Migfilin基因的克隆和真核表达载体的构建

目的 克隆Migfilin全长cDNA,构建Flag-migfilin真核表达载体并进行序列测定,为研究migfilin与疾病的关系奠定基础.方法 根据人全长migfilin cDNA序列,设计PCR引物,以人前列腺癌cDNA文库为模板,利用PCR技术克隆migfilin全长cDNA.将扩增出的目的基因克隆至pCR 2.1载体,经Xba I/Hind Ⅲ双酶切后进行重组质粒鉴定,DNA序列测定.将测序正确的目的基因克隆至Flag载体并鉴定.结果 成功克隆了migfi-lin全长cDNA,序列测定表明与已知migfilin基因序列一致.在此基础上,构建了flag-migfilin真核表达载体并进行了鉴定.结论 Flag-migfilin真核表达载体构建成功,为深入探讨migfilin在大肠癌中的作用奠定了基础.

Cloning of human Migfilin full length cDNA and construction of mammalian expression vector

Objective To clone migfilin full length cDNA sequence, constructe mammalian expression vector Flag-migfilin and analyze the sequence of migfilin. Methods Based on human migfilin cDNA sequence, we designed a pair of primers to amplify the full-length migfilin by PCR. The PCR product was subcloned into the cloning vector pCR2.1, and the right recombinant plasmids were identified by enzymatic cleavage followed by DNA sequence analysis. The right DNA sequence was subsequently subcloned into Flag tagged mammalian expression vector.Results We cloned human migfilin cDNA and analyzed its sequence, and then, constructed and identified mammalian expression vector Flag-migfilin successfully. Conclusion Mammalian expression vector Flag-migfilin was constructed successfully, setting up the foundation to investigate the role of migfilin in colon cancer.