BMPR-IB基因的克隆及其在绒山羊成纤维细胞中的表达

doi:10.11843/j.issn.0366-6964.2016.06.006

摘要/Abstract

摘要：

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72 h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1 550 bp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。

关键词: BMPR-IB基因, 真核表达载体, 绒山羊成纤维细胞, 表达

Abstract:

The research was conducted to clone BMPR-IB gene coding sequence of the Small-tailed 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-IB and other genes.The BMPR-IB gene was amplified by RT-PCR and the eukaryotic expression vector pEGFP-BMPR-IB was constructed by cloning BMPR-IB gene fragments into the pEGFP-N2 vector frame，which were followed by the transfer of recombinant plasmids into goat fibroblast cells by liposome Lipofectamine LTX&PLUS.After transfection for 48 and 72 h，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-IB gene 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-IB gene in transgenic cells were significantly higher than that in control group (P<0.01).The expression level of IGF-I gene in transfected cells 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 TLR4 decreased，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.

Key words: BMPR-IB gene, eukaryotic expression vector, cashmere goat fibroblast cells, expression

中图分类号:

S827
S813.3

孙洪新，王红娜，张英杰，刘月琴，陈晓勇，敦伟涛. BMPR-IB基因的克隆及其在绒山羊成纤维细胞中的表达[J]. 畜牧兽医学报, 2016, 47(6): 1124-1132.

SUN Hong-xin，WANG Hong-na，ZHANG Ying-jie，LIU Yue-qin，CHEN Xiao-yong，DUN Wei-tao. Cloning and Expression of BMPR-IB Gene in Cashmere Goat Fibroblast Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1124-1132.

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