弓形虫表面抗原SAG1 DNA疫苗的构建

目的　构建真核重组表达质粒 pVAX1-SAG1,并探讨其诱导的体液免疫应答。 方法　采用多聚酶链反应技术 ,从弓形虫RH株基因组DNA中扩增截短型的SAG1基因片断 ,并克隆至载体pMD18-T ,经菌落PCR鉴定和测序分析后 ,亚克隆至真核表达载体 pVAX1,构建重组真核表达质粒pVAX1-SAG1,并予菌落PCR和双酶切鉴定。以此为疫苗候选分子免疫小鼠 ,检测其诱导产生的抗体。结果　PCR扩增出SAG1基因的截短型片段 ,其大小约 780bp ;测序的阳性TA克隆除两处发生同义突变外 ,其余序列与原序列一致 ;TA克隆的插入片段被亚克隆到真核表达载体 pVAX1,构建了重组表达质粒pVAX1-SAG1;该质粒诱导小鼠产生了抗弓形虫抗原的抗体。 结论　成功构建了弓形虫表面抗原SAG1的DNA疫苗。

Construction of DNA vaccine with Toxoplasma gondii surface antigen SAG1

Objective To construct DNA vaccine with Toxoplasma gondii major surface antigen SAG1,and analyze the humoral immune response induced by pVAX1-SAG1in mice.Methods The sequence encoding truncated SAG1 was amplified by PCR,and was cloned into pMD18-T vector,positive TA clones were identified by colony PCR and sequencing with DNA Sequencer;the right gene fragment in positive TA clone was subcloned into pVAX1 to construct DNA vaccine of SAG1.The positive recombinant clone was also identified by colony PCR and digestion with restriction endonuclease.Mice were immunized with the DNA vaccine and its antibody was detected by ELISA.Results The gene fragment encoding truncated SAG1 was specifically amplified by PCR from Toxoplasma gondii genomic DNA,though there were two synonymous mutations in the insert of SAG1 in TA clone,others were coincident with the original sequence of SAG1 gene from GenBank;the recombinant plasmid pVAX1-SAG1 was constructed through subcloning the right insert of SAG1 gene into pVAX1 properly;mice immunized with pVAX1-SAG1 raised antibody to Toxoplasma antigen with a titer of 1∶100.Conclusion DNA vaccine encoding SAG1 has been successfully constructed.