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目的克隆、表达和纯化结核分枝杆菌(Mycobacterium tuberculosis,M.tb)体内诱导基因Rv0808,并制备多克隆抗体,研究其编码蛋白的抗原性,了解诊断应用价值。方法高保真PCR扩增Rv0808基因,克隆入表达载体pET-30a(+)后转化大肠杆菌BL21(DE3),测序正确的克隆用IPTG诱导表达。用镍柱纯化重组蛋白,然后用纯化蛋白免疫新西兰白兔,制备和纯化多克隆抗体。将重组蛋白分别与多克隆抗体和结核病患者血清进行Western blot鉴定,并尝试用多克隆抗体进行免疫组化染色检测巨噬细胞中M.tb。结果成功构建了pET-30a(+):Rv0808重组表达载体。经过重组蛋白的可溶性表达及纯化后,SDS-PAGE结果显示在66kD处有一单一蛋白条带。将此蛋白免疫新西兰白兔后获得了效价为6400的多克隆抗体。重组蛋白与多克隆抗体可以发生免疫反应,但却不能检测出结核患者血清中的相应抗体。用此多克隆抗体也无法检测出巨噬细胞中的M.tb。结论结核分枝杆菌Rv0808编码蛋白具有良好的免疫反应性和免疫原性,但可能对于结核病的诊断价值较小。
Objective To clone, express and purify the gene Rv0808 induced by Mycobacterium tuberculosis (M.tb) in vivo and to prepare polyclonal antibodies to study the antigenicity of the encoded protein and to understand its diagnostic value. Methods High-fidelity PCR amplification of Rv0808 gene was cloned into the expression vector pET-30a (+) and transformed into E. coli BL21 (DE3). The correct clones were induced to express by IPTG. The recombinant protein was purified by nickel column, and then the purified protein was used to immunize New Zealand white rabbits to prepare and purify the polyclonal antibody. The recombinant proteins were identified by Western blot with the polyclonal antibody and the serum of patients with tuberculosis, and the detection of M.tb in macrophages was performed by immunohistochemistry with polyclonal antibody. Results The pET-30a (+): Rv0808 recombinant expression vector was successfully constructed. After soluble expression and purification of the recombinant protein, SDS-PAGE showed a single protein band at 66 kD. The protein was immunized New Zealand white rabbit polyclonal antibody titer of 6400. Recombinant protein and polyclonal antibodies can occur immune response, but can not detect the corresponding antibodies in the serum of patients with tuberculosis. This polyclonal antibody also failed to detect M.tb in macrophages. Conclusion Mycobacterium tuberculosis Rv0808 protein has good immunoreactivity and immunogenicity, but may be of less diagnostic value for tuberculosis.