pcDNA3.1(+)-MCP-1和pcDNA3.1(+)-Gro α真核表达质粒的构建及鉴定

Construction and identification of eukaryotic expression plasmid pcDNA3.1(+)-MCP-1 and pcDNA3.1(+)-Gro α

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.

AIM: To construct and identify the eukaryotic expression plasmid for rat MCP-1 and Groα. METHODS: Acceding to the published MCP-1 Groa cDNA sequence in GeneBank, a pair of primers were respectively designed and synthesized. The total RNA was isolated froml rats with acute pancreatitis. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the product was cloned into pGEM-T easy vector using TA cloning followed by Bam H Ⅰ and Eco R Ⅰ digestion. The target sequences were then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1(+). The recombinants were finally sequenced and identified by restrictive endo nuclease digestion. RESULTS: pcDNA3.1(+)-MCP-1 and pcDNA3.1(+)-Groα eukaryotic expression vectors were successfully constructed, and they were identified by PCR, double restrictive endonu clease digestion and sequence analysis. The target fragment MCP-1 was the same as AF058786 in GenBank and the fragment Groa was different from NM_030845 (nt92-nt94) in GenBank. Repeated tests confirmed that NM_030845 (nt21-nt23) in GenBank was not correct. CONCLUSION: The MCP-1 and Groa eukaryotic expression vectors are successfully constructed and identified.