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目的:构建并鉴定真核表达质粒pcDNA3.1(+)-MCP-1和pcDNA3.1(+)-Groα. 方法:据GeneBank中大鼠MCP-1和GroαcDNA序列设计并合成引物,提取急性胰腺炎模型大鼠总RNA,RT-PCR 扩增,并将扩增产物TA克隆至pGEM-T easy载体,然后分别双酶切pGEM-MCP-1和pGEM-Groα回收目的片段再克隆至真核表达载体pcDNA3.1(+). 结果:pcDNA3.1(+)-MCP-1和pcDNA3.1(+)-Gro α真核表达质粒构建完成后,用限制性内切酶、PCR及DNA序列分析等多种方法进行鉴定,证实其构建成功. 结论:pcDNA3.1(+)-MCP-1和pcDNA3.1(+)-Groα真核表达质粒构建成功,为进一步研究该真核表达质粒的免疫保护效果及制备急性胰腺炎pcDNA3.1(+)-MCP-1和pcDNA3.1(+)-GroαDNA疫苗奠定了基础.
OBJECTIVE: To construct and identify eukaryotic expression plasmids pcDNA3.1 (+) - MCP-1 and pcDNA3.1 (+) - Groα.Methods: According to the sequences of rat MCP-1 and Groα cDNA in GeneBank, primers were designed and synthesized, The total RNA was amplified by RT-PCR from rat model of inflammation. The amplified product was cloned into pGEM-T easy vector and then double digested with pGEM-MCP-1 and pGEM-Groα respectively. After pcDNA3.1 (+) - MCP-1 and pcDNA3.1 (+) - Gro α eukaryotic expression plasmids were constructed, the restriction endonucleases, PCR and DNA sequence analysis The results showed that the eukaryotic expression plasmid pcDNA3.1 (+) - MCP-1 and pcDNA3.1 (+) - Groα were constructed successfully. To further study the immunity of the eukaryotic expression plasmid Protection and preparation of acute pancreatitis pcDNA3.1 (+) - MCP-1 and pcDNA3.1 (+) - Gro? DNA vaccine laid the foundation.