猪hnRNPK蛋白与酪氨酸蛋白激酶c-Src的互作分析  

Interaction Analysis between Porcine hnRNPK and Tyrosine Protein Kinase c-Src

作　　者：梁小娟[1,2] 徐海霞 李瑞[1] 张朋朋 徐永杰[1,3] 

机构地区：[1]信阳师范学院生命科学学院,信阳464000 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]信阳师范学院大别山农业生物资源保护与利用研究院,信阳464000

出　　处：《畜牧兽医学报》2018年第2期243-252,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：NSFC-河南人才培养联合基金(U1204326);河南省青年骨干教师资助计划(2015GGJS-139);信阳师范学院南湖学者计划(2016)

摘　　要：旨在探讨猪不均一核糖核蛋白K(hnRNPK)与含SH3结构域的非受体型酪氨酸激酶(c-Src)之间的互作关系。采用PCR和酶切连接的方法,构建pGBKT7-hnRNPK、pGADT7-c-Src、pGADT7-ΔSH3、pGADT7-ΔSH2、pGADT7-ΔPTKc和pGADT7-SH3酵母双杂交载体,用PEG-LiAc法转化到酵母菌株AH109中,鉴定其自激活活性,并通过酵母双杂交方法从体内验证猪hnRNPK蛋白与c-Src不同缺失体之间的相互作用;采用DNA重组技术构建pET28a-hnRNPK与pGEX-6p1-c-Src、pGEX-6p1-ΔSH3、pGEX-6p1-ΔSH2、pGEX-6p1-ΔPTKc和pGEX-6p1-SH3蛋白表达载体,转化E.coli BL21经IPTG诱导表达目的蛋白,利用亲和层析纯化法纯化融合蛋白,通过GST pull-down试验从体外验证猪hnRNPK蛋白与c-Src不同缺失体之间的相互作用。结果表明,成功构建了猪hnRNPK与c-Src不同缺失体的酵母双杂交重组质粒,转化酵母细胞发现各质粒均无自激活活性;在酵母中,与阴性对照相比,共转化pGBKT7-hnRNPK与pGADT7-c-Src或pGADT7-ΔPTKc或pGADT7-SH3的酵母菌落在SDTrp-Leu-His-Ade四缺培养基上生长良好,表明在酵母中hnRNPK与c-Src激酶N端的SH3结构域之间存在直接的相互作用;成功构建了猪hnRNPK与c-Src不同缺失体的融合表达质粒,经IPTG诱导表达及纯化获得了可溶性His-hnRNPK、GST-c-Src、GST-ΔSH3、GST-ΔSH2、GST-ΔPTKc和GST-SH3融合蛋白,GST pull-down试验表明,hnRNPK与c-Src N端存在较强的相互作用,与SH2和PTKc结构域相互作用则较弱。本研究揭示了猪hnRNPK蛋白可与c-Src蛋白的N-端的SH3结构域互作,有助于进一步研究它们的调节位点,为深入探讨hnRNPK与c-Src相互调控关系,进而阐明其生物学功能奠定了基础。This experiment was conducted to investigate the interaction between porcine heterogeneous nuclear ribonucleoprotein K（hnRNPK）protein and non-receptor tyrosine protein kinase（c-Src）containing SRC Homology（SH3）domain.The yeast two-hybrid expressing vectors pGBKT7-hnRNPK,pGADT7-c-Src,pGADT7-ΔSH3,pGADT7-ΔSH2,pGADT7-ΔPTKc and pGADT7-SH3 were constructed using PCR,enzyme digestion and ligation methods,and then transformed into the yeast strain AH109 by PEG-LiAc method,and self-activation activity of baitand prey proteins in yeast cells were tested.Furthermore,the interaction between porcine hnRNPK and different mutants of c-Src protein were analyzed using yeast two-hybrid assay in vivo.With the DNA recombinant technology,the plasmids pET28 a-hnRNPK,pGEX-6 p1-c-Src,pGEX-6 p1-ΔSH3,pGEX-6 p1-ΔSH2,pGEX-6 p1-ΔPTKc and pGEX-6 p1-SH3 were constructed.Then the plasmids were transformed into E.coli BL21 and induced to express the target proteins by IPTG.GST and His tagged fusion proteins were purified by affinity chromatography.The binding of porcine hnRNPK and different mutants of c-Src protein were verified via GST pulldown assay in vitro.The results showed that the yeast two-hybrid expressing vectors of porcine hnRNPKand different mutants of c-Src were successfully constructed.The bait and prey plasmids were transformed into yeast cells and proved to have no self-activation activity in yeast cells.In yeast,the yeast colonies co-transformed with pGBKT7-hnRNPK and pGADT7-c-Src or pGADT7-ΔPTKc or pGADT7-SH3 grew well in SD-Trp-Leu-His-Ade medium compared with negative controls,indicating that porcine hnRNPK directly interacted with SH3 domain at c-Src protein Nterminal in yeast.The expression vectors of porcine hnRNPK and different mutants of c-Src were successfully constructed,and the soluble His-hnRNPK,GST-c-Src,GST-ΔSH3,GST-ΔSH2,GST-ΔPTKc and GST-SH3 fusion proteins were obtained via induction expression by IPTG and purification.The further GST pull-down assay showed strong interaction between porcine

关 键 词：猪 hnRNPK C-SRC SH3结构域 酵母双杂交 GST-Pull DOWN 

分 类 号：S828.2[农业科学—畜牧学]