胞并经IPTG诱导表达出分子量约17 ku的融合蛋白。该蛋白以包涵体形式存在,用Ni2+离子亲和柱纯化。结果显示,获得高表达量的重组纯化蛋白。纯化的17 ku AJ-MBL进行红细胞凝集试验检测其生物学活性。凝集试验结果显示,AJ-MBL凝集最小浓度为10μg/mL。本实验成功构建了AJ-MBL原核表达质粒,并在大肠杆菌中高效表达了17 ku AJ-MBL蛋白,该蛋白具有良好的生物学活性。
To further study the functional characteristics of Apostichopus japonicus mannan-binding lectin（AJ-MBL）and to improve the natural immunity of A.japonicus,a prokaryotic vector of AJ-MBL gene CDS region was constructed.The fusion protein was expressed and purified in prokaryotic system and its bioactivity was studied.The coding sequence of AJ-MBL was amplified by RT-PCR method.After being identified by the restriction digestion and sequencing,the 498 bp of AJ-MBL gene was inserted into pET28a plasmid to yield an identified recombinant plasmid pET-AJ-MBL,which was used to transform the competent expressive cells of E.coli BL21（DE3）.After induction with IPTG,samples analysis results revealed that a fusion protein of approximately 17 ku was yielded,and it occurred in the form of inclusion bodies,and could be purified with Ni2＋ affinity chromatography.The results showed highly expressed fusion protein was acquired.The purified 17-ku AJ-MBL underwent hemagglutination assay to test its bioactivity,the results showed that the minimum hemagglutination concentration was 10 μg/mL.These results indicated that the CDS domain of AJ-MBL had high expressed and the fusion protein（17 ku AJ-MBL）was highly bioactivity.
出处
《水产学报》
CAS
CSCD
北大核心
2011年第8期1166-1171,共6页
Journal of Fisheries of China
基金
辽宁省教育厅团队项目(2008T021)
关键词
刺参
甘露糖结合凝集素
原核表达
蛋白纯化
红细胞凝集试验
Apostichopus japonicus
mannan-binding lectin
prokaryotic expression
purification
hemagglutination assay
