人肺新型抗蛋白酶性保护因子elafin cDNA的克隆、鉴定及真核表达载体的构建

The cloning and identification of human new type antiprotease protector elafin gene cDNA and construction of pEGFP-N1-elafin eukaryotic expression vector

目的克隆人肺新型抗蛋白酶性保护因子elafin基因cDNA,构建该基因的真核高效表达载体。方法抽提人肺总RNA进行RT-PCR法扩增,产物由pUCm-T载体装载、DNA测序确定后,用双酶切将Elafin cDNA定向克隆到绿色荧光蛋白表达载体pEGFP-N1,转化于大肠杆菌DH5α,筛选阳性克隆,双酶切鉴定重组质粒。结果经RT-PCR获得400b的产物,经DNA测序,确定所扩增片段为人elafin基因cDNA,进而成功筛选出真核表达载体pEGFP-N1-elafin。结论成功构建人elafin基因cDNA真核表达载体,为进一步深入研究慢性阻塞性肺疾病的发病机制奠定了技术基础。

Objective To clone human new type antiprotease protector （elafin）gene cDNA, and construct its highly efficient eukaryotic expression vector. Methods Total RNA extracted from human lung tissue was amplified with reverse transcription-polymerase chain reaction （RT-PCR）, its production was cloned into pUCm-T to definite DNA sequencing. Elafin cDNA was oriented to subelone into green fluorescent protein expression vector pEGFP-N1 transfected E. Coli DH5α to screen the positive clone and to identify the recombinant plasmid by double enzyme digestion. Results A fragment about 400 bp was obtained through RT-PCR and proved as human elafin gene cDNA according to its DNA sequencing, and further filtered the expression vector pEGFP-N1-elafin. Conclusions Elafin gene cDNA eukaryotic expression vector could be successfully constructed, which finds a technological base to the further fundamental study on the pathogeny of chronic obstructive pulmonary diseases.