摘要
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 make high efficiency expression of the SAG2 gene from Toxoplasma gondii RH strain in E.coli and study the antigenicity of the expressed product. Methods The SAG2 gene fragment of T.gondii RH strain amplified by PCR method from genome DNA was cloned into the pMD-18T vector and transformed into E.coli DH5α. After nucleotide sequencing, the SAG2 gene fragment was subcloned into the expression vector pET23a with correct orientation and transformed into E.coli DH5α. The plasmid from the correct clone identified by PCR method and endonuclease digestion was transformed into E.coli BL21(DE3) and induced for expression. The expressed product was studied by SDS\|PAGE and Western blot. \ Results \ 502 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100% homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about \{Mr 19 000\}. Western blotting indicated that the antigenicity of the protein was specific. \ Conclusion \ The plasmid pET 23a\|SAG2 was constructed and a high efficiency expression of the SAG2 gene from T.gondii RH strain was made. The expressed product shows a specific antigenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2004年第4期231-234,共4页
Chinese Journal of Parasitology and Parasitic Diseases
