登革2型病毒非结构蛋白NS4B的原核表达、纯化及多克隆抗体制备

Protokaryotic Expression and Purification of Nonstructural Protein NS4B of Dengue Virus Type 2 and Preparation of Polyclonal Antibody Against NS4B

目的:原核表达、纯化登革2型病毒非结构蛋白NS4B,并制备其多克隆抗体,以研究其结构与功能。方法:扩增编码登革2型病毒NS4B的24～238位氨基酸残基的基因序列,并将其克隆到原核表达载体pGEX-4T-1,转化大肠杆菌BL21(DE3),IPTG诱导表达;采用蛋白浸提方法从SDS-PAGE胶中回收融合蛋白;用纯化后的融合蛋白免疫BALB/c鼠制备多克隆抗体,采用间接免疫荧光法检测抗体效价。结果:原核表达了NS4B-GST融合蛋白,并获得了其多克隆抗体,抗体效价为1∶800。结论:登革2型病毒NS4B的24～238位氨基酸残基可诱导小鼠产生具有较高效价和特异性的多克隆抗体,这为研究NS4B的结构与功能奠定了基础。

Objective： To protokaryotic express and purify the nonstructural protein NS4B of dengue virus type 2, and to prepare the polyclonal antibody against NS4B. Methods： The gene for the area that includes amino acids 24 to 238 of NS4B was amplified, then the fragment was cloned into plasmid pGEX-4T-1 to construct the expression ptasmid. The recombinant NS4B-GST fusion protein was expressed after the expression plasmid was transformed into E.coli BL21 （DE3） and induced by IPTG. The fusion protein was detected by SDS-PAGE, and eluted from polyacrylamide gels. Then anti- NS4B antiserum was prepared in mice against NS4B and the antibody titer was determined by immunofluorescence assay. Results： NS4B of dengue virus type 2 as a GST fusion protein was successfully expressed. The polyclonal antibody against NS4B was prepared, and the antibody titer is 1：800. Conclusion： The amino acids 24 to 238 of NS4B of dengue virus type 2 can induce the effective and specific polyclonal antibody in mice. It makes some basis for the further understand of the structure and the function on NS4B of dengue virus.