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[目的]构建果实特异性启动子驱动的含伯氏疟原虫裂殖子表面蛋白PbMSP4/5(Plasmodium berghei mero-zoite surface protein4/5)基因的植物表达载体,进一步提高目的基因的表达量,为研制有效的转基因植物疟疾疫苗打下基础。[方法]分别以提取的番茄和伯氏疟原虫的基因组DNA为模板,通过SOEPCR,即重叠区扩增基因拼接法(gene splicing by overlap extension)拼接番茄果实特异表达启动子E8的核心序列(约1.11Kb)及PbMSP4/5基因(704bp),拼接后的序列为EM。用EcoRI和HindIII分别双酶切EM序列及表达载体pCAMBIA1302,连接转化,得到的重组质粒pCAMBIA1302-EM用电击法转化根癌农杆菌GV3103。[结果]重组质粒经PCR及酶切鉴定证明已成功转化。[结论]本实验成功构建了番茄果实特异性启动子驱动PbMSP4/5基因的植物表达载体。
[Objective] The aim of this study was to construct a plant-specific expression vector containing the Plasmodium berghei mero-zoite surface protein 4/5 gene driven by fruit-specific promoter and to further improve the expression of the target gene The development of effective transgenic malaria vaccine lay the foundation. [Method] The genomic DNAs of tomato and Plasmodium berghei were respectively used as templates to splicing the core sequence of tomato fruit-specific promoter E8 by SOEPCR (gene splicing by overlap extension) 1.11Kb) and PbMSP4 / 5 gene (704bp). The spliced sequence was EM. The EcoRI and HindIII double restriction enzyme digestion of the EM sequence and the expression vector pCAMBIA1302, connected and transformed, the resulting recombinant plasmid pCAMBIA1302-EM was electroporated into Agrobacterium tumefaciens GV3103. [Result] The recombinant plasmids were successfully transformed by PCR and restriction enzyme digestion. [Conclusion] The present study successfully constructed a plant expression vector for tomato fruit-specific promoter-driven PbMSP4 / 5 gene.