摘要
目的克隆大鼠转化生长因子β1（TGFβ1）基因并转染肝星状细胞（Hepatic stellate cells，HSC），筛选稳定高表达TGF β1的阳性克隆。方法采用逆转录-聚合酶链式应（RT-PCR）从大鼠成骨细胞中扩增出特异性片段，经酶切鉴定证实为大鼠TGFβ1 cDNA，将此片段插入pcDNA3．1（＋）表达载体，构建得到pcDNA3．1（＋）-TGFβ1重组质粒。应用阳离子脂质体介导的基因转移技术将TGFβ1表达载体导入大鼠HSC，经G418筛选高表达TGFβ1的阳性克隆，用荧光定量PCR法及Western blot法检测TGFβ1 mRNA及蛋白的表达。结果重组真核表达载体pcDNA3.1（＋）-TGFβ1经限制性内切酶BamHI酶切，电泳后显示的TGFβ1目的片段和5．4kb的pcDNA3．1（＋）载体片段，测序证实酶切片段与GenBank中登记的TGFβ1全长序列相同，证实pcDNA3.1（＋）-TGFβ1真核表达载体构建成功，转染HSC并经G418筛选出阳性克隆后，TGFβ1 mRNA及蛋白的表达明显增高（P〈0．05）。结论重组TGFβ1真核表达载体构建正确，并可成功转染HSC，经G418筛选可获得稳定表达TGFβ1的HSC阳性克隆。
Objective To clone the gene of transforming growth factor- β1 （ TGF- β1 ） and transfect it into hepatic stellate cells （ HSC ） to screen positive clones that stably express TGF- [3 l- Methods The specific fragment was amplified from rat osteoblasts by reverse transcription polymerase chain reaction （RT-PCR） , confirmed by enzyme digestion of rat TFG- β1 cDNA, and inserted into the pcDNA3.1 （ ＋ ） expression vector, the pcDNA3.1 （ ＋ ） -TGF [3 l recombinant plasmid was constructed. The TFG β1 expression vector was introduced into HSC by cationic liposome- mediated gene transfer technique. Positive clones with high expression of TGF β1 were screened by G418.The expression of TGB β1 mRNA and protein was detected by fluorescence quantitative PCR and Western blot. Results The recombinant eukaryotic expression vector pcDNA3.1 （ ＋ ） -TGF β1 was digested with restriction endonuclease BamHI. The target fragment of TGF β1 and the pcDNA3.1 （ ＋ ） vector fragment of 5.4kb were shown by electrophoresis. Compared with the full-length TGF β1 sequence registered in Genbank, the eukaryotic expression vector of pcDNA3.1 （ ＋ ） - TGF β1 was successfitlly constructed. After transfection of HSC and positive clones screened by G418, the expression of TGF β1 mRNA and protein was significantly increased （ P〈0.05 ） . Conclusions The recombinant TGF β1 eukaryotic expression vector is constructed correctly and transfected into HSC successfully. HSC positive clones stably expression TGF β1 can be obtained through G418 screening.
出处
《浙江临床医学》
2018年第7期1177-1179,共3页
Zhejiang Clinical Medical Journal
关键词
转化生长因子Β1
分子克隆
肝星状细胞
Transforming growth factor beta 1 Molecular cloning Hepatic stellate cells
