BMPR-IB基因的克隆及其在绒山羊成纤维细胞中的表达

Cloning and Expression of BMPR-IB Gene in Cashmere Goat Fibroblast Cells

本研究旨在克隆BB基因型小尾寒羊的BMPR-IB基因编码区,构建重组载体,瞬时转染绒山羊成纤维细胞,并对BMPR-IB等基因的表达情况进行检测。采用RT-PCR方法扩增BMPR-IB基因完整编码区,构建真核表达载体pEGFP-BMPR-IB,经脂质体Lipofectamine LTX&PLUS介导转染绒山羊成纤维细胞,并分别于转染后48和72h收集细胞,分别提取RNA和蛋白,利用RT-PCR和Western blot方法检测相关基因的表达情况。结果扩增得到了包含BMPR-IB基因完整编码区全长在内的1 550bp片段,与已知序列高度同源;Real-time PCR检测结果均表明,转基因组细胞中BMPR-IB表达量显著高于空白对照组(P<0.01),IGF-Ⅰ基因表达量也显著上调(P<0.01),TLR4、IFN、MHC、PNRP、GDF5、INH基因的表达量显著降低(P<0.01);Western blot检测表明,转染组BMPR-IB、IGF-I的表达有所增加,BMP4、TLR4的表达略有降低,但差异均不显著(P>0.05)。本研究成功实现了小尾寒羊BMPR-IB基因在山羊成纤维细胞中的表达,为转BMPR-IB基因阳性细胞株和细胞系的建立提供了基础;研究表明BMPR-IB(BB型)基因的过表达能上调IGF-Ⅰ基因的表达,下调TLR4基因的表达。

The research was conducted to clone BMPR-IB gene coding sequence of the Smalltailed Han sheep with BB genotype,construct the recombinant eukaryotic expression vectorwhich was transiently transfected into cashmere goat fibroblast cells,and detect the expression of BMPR-IBand other genes.The BMPR-IBgene was amplified by RT-PCR and the eukaryotic expression vector pEGFP-BMPR-IB was constructed by cloning BMPR-IB gene fragments into the pEGFP-N_2 vector frame,which were followed by the transfer of recombinant plasmids into goat fibroblast cells by liposome Lipofectamine LTXPLUS.After transfection for 48 and 72h,transfection cells were collected to extract RNA and total protein.Real-time quantitative PCR and Western blot approachs were used to identify the expression level of target genes and other related genes.The results showed that BMPR-IBgene which was amplified including CDS region was about 1 550 bp in length,and highly homologous with the published sequences.Real-time PCR detection results showed that the expression of BMPR-IBgene in transgenic cells were significantly higher than that in control group（P〈0.01）.The expression level of IGF-I gene in transfectedcells were significantly higher than that in control group（P〈0.01）,while the expression of TLR4,IFN,MHC,PNRP,GDF5 and INH genes were significantly lower（P〈0.01）.Western blot detection results showed that the expression of BMPR-IB and IGF-I in transgenic cells was higher than that in control group,although the expression of BMP4 and TLR4decreased,the difference did not reach significant level（P〉0.05）.The result showed that expression vector of BMPR-IB was constructed successfully and the expression of BMPR-IB gene in goat fibroblast cells was realized,which provide the basis for the preparation of positive cell strains,cell lines and transgenic animals.It is also concluded that the over-expression of BMPR-IB gene up-regulates the IGF-I expression and down-regulates the TLR4 expression in transfected cells.