酵母双杂交筛选与小鼠维生素D受体互作的蛋白
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摘要
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,VDRgene 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 Hind Ⅲ digestion 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.
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,VDRgene 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 Hind Ⅲ digestion 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.
引文
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[23]ASSIMON V A,SOUTHWORTH D R,GESTWICKI JE.Specific binding of tetratricopeptide repeat(TPR)proteins to heat shock protein 70(Hsp70)and heat shock protein 90(Hsp90)is regulated by affinity and phosphorylation[J].Biochemistry,2015,54(48):7120-7131.
[24]BALSEVICH G,HUSL A S,MEYER C W,et al.Stressresponsive FKBP51regulates AKT2-AS160signaling and metabolic function[J].Nature Communications,2017,8(1):1725.
[25]LUO K,LI Y,YIN Y,et al.USP49negatively regulates tumorigenesis and chemoresistance through FKBP51-AKTsignaling[J].Embo Journal,2017,36(10):1434-1446.
[26]NELSON D R,KOYMANS L,KAMATAKI T,et al.P450superfamily:update on new sequences,gene mapping,accession numbers and nomenclature[J].Pharmacogenetics,1996,6(1):1-42.
[27]TIAN J H,XUE B,HU J H,et al.Exogenous substances regulate silkworm fat body protein synthesis through MAPK and PI3K/Akt signaling pathways[J].Chemosphere,2017,171(3):202-207.
[28]赵伊英,武万锋,汪小东,等.棉铃虫细胞色素P450家族CYP6AE14基因克隆、序列分析及蛋白原核表达[J].西北农业学报,2012,21(12):181-187.ZHAO Y Y,WU W F,WANG X D,et al.Cloning,sequence analysis and prokaryotic expression of cytochrome P450 CYP6AE14 gene from Helicover pa armigera[J].Acta Agriculturae Boreali-occidentalis Sinica,2012,21(12):181-187.
[29]张立群,巴拉,玉斯日古楞,等.细胞色素P450 2J亚家族研究进展[J].动物医学进展,2014,35(8):84-89.ZHANG L Q,BA LA,YUSIRIGULENG,et al.Advance in cytochrome P4502Jsubfamily[J].Progress in Veterinary Medicine,2014,35(8):84-89.
[30]MESSINA A,NENCIONI S,GERVASI PG,et al.Molecular cloning and enzymatic characterization of sheep CYP2J[J].Xenobiotic,2010,40(2):109-118.
[2]SAYEM A,GIRIBABU N,KARIM K,et al.Differential expression of the receptors for thyroid hormone,thyroidstimulating hormone,vitamin D and retinoic acid and extracellular signal-regulated kinase in uterus of rats under influence of sex-steroids[J].Biomedicine&Pharmacotherapy,2018,100:132-141.
[3]CHRISTAKOS S,DHAWAN P,VERSTUYF A,et al.Vitamin D:metabolism,molecular mechanism of action,and pleiotropic effects[J].Physiological Reviews,2016,96(1):365-408.
[4]HUANGY F,WUY H,CHENGW F,et al.Vitamin D-binding protein enhances epithelial ovarian cancer progression by regulating the insulin-like growthfactor-1/Akt pathway and vitamin D receptor transcription[J].Clinical Cancer Research,2018,24(13):3217-3228.
[5]HE L,LIU T,SHI Y,et al.Gut epithelial vitamin D receptor regulates microbiota-dependent mucosal inflammation by suppressing intestinal epithelial cell apoptosis[J].Endocrinology,2018,159(2):967-979.
[6]YUAN J,GUO X,LIU Z.et al.Vitamin D receptor activation influences the ERK pathway and protects against neurological deficits and neuronal death[J].International Journal of Molecular Sciences,2018,41(1):364-372.
[7]SHANG M,SUN J.Vitamin D/VDR,probiotics,and gastrointestinal diseases[J].Current Medicinal Chemistry,2017,24(9):876-887.
[8]李爱梅,张伟,张浩,等.维生素D/维生素D受体、自噬与炎症相关疾病[J].中南大学学报(医学版),2017(8):979-985.LI A M,ZHANG W,ZHANG H,et al.Vitamin D/vitamin D receptor,autophagy and inflammation relevant diseases[J].Journal of Central South University(Medical Sciences),2017,42(8):979-985.
[9]KWIECINSKI G,PETRIE GIand DELUCA H F.Vitamin D is necessary for reproductive functions of the male rat[J].Journal of Nutrition,1989,119(5):741-744.
[10]路宏朝.FGF13影响小鼠骨骼肌增殖与分化的机制研究[D].陕西杨凌:西北农林科技大学,2016.LU H ZH.FGF13regulates proliferation and differentiation of skeletal muscle in mice[D].Yangling Shaanxi:Northwest A&F University,2016.
[11]PIKE J,MEYER M,LEE S,et al.The vitamin D receptor:contemporary genomic approaches reveal new basic and translational insights[J].Journal of Clinical Investigation,2017,127(4):1146-1154.
[12]SAITO Y,NAKAGAWA T,KAKIHANA A,et al.Yeast two-hybrid and one-hybrid screenings identify regulators of hsp70gene expression[J].Journal of Cellular Biochemistry,2016,117(9):2109-2117.
[13]READ J,BRENNER S.Yeast two-hybrid system[J].Encyclopedia of Genetics,2001:2164-74.
[14]CALEJO A I,TASK N K.Targeting protein-protein interactions in complexes organized by Akinase anchoring proteins[J].Frontiers in Pharmacology,2015,6(192):DOI:10.3389/fphar.2015.00192.
[15]KRITZER MD,LI J,DODGEKAFKA K,et al.AKAPs:the architectural underpinnings of local cAMP signaling[J].Journal of Molecular&Cellular Cardiology,2012,52(2):351-358.
[16]VIZEL R,HILLMAN P,ICKOWICZ D,et al.AKAP3degradation in sperm capacitation is regulated by its tyrosine phosphorylation[J].Biochimica et Biophysica Acta,2015,1850(9):1912-1920.
[17]XU K,YANG L,ZHAO D,et al.AKAP3synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis[J].Biology of Reproduction,2014,90(6):119.
[18]JOHNSON L R,FOSTER J A,HAIGLADEWIG L,et al.Assembly of AKAP82,aprotein kinase A anchor protein,into the fibrous sheath of mouse sperm[J].Developmental Biology,1997,192(2):340-350.
[19]KERNS K,MORALES P,SUTOVSKY P.Regulation of sperm capacitation by the 26Sproteasome:an emerging new paradigm in spermatology1[J].Biology of Reproduction,2016,94(5):117.
[20]HONG J,KIM S T,TRANGUCH S,et al.Deficiency of co-chaperone immunophilin FKBP52compromises sperm fertilizing capacity[J].Reproduction,2007,133(2):395-403.
[21]张国铸,黄灿华,周鹏,等.FKBP6基因单核苷酸多态性与原发性无精症相关研究[J].现代生物医学进展,2015,15(9):1640-1642.ZHANG G ZH,HUANG C H,ZHOU P,et al.Investigation of single nucleotide polymorphisms(SNPs)in gene FKBP6 and its association with idiopathic azoospermia[J].Progress in Modern Biomedicine,2015,15(9):1640-1642.
[22]CRIADO-MARRERO M,REIN T,BINDER E B,et al.Hsp90and FKBP51:complex regulators of psychiatric diseases[J].Philosophical Transactions of the Royal Society of London,2018,373(1738):DOI:10.1098/RSTB.2016.0532.
[23]ASSIMON V A,SOUTHWORTH D R,GESTWICKI JE.Specific binding of tetratricopeptide repeat(TPR)proteins to heat shock protein 70(Hsp70)and heat shock protein 90(Hsp90)is regulated by affinity and phosphorylation[J].Biochemistry,2015,54(48):7120-7131.
[24]BALSEVICH G,HUSL A S,MEYER C W,et al.Stressresponsive FKBP51regulates AKT2-AS160signaling and metabolic function[J].Nature Communications,2017,8(1):1725.
[25]LUO K,LI Y,YIN Y,et al.USP49negatively regulates tumorigenesis and chemoresistance through FKBP51-AKTsignaling[J].Embo Journal,2017,36(10):1434-1446.
[26]NELSON D R,KOYMANS L,KAMATAKI T,et al.P450superfamily:update on new sequences,gene mapping,accession numbers and nomenclature[J].Pharmacogenetics,1996,6(1):1-42.
[27]TIAN J H,XUE B,HU J H,et al.Exogenous substances regulate silkworm fat body protein synthesis through MAPK and PI3K/Akt signaling pathways[J].Chemosphere,2017,171(3):202-207.
[28]赵伊英,武万锋,汪小东,等.棉铃虫细胞色素P450家族CYP6AE14基因克隆、序列分析及蛋白原核表达[J].西北农业学报,2012,21(12):181-187.ZHAO Y Y,WU W F,WANG X D,et al.Cloning,sequence analysis and prokaryotic expression of cytochrome P450 CYP6AE14 gene from Helicover pa armigera[J].Acta Agriculturae Boreali-occidentalis Sinica,2012,21(12):181-187.
[29]张立群,巴拉,玉斯日古楞,等.细胞色素P450 2J亚家族研究进展[J].动物医学进展,2014,35(8):84-89.ZHANG L Q,BA LA,YUSIRIGULENG,et al.Advance in cytochrome P4502Jsubfamily[J].Progress in Veterinary Medicine,2014,35(8):84-89.
[30]MESSINA A,NENCIONI S,GERVASI PG,et al.Molecular cloning and enzymatic characterization of sheep CYP2J[J].Xenobiotic,2010,40(2):109-118.