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目的 构建原核重组表达质粒pET2 3a SAG2 ,并在大肠埃希菌中实现高效表达 ,以及检测表达产物的抗原性。 方法 PCR扩增SAG2编码基因目的片段 ,琼脂糖凝胶电泳回收纯化 ,克隆至 pMD18 T载体 ,转化大肠埃希菌DH5α。测序后亚克隆至表达质粒载体 pET2 3a ,构建重组表达质粒 pET2 3a SAG2 ,转化大肠埃希菌DH5α。筛选阳性克隆 ,经限制性酶切分析鉴定后 ,转化大肠埃希菌BL2 1(DE3 ) ,以异丙基 β D 硫代半乳糖苷诱导表达。十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)与免疫印迹分析表达产物。 结果 PCR扩增出约 5 0 0bp的SAG2编码基因目的片段 ,与预期片段大小相符 ,经测序鉴定无基因突变 ;所构建的 pET2 3a SAG2重组表达质粒阳性克隆经PCR与双酶切鉴定 ,与预期结果一致 ;含有pET2 3a SAG2重组质粒的大肠埃希菌BL2 1(DE3 )诱导后得到了高效表达 ,SDS PAGE显示表达产物约Mr 190 0 0 ;免疫印迹结果表明表达产物具有良好的抗原性。 结论 成功构建了pET2 3a SAG2表达质粒 ,实现了全长成熟SAG2蛋白在大肠埃希菌中的高效表达 ;表达产物具有良好的抗原性。
Objective To construct prokaryotic recombinant plasmid pET2 3a SAG2 and express it in Escherichia coli, and to detect the antigenicity of the expressed product. Methods The target fragment of SAG2 gene was amplified by PCR, purified by agarose gel electrophoresis, cloned into pMD18 T vector and transformed into Escherichia coli DH5α. After sequencing, the recombinant plasmid pET2 3a was subcloned into expression vector pET2 3a to construct Escherichia coli DH5α. Positive clones were screened and identified by restriction analysis. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl β D thiogalactoside. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot analysis of expression products. Results The target fragment of about 500 bp SAG2 gene was amplified by PCR, which was consistent with the size of the expected fragment. The gene was identified by sequencing. The positive recombinant plasmid pET2 3a SAG2 was identified by PCR and double enzyme digestion, The results showed that the recombinant plasmid pET2 3a SAG2 was highly expressed in E. coli BL21 (DE3) induced by SDS-PAGE and the expressed product was about 190 × 10 SDS-PAGE. Western blotting showed that the expressed product had good antigenicity. Conclusion The pET2 3a SAG2 expression plasmid was successfully constructed and the full-length mature SAG2 protein was highly expressed in Escherichia coli. The expressed product has good antigenicity.