噬菌体展示布鲁氏菌蛋白文库构建及差减筛选融合蛋白

目的建立噬菌体展示羊型布鲁氏菌16M株表面蛋白文库,差减筛选具有良好特异性、亲和力及反应原性的蛋白,为确定新型诊断用抗原奠定基础。方法利用噬菌粒载体pYW01构建重组羊型布鲁氏菌16M株基因文库,辅助噬菌体VCSM13感染基因文库进而构建噬菌体展示羊型布鲁氏菌16M蛋白文库。利用PEG纯化后,扩增蛋白文库。随机挑取单克隆,提取质粒,测序鉴定蛋白文库。通过差减筛选,选择具有良好特异性、亲和力及反应原性的表面蛋白,为确定诊断用抗原奠定基础。结果通过DNA重组技术,构建羊型布鲁氏菌16M基因文库,库容量达到10~8pfu/mL,并且随机性良好。利用辅助噬菌体VCSM13包装基因文库,随机挑取蛋白文库中单克隆,提取质粒,经测序鉴定,成功构建噬菌体展示羊型布鲁氏菌16M株表面蛋白文库。利用免疫血清及感染血清对噬菌体展示蛋白文库进行差减淘选,筛选出6个噬菌体融合蛋白。经Western-blot分析6个噬菌体融合蛋白均具有较好的反应原性及特异性。结论成功构建噬菌体展示布鲁氏菌蛋白文库,差减筛选出6个具有较好反应原性及特异性的融合蛋白,为后续新型血清学诊断制剂筛选奠定基础。

Phage display proteins library of Brucella and subtractive scanning fusion protein

We established a phage display surface protein library of Brucella melitensis 16M strain and to lay a foundation for the screening of a new diagnostic antigen. Recombinant Brucella melitensis 16M strain gene library was constructed by using phagemid vector pYW01. The protein library of B. melitensis 16M strain was constructed infected by helper phage VCSM13. Purified by PEG and enlarged library, the plasmid DNA was randomly extracted from the library to sequence and analyze. The novel proteins are identified by subtractive scanning of the protein library with vaccine serum and infected serum. Complete-enzyme-linked immunosorbent assay (c-ELISA) and Western blot were used to evaluate novel serodiagnostic antigens. B. melitensis 16M strain gene library was constructed by DNA recombination. The library's capacity was 10⁸ pfu / mL with performance randomness. Gene library was infected by helper phage VCSM13 to generate protein library. The plasmid DNA of protein library were randomly extracted to sequence and analyze. The result reveals that phage display protein library of B. melitensis 16M strain was successfully constructed. Six fusion proteins were identified with subscribe scanning and verified by c-ELISA and Western blot.