摘要
目的构建重组质粒pGEX6p-2/Cpn0425,在E.coli BL21中进行诱导表达,纯化获得重组融合蛋白Cpn0425,并评价其抗原性。方法 PCR扩增肺炎嗜衣原体Cpn0425蛋白编码基因,构建pGEX6p-2/Cpn0425重组质粒,测序正确后在E.coli BL21中诱导表达,用SDS-PAGE和Western-Blot进行分析和鉴定。结果构建了重组质粒pGEX6p-2/Cpn0425,并在E.coli BL21菌中高效表达出Mr约为50kDa的GST-Cpn0425目的蛋白,超声裂解后SDS-PAGE分析目的蛋白在菌体细胞内以可溶性形式表达,经采用默克Novagen的GST纯化树脂纯化获得纯度在95%以上的重组蛋白。结论获得了Cpn0425基因片段,并在E.coli BL21中成功表达,GST-Cpn0425具有良好的抗原性。
The aim of this study is to construct the recombinant protein of Cpn0425fromChlamydophila pneumoniae and induce its expression in E.coli,meanwhile assess the antigenicity of protein Cpn0425.The gene of Cpn0425was amplified by PCR,which was used to construct the recombinant plasmid of pGEX6p-2/Cpn0425,and then the products were identified and analyzed by SDS-PAGE and Western blot.The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2prokaryotic expression vector.The similarity was 100% between the inserted gene and Cpn0425gene reported in GenBank by BLAST analysis.The SDS-PAGE demonstrated that the GST-Cpn0425 recombinant protein with a relative molecular weight about 50kDa was expressed after the E.coli BL21contained recombinant plasmid was induced and mainly existed in the pattern of solubility.Its purity reached up to 95%after purification with glutathione S-transferase(GST)resin chromatography of Novagen.In conclusion,we obtain Cpn0425gene of Ct and express it successfully in E.coli BL21.The GST-Cpn0425has a good antigenicity.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2012年第11期1061-1064,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金(No.30870134/C010902)资助~~
