猪hnRNPK蛋白与酪氨酸蛋白激酶c-Src的互作分析

doi:10.11843/j.issn.0366-6964.2018.02.003

摘要/Abstract

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SD-Trp-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。

Abstract:

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 bait and 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 pET28a-hnRNPK, pGEX-6p1-c-Src, pGEX-6p1-ΔSH3, pGEX-6p1-ΔSH2, pGEX-6p1-ΔPTKc and pGEX-6p1-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 pull-down assay in vitro. The results showed that the yeast two-hybrid expressing vectors of porcine hnRNPK and 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 N-terminal 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 hnRNPK protein and N-terminal of c-Src protein, but weak interaction between hnRNPK protein and SH2, PTKc domains of c-Src protein. The results indicate that porcine hnRNPK protein can interact with the N-terminal domain (SH3 domain) of c-Src protein, which is convenient for studying the regulation sites of porcine c-Src protein, and find a technical platform for researching the mechanism of hnRNPK protein regulated kinase activity of c-Src protein.

中图分类号:

S828.2

梁小娟, 徐海霞, 李瑞, 张朋朋, 徐永杰. 猪hnRNPK蛋白与酪氨酸蛋白激酶c-Src的互作分析[J]. 畜牧兽医学报, 2018, 49(2): 243-252.

LIANG Xiao-juan, XU Hai-xia, LI Rui, ZHANG Peng-peng, XU Yong-jie. Interaction Analysis between Porcine hnRNPK and Tyrosine Protein Kinase c-Src[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 243-252.

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