弓形虫SAG1-MIC3融合基因真核表达载体的构建与鉴定

Construction and identification of recombinant plasmid containing SAG1 and MIC3 from Toxoplasma gondii

目的构建编码弓形虫RH株膜表面蛋白1(SAG1)和微线体蛋白3(MIC3)的重组真核表达载体pcDNA3.1-SAG1-MIC3并鉴定,为弓形虫疫苗研制作准备。方法采用PCR技术从弓形虫基因组DNA中分别扩增SAG1和MIC3基因片段,分别克隆入pMD18-T载体,并对重组入外源基因的质粒通过PCR、酶切和测序鉴定;采用亚克隆技术将SAG1和MIC3基因克隆至真核表达载体pcDNA3.1(-),经含氨苄青霉素的LB平板筛选阳性重组质粒pcDNA3.1-SAG1-MIC3,PCR、酶切和测序鉴定;采用脂质体法将重组载体转染Hela细胞,经RT-PCR法检测转染细胞转录情况。结果MIC3和SAG1基因的TA-cloning经PCR和酶切鉴定,大小分别为933bp和789bp,与预期值一致;pcNDA3.1-SAG1-MIC3经酶切鉴定,目的片段约为1722bp,与SAG1-MIC3长度相当;测定重组载体的核苷酸序列,与GenBank中的相应序列100%同源;PCR验证载体携带的SAG1-MIC3融合基因在Hela细胞中转录生成mRNA。结论成功构建重组真核表达载体pcDNA3.1-SAG1-MIC3,为弓形虫核酸疫苗的研制奠定了基础。

Objective To construct and identify an eukaryotic expression recombinant plasmid pcDNA3.1-SAG1-MIC3 for the further development of multiantigenic vaccine against toxoplasmosis. Methods Two gene fragments were amplified by PCR with primers that were designed according to the published gene sequence of SAG1 ; MIC3 from Toxoplasma gondii R H strain, identified and then subcloned into pMD18-T simple vector, then subcloned into pcDNA3.1 （- ） to gen- erate eukaryotic expression plasmid pcDNA3. 1-SAG1 MIC3. Ampicilin resistant transformants were selected and identified by PCR, enzyme digestion and DNA sequencing analysis. The recombinant plasmid was transfected into Hela cells by LipofectamineTM 2000. Then, detect the expression by RT-PCR. Results Enzyme digestion and PCR analysis of the gene SAG1 and MIC3 T A clone showed the length of fragment was about 933 bp and 789 bp, which fits the expected re sult. Enzyme digestion of pcNDA3. 1 SAC-1-MIC3 showed the length of fragment was 1 722 bp, corresponding to the length of SAG1 MIC3. The sequence analysis demonstrated that the sequence identities were 100% between recombinant SAG1-MIC3 gene and that from GenBank. RT-PCR analysis of cells transfected gene fragment display positive bands. Conclusion The recombinant eukaryotic expression plasmid of pcDNA3. 1-SAG1 MIC3 has been successfully obtained, building a foundation for the further studies on the vaccine development for T. gondii infection.