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

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

【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1.35kbpsbA和1.5kbtrnK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。

[ Objective] In order to produce edible plant vaccines in forage legume, a site-specific integration vector harboring the E2 gene which codes for main antigen of Hog Cholera Virus was constructed to transfer chloroplast genome of Lotus corniculatus. [ Method ] Homologous recombination sequences was selected by analyzing the chloroplast genome sequence of Lotus corniculatus with molecular biology software BLAST （Basic Local Alignment Search Tool） and DNAMAN. Plastid transformation vector was constructed and identified via PCR and molecular cloning. [Result] An optimal site was selected to integrate exogenous genes into chloroplast genome of Lotus corniculatus. Primers were designed to amplify chloroplast DNA fragments. A plastid transformation vector of Lotus corniculatus was constructed based on cloned fragments. The constructed vector, pAKE2, contains two neighboring DNA fragments of 1.35 kb and 1.5 kb as targeting sequences in the region ofpsbA and trnK genes respectively for chloroplast transformation of Lotus corniculatus. The E2 gene cassette, which was driven by strong chloroplast promoter Prrn and terminated by psbA gene terminator in tobacco, was inserted between the two neighboring DNA fragments, pAKE2 carries the aadA cassette that was used for spectinomycin resistance selection of transformants. [Conclusion] PCR amplification results 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.