弓形虫SAG1基因在HeLa细胞表达的研究

Study on the expression of toxoplasma gondii SAG1 gene in hela

目的 :研究弓形虫 SAG1基因在 He L a细胞中的表达 ,为研制弓形虫疫苗奠定基础。方法 :采用磷酸钙 -DNA共沉淀转化法将重组质粒 pc DNA3- SAG1导入 He L a细胞 ,用 G418进行选择培养 ,筛选出表达阳性的细胞克隆 ,再通过培养和加压 ,建立稳定分泌 SAG1抗原的阳性 He L a细胞克隆株 ,分离表达产物 ,进行 SDS- PAGE、Western blot分析。结果 :采用钙沉淀法成功地将重组质粒 SAG1导入 He L a细胞 ,通过 G418选择和加压培养 ,获得稳定分泌 SAG1抗原的阳性 He L a细胞 ,表达的蛋白经 SDS- PAGE、Western blot分析 ,显示其分子量 30 k Da蛋白 ,与理论值相符。结论 :成功构建重组质粒 pc DNA3- SAG1,建立稳定分泌 SAG1抗原的阳性 He L

Objective:To study the SAG1 gene of the main surface antigen of toxoplasma gondii in HeLa,and to lay a foundation of further study on the vaccine development of Toxoplasma gondii.Method:Using the calcium phosphate-DNA coprecipitation technique,the recombinant plismid pcDNA3-SAG1 was transformed into mammalian cell line of human HeLa cells.The transformants were selected in medium containing 200 ug/mL of antibiotic G418.For cell cloning,individual colonies were randomly isolated,picked up and grown into confluent culture.The colonies were cultured and subcultured at the high pressure of G418(800ug/ mL) and therefore chosen to the expression of SAG1.The expressed protein was isolated and analyzed by SDS-PAGE and Western blot.Result:The recombinant plasmid pcDNA3-SAG1 was successfully transformed into human HeLa cell line.Transformed colonies of G418-resistant cells were obtained after transfection with recombinant plasmid carrying the SAG1 gene.High transformation efficiencies were obtained.The G418-resistance phenotype was stable under selective condition.SDS-PAGE analysis indicted that the molecular weight of the expressed protein of SAG1 was about 30kDa,similar to the predicted value.Conclusion:Successfully constructing eukaryotic expression plasmid pcDNA3-SAG1,Stable expression of SAG1 in human HeLa cells line was established.