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目的 构建弓形虫表面抗原SAG2的DNA疫苗载体 ,并在Vero细胞中表达。 方法 设计 1对引物 ,从弓形虫RH株速殖子基因组DNA中扩增SAG2全长编码基因 ,构建 pVAX1 SAG2真核表达重组质粒。以限制性内切酶KpnⅠ和EcoRⅠ进行双酶切、PCR鉴定 ,纯化后进行测序鉴定。脂质体介导法瞬时转染Vero细胞 ,同时以 pVAX1为对照 ,48h后收集细胞 ,Western blot鉴定。 结果 从弓形虫RH株DNA中扩增出了 5 77bp的SAG2基因 ,构建了真核表达载体 pVAX1 SAG2 ,在质脂体介导下转染Vero细胞 ,质粒DNA成功的转染到细胞中。通过Westen blot分析 ,细胞裂解液样品有 1条可被弓形虫免疫血清所识别的约 17ku大小的条带 ,与预计大小一致。 结论 真核表达载体pVAX1 SAG2在Vero细胞中有一定表达 ,且有一定的活性。
Objective To construct the DNA vaccine vector of Toxoplasma gondii surface antigen SAG2 and express it in Vero cells. Methods A pair of primers was designed to amplify the full-length SAG2 gene from Toxoplasma gondii RH strain tachyzoites genomic DNA to construct the eukaryotic expression recombinant plasmid pVAX1 SAG2. Restriction enzyme Kpn Ⅰ and EcoR Ⅰ double digestion, PCR identification, purification and sequencing identification. Vero cells were transiently transfected by liposome-mediated method, pVAX1 was used as a control, and cells were collected after 48h and identified by Western blot. Results A 5 77 bp SAG2 gene was amplified from DNA of T. gondii RH strain. The eukaryotic expression vector pVAX1 SAG2 was constructed and transfected into Vero cells by liposomes. The plasmid DNA was successfully transfected into the cells. By Westen blot analysis, the cell lysate sample has a band of approximately 17 ku, which is recognized by Toxoplasma gondii immune serum and is consistent with the expected size. Conclusion The eukaryotic expression vector pVAX1 SAG2 is expressed in Vero cells with certain activity.