[Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2].

: [摘要] 目的 构建pEGFP-N1-HBsAg-ROP2重组质粒, 并转染HEK293T细胞进行表达鉴定, 为弓形虫病核酸疫苗的研 制奠定基础。方法 根据HBsAg基因序列和pcDNA3-p30-ROP2重组质粒酶切位点设计引物, PCR扩增HBsAg基因, 经 酶切、连接、转化, 利用HBsAg基因替换p30基因, 构建pcDNA3-HBsAg-ROP2重组质粒; 经Hind Ⅲ和Kpn Ⅰ双酶切, 将 HBsAg-ROP2片段与pEGFP-N1真核表达载体相连, 构建pEGFP-N1-HBsAg-ROP2重组表达质粒, 转染HEK293T细胞, 观 察其蛋白表达水平。结果 HBsAg片段PCR产物约700 bp, 与理论值相符; 构建pcDNA3-HBsAg-ROP2重组质粒, 双酶切 电泳后得到约5.4 kb和1.9 kb的两条带, 与预期结果相符。pEGFP-N1-HBsAg-ROP2双酶切后产生约4.7 kb和1.9 kb的条 带, 经测序鉴定, 与GenBank发表的序列同源性为99.84%。目的基因已成功转染入HEK293T细胞中, 且正确表达, 蛋白 浓度为3.08 mg/ml。结论 成功构建pEGFP- N1-HBsAg-ROP2重组表达质粒, 转染HEK293T细胞能正确表达。. METHODS: According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites, the HBsAg gene was amplified by PCR. The HBsAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene. The HBsAg-ROP2 fragment was amplified by PCR and digested with Hind Ⅲ and Kpn Ⅰ to clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2. The expression vector was transfected into HEK293T cells based on the identification of PCR amplification, restriction endonucleases and sequencing. RESULTS: The PCR product of HBsAg was about 700 bp, which was consistent with the theoretical value. Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3HBsAg-ROP2 recombinant plasmid. The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment. The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank. The target plasmid was successfully transfected into HEK293T cells, and the expression was correct, the protein concentration was 3.08 mg/ml. CONCLUSIONS: The recombinant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.