弓形虫表面抗原SAG2基因的克隆表达纯化及鉴定

目的构建弓形虫表面抗原2(SAG2)基因重组质粒并在大肠埃希菌中表达。方法根据SAG2基因序列设计并合成引物,用PCR法从弓形虫基因组DNA中扩增SAG2基因片段,再克隆到p GEX-4T载体中,构建重组质粒。重组质粒经酶切鉴定并测序后,在大肠埃希菌BL21中诱导表达,产物经SDS-PAGE分析并纯化,以Western blotting分析其反应原性。结果 SAG2基因PCR产物大小约为561 bp,与预期相符。重组质粒经酶切及PCR鉴定构建成功,测序结果与已知序列吻合。重组质粒转化菌经IPTG诱导后表达的SAG2融合蛋白分子量约为47 ku,该蛋白可被GST标签抗体识别。结论成功重组了弓形虫SAG2基因,表达蛋白具有反应原性。

Cloning,expression, purification and identification of Toxoplasma gondii SAG2 gene in Escherichia coli

Objective Objective To construct a recombinant plasmid containing surface antigen 2 （SAG2）gene of Toxoplasma gondii and express it in Escherichia coli. Methods Methods The truncated SAG2 gene was amplified from the genomic DNA of T. gondii RH strain and cloned into plasmid pGEX?4T. Then the recombinant pGEX?4T?SAG2 was induced by IPTG and expressed in E. rich? ia coli BL21. The expressed proteins were analyzed by SDS?PAGE and purified，and the immunogenicity of the product was ana? lyzed by Western blotting. Results Results The amplified SAG2 gene was about 561 bp，which was accorded to the expectation. The re? combinant plasmid was constructed successfully by digested with double restriction enzyme and confirmed with DNA sequenc? ing. SDS?PAGE and Western blotting showed the molecular weight of SAG2 fusion protein was about 47 ku，and the protein could be identified by GST?tag antibody. Conclusion Conclusion The truncated SAG2 gene of T. gondii has been successfully cloned and expressed in E. coli BL21 cells，and the recombinant protein has immunogenicity.