Expression of Cyanovirin-N in E.coli and preparation of polyclonal antibody to Cyanovirin-N
- 生命科学－已发表论文 
目的:构建抗病毒蓝藻蛋白-N(Cyanovirin-N,CV-N)表达载体并在大肠杆菌中稳定表达,并进一步制备用于检测转CV-N基因表达产物的特异抗体。方法:以已构建的pMD(CV-N)为模板,设计引物,利用PCR技术扩增出CV-N基因,将成熟CV-N的编码框序列构建到原核表达载体pET-His中,氨苄青霉素平板筛选及PCR、酶切鉴定,挑选出阳性克隆进行扩增,DNA测序正确后,转化大肠杆菌BL21(DE3)进行IPTG诱导表达。SDS-PAGE以及MALDI-TOF/MS分析鉴定,采用透析法重折叠复性,亲和层析分离纯化得到分子量为12700的带有组氨酸标签的重组CV-N融合蛋白。用亲和层析分离纯化后的CV-N融合蛋白作为抗原,采用皮下多点注射免疫的方法免疫昆明小鼠,经过31天的免疫,获得抗CV-N的多克隆抗体。结果:成功地获得了CV-N基因,并且以包涵体的形式表达于大肠杆菌中,表达量高达42.0%。用间接酶联免疫吸附(ELISA)方法,测得抗体效价超过1∶8000,Westernblot检测结果显示,该抗体能特异性地与CV-N抗原产生明显免疫亲和反应。结论:建立原核高效稳定表达CV-N系统,获得具有生物学活性的CV-N蛋白,所制备的抗体具有很好的灵敏性和特异性,为进一步研究其功能奠定基础。Objective:To construct Cyanovirin-N(CV-N) expression plasmid pET-CVN and express the protein in E.coli, and to prepare specific antibody against Cyanovirin-N.Methods:In this study, the encoding sequence of Cyanovirin-N was obtained by PCR. The expression plasmid pET-CVN was constructed by inserting the encoding sequence of CV-N into pET-His containing T7 promoter and then BL21(DE3)strain was transformed. Expression of CV-N was induced by IPTG at 30℃ for 8 h. Cyanovirin-N was refolded by using dialysis method. The target protein was purified through Ni2+-NTA Agarose Fast Flow. The purified Cyanovirin-N was used as an antigen to immunize rats,and the polycolonal antibody to Cyanovirin-N was obtained.Results:Cyanovirin-N was cloned and expressed in E.coli successfully. SDS-PAGE analysis showed that the Cyanovirin-N was expressed in the form of insoluble inclusion body,and the expression amount of Cyanovirin-N was about 42.08% of total protein. With ELISA detection the titer of antibody was more than 1∶8 000, Western blot analysis showed that the antibody can bind with Cyanovirin-N specifically.Conclusion:Cyanovirin-N expression plasmid pET-CVN has been constructed, Cyanovirin-N with bioactivity has been obtained. The prepared antibody against Cyanovirin-N has well specificity and reactivity and can be used to detect the expression products by recombinant strains of E.coli.The work lays a foundation further study of its functions.