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目的 构建 pcDNA3-HBsAg -GRA1真核表达质粒 ,为提高弓形虫DNA疫苗保护性做出新的探索 ,并期望“一 / +-苗两用”。方法 采用PCR分别扩增HBsAg、GRA1;经BclⅠ、EcoRⅠ双酶切HBsAg ;EcoRⅠ、XhoⅠ双酶切GRA1;BamHⅠ、XhoⅠ双酶切载体 pcDNA3 0 ;HBsAg、GRA1、pcDNA3 0双酶切纯化产物与T4DNA连接酶在 2 2℃连接过夜 ,用双酶切及双脱氧末端终止法测序鉴定重组质粒。结果 成功构建pcDNA3-HBsAg -GRA1真核表达质粒。结论 为进一步研究 pcD NA3-HBsAg -GRA1诱导的保护性免疫奠定了基础。
Objective To construct eukaryotic expression plasmid pcDNA3-HBsAg-GRA1 and to explore new ways to improve the DNA vaccine protection against Toxoplasma gondii. Methods HBsAg and GRA1 were amplified by PCR respectively. HBsAg was digested by BclⅠand EcoRⅠ, GRA1 was digested by EcoRⅠand XhoⅠ, and pcDNA3 0 was digested by BamHⅠand XhoⅠ. The purified product was digested by T4 DNA The ligase was ligated overnight at 22 ° C, and the recombinant plasmids were identified by double digestion and dideoxy terminator sequencing. Results The pcDNA3-HBsAg-GRA1 eukaryotic expression plasmid was successfully constructed. Conclusion This study laid the foundation for further study of protective immunity induced by pcD NA3-HBsAg-GRA1.