弓形虫真核表达质粒pcDNA3/GRA1的构建及序列测定

目的 :构建弓形虫致密颗粒抗原 1 (GRA1 )真核表达质粒 ,为进一步开展DNA疫苗的保护性研究打下基础。方法 :采用PCR扩增出编码GRA1目的基因 ,用EcoRⅠ /XhoⅠ分别对扩增产物和真核表达质粒pcDNA3进行双酶切 ,将GRA1定向克隆到pcDNA3EcoRⅠ /XhoⅠ位点 ;对重组质粒进行PCR ,双酶切初步鉴定后做序列测定。结果 :特异扩增出预计的GRA1片段 ,大小为 5 73bp ;扩增产物双酶切后成功连接到pcDNA3中 ,经PCR ,双酶切及序列测定结果表明重组质粒中含有GRA1读框。结论 :成功构建弓形虫GRA1真核表达质粒。

Construction and sequencing of recombinant plasmid pcDNA3/ GRA1 from Toxoplasma gondii

Objective To construct a mammalian expression plasmid pcDNA3/ GRA1 to express dense granules antigen 1 (GRA1)of Toxoplasma gondii ,and to lay a foundation for further studying the protective immunity of pcDNA3/GRA1 as a DNA vaccine. Methods The GRA1 opening reading frame (ORF) was amplified with two specific primers. The ORF and plasmid pcDNA3 were digested with Eco R I and Xho I respectively and the ORF was ligated into the pcDNA3 at polylinker. The recombinant vector pcDNA3/GRA1 was characterized by PCR, restriction enzyme digestion,and sequencing analysis.Results The expected ORF, 573 bp long, was amplified by PCR , and inserted into plasmid pcDNA3. PCR, restriction enzyme digestion and sequencing analysis showed that pcDNA3/GRA1 contained GRA1 ORF with the right orientation. Conclusion The mammalian expression vector pcDNA3/GRA1 is successfully constructed.