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

Abstract

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.

CLC Number:

S827
S813.3

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

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