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目的构建pcDNA3-HBsAg-p30-ROP2多基因重组表达载体,并对其进行初步鉴定。方法根据重组体pcDNA3-p30-ROP2酶切位点和乙型肝炎表面抗原(HBsAg)基因序列等因素设计合成引物,扩增HBsAg目的基因片段,再应用酶切、连接等分子生物学技术将HBsAg目的基因克隆至pcDNA3-p30-ROP2表达载体中。应用聚合酶链反应(PCR)初筛,再采用酶切、测序等技术对构建的重组表达载体pcDNA3-HBsAg-p30-ROP2进行鉴定。结果 PCR扩增出HBsAg基因片段,构建了pcDNA3-HBsAg-p30-ROP2多基因真核表达载体。PCR与酶切结果显示,该基因片段大小均与理论值相符;测序结果显示该重组表达载体包含了p30-ROP2和HBsAg目的基因的完整序列。结论成功构建了多基因重组表达载体pcDNA3-HBsAg-p30-ROP2,为进一步研究多基因核酸疫苗奠定了基础。
Objective To construct pcDNA3-HBsAg-p30-ROP2 multi-gene recombinant expression vector and preliminary identification. Methods According to the recombinants pcDNA3-p30-ROP2 restriction sites and hepatitis B surface antigen (HBsAg) gene sequences and other factors designed primers to amplify HBsAg gene fragment, and then applied enzyme digestion, ligation and other molecular biology techniques to HBsAg The target gene was cloned into pcDNA3-p30-ROP2 expression vector. The recombinant plasmid pcDNA3-HBsAg-p30-ROP2 was identified by polymerase chain reaction (PCR) screening and enzyme digestion, sequencing and other techniques. Results HBsAg gene fragment was amplified by PCR and eukaryotic expression vector pcDNA3-HBsAg-p30-ROP2 was constructed. PCR and digestion results showed that the size of the gene fragment was consistent with the theoretical value; sequencing showed that the recombinant expression vector contains the complete sequence of the target gene p30-ROP2 and HBsAg. Conclusion The recombinant plasmid pcDNA3-HBsAg-p30-ROP2 was successfully constructed, which laid the foundation for further study of multi-gene nucleic acid vaccine.