酵母双杂交筛选与小鼠维生素D受体互作的蛋白

Screening for VDR Interacting Proteins by Yeast Two-hybrid System

通过酵母双杂交实验技术筛选小鼠VDR相互作用的蛋白质,评价不同蛋白与VDR的结合活性,以期获得基于互作影响雄性生殖能力基因。扩增小鼠VDR完整CDS序列,克隆至诱铒表达载体pGBKT7,经菌落PCR、DNA测序鉴定,获得重组载体pGBKT7-VDR。随后将重组载体转化至酿酒酵母AH109,并进行毒性和自激活检测;然后与含小鼠cDNA文库的Y187酵母杂交,获得阳性克隆,经HindⅢ酶切,测序分析VDR互作候选蛋白的核酸序列,再检测β-半乳糖苷酶活性,评价候选蛋白与VDR作用力的强弱。结果表明,从小鼠cDNA文库中获得12个与VDR互作的候选蛋白。初步检测到12个蛋白可能与VDR互作,其中3个蛋白因子可能与VDR结合并调控蛋白激酶磷酸化或去磷酸化以影响精子形成、发育和精子活力。

Mouse VDR-interacting proteins were screened by the yeast two-hybrid assay technique,and the binding activity between VDR and screened proteins was evaluated to obtain candidate genes involved in male reproductive ability via protein interactions.Firstly,the complete CDS of VDR of mouse was amplified by PCR.Then,using the gene recombination technology,VDR gene was inserted into the bait expression vector.It proved that the recombinant plasmid was constructed successfully by colony PCR and DNA sequencing.Then pGBKT7-VDR was transformed into yeast strain AH109,and it was verified that the bait protein was non-toxic and had a self-activation phenomenon.Protein optimization cut the self-activation domain of VDR.Finally,the positive clones were screened from the Y187 yeast two-hybrid prey protein cDNA library of the mouse.The nucleic acid sequence of the VDR interaction protein was analyzed by Hin dⅢdigestion and sequencing.The strength of the protein interacting with the VDR was determined by the ONPG assay.Twelve candidate proteins interacting with VDR were obtained from mouse cDNA library.Three proteins of twelve candidate proteins interacting with VDR,may interact with VDR to regulate protein kinase phosphorylation or dephosphorylation,which affected sperm formation,development and sperm motility.