猪瘟病毒E2基因在百脉根叶绿体基因组中定点整合载体的构建

 【目的】构建猪瘟病毒主要抗原E2基因在百脉根叶绿体基因组中定点转化载体，为百脉根叶绿体的转化及用叶绿体生产动物可直接食用疫苗奠定基础。【方法】通过用BLAST、DNAMAN等分子生物学分析软件对百脉根叶绿体基因组序列进行分析选定同源重组片段，采用PCR、分子克隆技术进行载体构建和鉴定。【结果】在百脉根叶绿体基因组中选择合适的外源基因整合位点，设计引物用PCR扩增叶绿体同源片段，将同源片段克隆后，构建百脉根特异的叶绿体转化载体；构建的百脉根叶绿体定点转化载体pAKE2以扩增的1.35 kb psbA 和1.5 kb trnK两段相邻叶绿体 DNA为定点整合外源基因的同源重组片段，包含以叶绿体基因特异强启动子Prrn和终止子TpsbA为调控序列的E2基因表达盒和aadA壮观霉素抗性基因表达盒。【结论】经PCR和酶切验证，所构建的转化载体符合预期设计。叶绿体转化及后续工作目前正在进行之中。

Site-specific Integration Vector Construction of E2 Gene in Hog Cholera Virus for Chloroplast Transformation of Lotus corniculatus Genome

The objective of this research is to construct a site-specific integration vector harboring the E2 gene of Hog Cholera Virus, the gene coding for the main antigen, for transformation of chloroplast genome of Lotus corniculatus, which is the groundwork for producing edible plant vaccines in the chloroplast of this forage. The optimal site was selected for integration of exogenous genes into the chloroplast genome of Lotus corniculatus, and primers were designed and used to amplify chloroplast DNA fragments. After cloning of these fragments, the plastid transformation vector of Lotus corniculatus was constructed. The constructed vector, the pAKE2, contains two neighboring DNA fragments of 1.35 kb and 1.5 kb in the region of psbA and trnK genes, respectively, as targeting sequences for the chloroplast transformation of Lotus corniculatus, as well as the E2 gene cassette, which is inserted between the strong chloroplast promoter Prrn and the terminator of psbA gene form tobacco, and the aadA cassette, which is used for spectinomycine resistance selection of transformants. The results of PCR amplification and restriction enzyme digestion analysis showed that the constructed vector is in full accordance with what was desired. The transformation and following works are in progress.