MCP-1真核表达载体的构建和鉴定  

Construction and identification of eukaryotic expression plasmid MCP-1

作　　者：齐向明[1] 张培[1] 吴永贵[1] 罗庆礼[2] 

机构地区：[1]安徽医科大学第一附属医院肾内科,合肥230022 [2]人畜共患病安徽省重点实验室重要遗传病基因资源利用教育部省部共建重点实验室,合肥230022

出　　处：《安徽医学》2010年第3期205-207,共3页Anhui Medical Journal

基　　金：安徽省自然科学基金项目(编号:070413100)

摘　　要：目的构建大鼠MCP-1真核表达载体pcDNA3.1(+)-MCP-1。方法据Genebank中大鼠MCP-1cDNA序列设计并合成特异性引物,提取糖尿病模型大鼠肾脏总RNA,利用PCR技术扩增出MCP-1全编码区基因片段,并将扩增产物TA克隆至pMD-18T载体,然后分别双酶切pMD-18T-MCP-1回收目的片段再克隆到真核表达载体pcDNA3.1(+)中。结果PCR扩增得到大鼠成熟MCP-1的cDNA片段大小为500 bp,重组子利用限制性内切酶进行酶切、PCR鉴定和DNA序列分析得到真核表达载体pCDA3.1(+)-MCP-1。结论成功构建了大鼠MCP-1真核表达载体,为进一步研究MCP-1的DNA疫苗奠定基础提供了条件。Objective To construct and identify the eukaryotic expression plasmid for rat pcDNA3.1（＋）-MCP-1.Methods Accord to the published MCP-1 Groa cDNA sequence in Genebank,a pair of primers were respectively designed and synthesized.The total RNA was isolated from rats with diabetic nephropathy.After amplification with reverse transcription polymerase chain reaction（RT-PCR）,the product was cloned into pMD-18T vector using TA cloning.The target sequences was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1（＋）.The recombinants were finally sequenced and identified by restrictive endonuclease digestion.Results pcDNA3.1（＋）-MCP-1 eukaryotic expression vectors was successfully constructed,and it was identified by PCR,double restrictive endonuclease digestion and sequence analysis.The target fragment MCP-1 was the same as M57441 in Genbank.Conclusion The MCP-1 eukaryotic expression vectors was successfully constructed and identified.

关 键 词：DNA疫苗 单核细胞趋化蛋白-1 构建 鉴定 

分 类 号：R587.2[医药卫生—内分泌] R346[医药卫生—内科学]