构建pcDNA3.1/Hygro(+)-LeftyA真核表达载体及Lefty A的稳定表达细胞系

Construction of eukaryotic expression vector pcDNA3.1/Hygro(+)-Lefty A and establishment of cell line stably expressing Lefty A

背景:Lefty基因可能通过拮抗转化生长因子β1信号通路发挥抗肾纤维化作用。目的:为验证其抗纤维化作用,构建pcDNA3.1/Hygro(+)-LeftyA真核表达载体,并建立LeftyA稳定表达细胞系。设计、时间及地点:基因克隆构建,单一样本观察,于2008-04/07在武汉大学人民医院中心实验室完成。材料:人肾小管上皮细胞株购于中国协和医科大学细胞库;真核表达载体pcDNA3.1/Hygro(+)由美国纽约州石溪大学SiamakTabibzadeh教授惠赠;LeftyA克隆载体pCMV-SPORT6购自武汉晶赛公司。方法:以pCMV-SPORT6为模板经PCR反应获得LeftyA编码区DNA,将其插入pcDNA3.1/Hygro(+)中构建LeftyA真核表达载体;通过LipofectamineTM2000将此重组质粒转染入人肾小管上皮细胞中,通过Hygromycin筛选挑取阳性表达克隆从而构建LeftyA稳定表达细胞系。主要观察指标:PCR扩增产物电泳鉴定结果。重组质粒的酶切鉴定结果。重组质粒基因测序结果。LeftyA稳定表达细胞系的筛选及其细胞内LeftyAmRNA表达。结果:pCMV-SPORT6经针对人LeftyA基因编码区序列的上下游引物PCR扩增后,10g/L琼脂糖凝胶电泳检测,在1.0kb条带处出现稍大于1.0kb的特异性扩增条带,与预期1.1kb目的片段大小相符。重组质粒分别被HindⅢ、BamHⅠ单酶切后所得片段大小一致,均为6.7kb,与重组质粒理论大小一致;经双酶切后得到大小分别为5.6kb和1.1kb的2条片段,与pcDNA3.1/Hygro(+)及LeftyA片段大小相符。将酶切鉴定阳性重组质粒测序,对测序结果进行生物信息学分析,发现与人LeftyA基因编码区序列完全一致。稳定转染LeftyA重组质粒的人肾小管上皮细胞高表达LeftyAmRNA。结论:pcDNA3.1/Hygro(+)-LeftyA真核表达载体构建成功,并建立了LeftyA稳定表达细胞系。

BACKGROUND： Lefty gene could attenuate renal fibrosis by inhibiting transforming growth factor-β1 （TGF-β1） signal transduction. OBJECTIVE： To construct eukaryotic expression vector pcDNA3.1/Hygro （＋）-Lefty A and to establish a cell line that can stably express Lefty A in order to verify the effects on inhibiting renal fibrosis. DESIGN, TIME AND SETTING： Gene cloning and single sample experiment was performed at Key Laboratory of Renmin Hospital of Wuhan University from April to July 2008. MATERIALS： Human renal tubule epithelial cells （HK-2 cell） were purchased from Peking Union Medical College, pcDNA3.1/Hygro（＋） was given by professor Siamak Tabibzadeh in Stony University as a gift. Clone vector of Lefty A （pCMV-SPORT6, GenBank Accession is BC035718） was purchased from Wuhan Genesil Biotechnology Co., Ltd. METHODS： The coding sequence of Lefty A was gained from pCMV-SPORT6 through PCR and then was used to construct eukaryotic expression vector in pcDNA3.1/Hygro（＋）. The recombinant pcDNA3.1/Hygro （＋）-Lefty A was transfected into human kidney tubular epithelial-2 cells （HK-2 cells） through LipofectamineTM 2000. After screening culture by Hygromycin, stable transfected cell line was established and the expression of Lefty A was identified by Hygromycin. MAIN OUTCOME MEASURES： The results identified by electrophoresis of PCR products, the results identified by recombinant enzyme incision, the results of recombinant gene sequence, the filtration of Lefty A stable expression cell line and the expression of mRNA Lefty A in cells. RESULTS： The coding sequence of Lefty A was gained from pCMV-SPORT6 by PCR using specific primers, and was confirmed by 10 g/L gel electrophoresis. There was a little bigger than 1.0 kb specific PCR band at the 1.0 kb of the bands. The results were consistent with 1.1 kb location. The recombinant plasmid was confirmed by Hind III, BamH I single enzyme digestion respectively, and each segment was 6.7kb. 5.6 kb and 1.1 kb segments were gained respectively after double digestion, and consistent with the segment of pcDNA3.1/Hygro（＋） and Lefty A. The positive recombinant plasmids were sequenced for bioinformatics analysis. The results were consistent with expectation. The high expression of Lefty A mRNA in transfected tubular epithelial cell line was found. CONCLUSION： The eukaryotic expression vectors of pcDNA3.1/Hygro（＋）-Lefty A were constructed successfully and the gene of Lefty A was stably expressed in stable transfected HK-2 cell line.