弓形虫SAG1、SAG3基因的克隆和复合基因SAG1/SAG3真核表达重组质粒的构建

Toxoplasma gondii SAG1,SAG3 Cloning and composite SAG1/SAG3 gene expression of recombinant plasmid

目的构建弓形虫表面抗原SAG1、SAG3复合基因的真核表达重组质粒,为弓形虫疫苗的研制作准备。方法提取弓形虫基因组DNA;用PCR技术扩增出表面抗原SAG1、SAG3的基因,再分别重组入pGEM-T克隆载体;将pGEM-SAG1和pGEM-SAG3重组质粒分别经酶切、纯化后定向亚克隆入pcDNA3.1(+)真核表达载体,经PCR、酶切及测序等方法对重组子进行鉴定。结果从弓形虫基因组DNA中扩增出SAG1、SAG3基因;构建了pGEM-SAG1、pGEM-SAG3克隆质粒;成功构建pcDNA3.1(+)-SAG1-SAG3真核表达复合基因质粒,测序表明目的基因定向正确连接。结论构建了pcDNA3.1(+)-SAG1-SAG3复合基因表达质粒,为今后研制弓形虫复合多价疫苗提供候选抗原奠定了实验基础。

Objective To construct eukaryotic expression recombinant of surface antigen SAG1 and SAG3 of Toxoplasma,preparate for further toxoplasmosis vaccine study. Methods extract the Toxoplasma＇ s genome DNA; amplify the surface antigen SAG1 and SAG3 genes by PCR technology,link two genes to pGEM - T cloning vector respectively; after enzyme digestion and purification take two genes subelone into pcDNA3. 1 （ ＋ ） eukaryofic expression vector,then by PCR.enzyme digestion and sequencing methods identify the recombinant plasmid. Results amplified SAG1 and SAG3 genes from toxoplasmosis genome; successfully constructed pGEM- SAG1 and pGEM- SAG3 cloning plasmids; constructed pcDNA3. 1 -SAG1 -SAG3 complex gene expression vector, DNA sequencing showed that connect the right direction. Conclusion constructed pcDNA3. 1 -SAG1 -SAG3 compound gene expression vector, to provide the candidate antigen and lay the experiment foundation for the next development Toxoplasmosis compound multiple vaccine.