弓形虫表面抗原SAG2的DNA疫苗载体构建及在Vero细胞中的表达

CLONING AND EUKARYOTIC EXPRESSION OF SAG2 GENE OF TOXOPLASMA GONDII

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 amplify the gene encoding surface antigen 2 (SAG2) of Toxoplasma gondii by polymerase chain reaction (PCR), to construct eukaryotic expression vector containing SAG2 gene, and to express SAG2 protein in Vero cell. W Methods The SAG2 gene fragment (577 bp) was amplified by PCR. After digestion with KpnⅠ and EcoRⅠ, the gene fragment was ligated to the eukaryotic expression vector pVAX1. Positive recombinant plasmid were screened, sequenced. pVAX1 and the recombinant plasmid was introduced into mammalian cells, Vero, by lipsome mediated transfection. The expressed protein was tested by Western blot analysis. W Results and Conclusion The eukaryotic recombinant plasmid, pVAX1 SAG2, was obtained by identification with restrictive enzymes and PCR. Western blot analysis revealed a strip about 17 ku in the sample of pVAX1 SAG2/Vero cell lysis which could be recognized by rabbit serum against T. gondii , indicating that the protein of interest was expressed transiently in the cells.