重组UK114融合蛋白的表达纯化及免疫组化定位
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摘要
[目的]UK114(山羊肝脏肿瘤抗原)是钙蛋白酶系统的主要成分钙蛋白酶ⅠCAPN1/S1(μ-calpain)的激活蛋白,本研究旨在利用原核表达系统体外制备重组UK114蛋白,获得融合蛋白制备抗体,对其进行组织定位,为深入研究其功能提供蛋白水平的证据。[方法]将重组质粒pGEX-4T-3-UK114转化入大肠杆菌BL21(DE3)中诱导表达、纯化,用凝血酶对GST-UK114融合蛋白进行酶切,制备抗UK114多克隆抗体,免疫组化分析重组UK114蛋白在组织中的分布情况。[结果]GST-UK114融合蛋白的分子量为40kD,酶切得到目标蛋白,其分子量为14kD;用UK114蛋白免疫家兔,得到了理想的高效价兔抗UK114多克隆抗血清,效价大于1∶125 000,抗血清经纯化得到兔抗UK114多克隆抗体,效价大于1∶25 000;Western blot分析结果显示,GST-UK114融合蛋白在约40kD处有一明显条带,融合蛋白酶切后在14kD附近出现一条带;免疫组织化学分析表明,免疫组化染色主要发生在细胞质中,山羊肝脏组织中肝小叶边缘区有肝细胞出现棕色的阳性着色,部分肝窦内皮细胞也有棕色的阳性着色。肾脏皮质中的肾小管上皮细胞有棕色的阳性着色出现,肾小球中没有出现着色。[结论]试验成功获得纯化后的重组UK114蛋白,制备的抗UK114抗体可以与正常山羊肝脏和肾脏组织中的UK114蛋白结合,为其后续分子伴侣特性以及抗肿瘤特性的研究提供一定的蛋白试验依据。
[Objective]UK114(Goat liver tumor antigen)is the activator of CAPN1/S1(μ-calpain),the main component of calpain enzyme system.The aim of this study was to prepare recombinant UK114 protein using in vitro prokaryotic expression system.The fusion protein was obtained and used for antibody preparation,and its tissue localization was determined to provide evidence at protein level for the further functional study.[Methods]Recombinant plasmid pGEX-4 T-3-UK114 was transformed into Escherichia coli BL21(DE3)to induce gene expression.Induced genes were purified,and anti-UK114 polyclonal antibody was prepared by digesting GST-UK114 fusion protein with thrombin.The distribution of recombinant UK114 protein in tissues was analyzed with immunohistochemistry.[Results]The MW of GST-UK114 fusion protein was determined at about 40 kD,and that of the target protein was about 14 kD through Thromibin's digestion.The UK114 protein was used to immunize the rabbits,and the ideal high titer of rabbit anti-UK114 polyclonal antiserum was obtained,which was higher than 1:125000.After purification,the rabbit anti-UK114 polyclonal antibody was obtained,and the titer was greater than 1:25000.Western blot analysis showed a clear band of GST-UK114 fusion protein at about 40 kD,and another band was found near14 kD after the fusion protease was cut.Immunohistochemical analysis indicated that immunohistochemical staining was mainly occurred in the cytoplasm of goat liver.Brown positive staining was observed on both hepatocytes in the marginal region of hepatic lobule and some sinusoidal endothelial cells in goat liver tissue.It also was detected on the tubule epithelial cells in the renal cortex,but not in the glomeruli.[Conclusion]The purified recombinant UK114 protein was successfully obtained,and the anti UK114 antibody could bind to UK114 protein in normal goat liver and kidney.Present research provides a basis for the further study of molecular chaperone and anti-tumor characteristics.
[Objective]UK114(Goat liver tumor antigen)is the activator of CAPN1/S1(μ-calpain),the main component of calpain enzyme system.The aim of this study was to prepare recombinant UK114 protein using in vitro prokaryotic expression system.The fusion protein was obtained and used for antibody preparation,and its tissue localization was determined to provide evidence at protein level for the further functional study.[Methods]Recombinant plasmid pGEX-4 T-3-UK114 was transformed into Escherichia coli BL21(DE3)to induce gene expression.Induced genes were purified,and anti-UK114 polyclonal antibody was prepared by digesting GST-UK114 fusion protein with thrombin.The distribution of recombinant UK114 protein in tissues was analyzed with immunohistochemistry.[Results]The MW of GST-UK114 fusion protein was determined at about 40 kD,and that of the target protein was about 14 kD through Thromibin's digestion.The UK114 protein was used to immunize the rabbits,and the ideal high titer of rabbit anti-UK114 polyclonal antiserum was obtained,which was higher than 1:125000.After purification,the rabbit anti-UK114 polyclonal antibody was obtained,and the titer was greater than 1:25000.Western blot analysis showed a clear band of GST-UK114 fusion protein at about 40 kD,and another band was found near14 kD after the fusion protease was cut.Immunohistochemical analysis indicated that immunohistochemical staining was mainly occurred in the cytoplasm of goat liver.Brown positive staining was observed on both hepatocytes in the marginal region of hepatic lobule and some sinusoidal endothelial cells in goat liver tissue.It also was detected on the tubule epithelial cells in the renal cortex,but not in the glomeruli.[Conclusion]The purified recombinant UK114 protein was successfully obtained,and the anti UK114 antibody could bind to UK114 protein in normal goat liver and kidney.Present research provides a basis for the further study of molecular chaperone and anti-tumor characteristics.
引文
[1]常泓,尹淑琴.钙蛋白酶系统[M].北京:中国农业科学技术出版社,2016:82-83.
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[7]Kiruthika J,Rajesh S,Ponniah A G,et al.Molecular cloning and characterization of acyl-CoA binding protein(ACBP)gene from shrimp Penaeus monodon exposed to salinity stress[J].Developmental and Comparative immunology.2013,40(1):78-82.
[8]Moretti D,Bello B D,Allavena G,et al.Calpains and cancer:Friends or enemies?[J].Archives of Biochemistry and Biophysics.2014,564:26-36.
[9]Siklos M,Benaissa M,Thatcher G R J.Cysteine proteases as therapeutic targets:does selectivity matter?A systematic review of calpain and cathepsin inhibitors[J].Acta Pharmaceutica Sinica B,.2015,5(6):506-519.
[10]Jacob U,Gaestel M,Engel K,et al.Small heat shock proteins are molecular chaperones[J].Journal of Biological Chemistry.1993,268(3):1517-1520.
[11]He Y,Huang H,Li L,et al.Changes of activated factors and activation of calpain in tilapia muscle during storage[J].Fisheries Science,2018,84(5):889-895.
[12]Carragher N O.Calpain inhibition:a therapeutic strategy targeting multiple disease states[J].Current Pharmaceutical Design,2006,12(5):615-638.
[13]Ray S K.Currently evaluated calpain and caspase inhibitors for neuroprotection in experimental brain ischemia[J].Current Medicinal Chemistry,2006,13(28):3425-3440.
[14]Liu X,Zeng J,Chen X,et al.Crystal structures of RidA,an important enzyme for the prevention of toxic side products[J].Scientific Reports,2016,6:30494.
[15]郑晖,蒋海鹰,颜亚晖.浅谈常规病理制片及免疫组化染色的质控问题[J].诊断病理学杂志,2003,10(2):119-120.
[16]龙小霞.酶消化及微波抗原修复在免疫组化中的应用[J].诊断病理学杂志,1995,2(2):115-116.
[2]Ono Y.,Sorimachi H.Calpains:an elaborate proteolytic system.Biochimicaand biophysica acta.2012,1824(1),224-236.
[3]Macqueen D J,Wilcox A H.Characterization of the definitive classical calpain family of vertebrates using phylogenetic,evolutionary and expression analyses[J].Open Biology,2014,4(4):130219.
[4]Kemp C M,Sensky P L,Bardsley R G,et al.Tenderness-An enzymatic view[J].Meat Science,2010,84(2):248-256.
[5]Huang F,Huang M,Zhang H,et al.Cleavage of the calpain inhibitor,calpastatin,during postmorterm ageing of beef skeletal muscle[J].Food Chemistry.2014,148:1-6.
[6]Niehaus T D,Gerdes S,Hodge H K,et al.Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114(Rid)protein family[J].BMC Genomics,2015,16(1):382.
[7]Kiruthika J,Rajesh S,Ponniah A G,et al.Molecular cloning and characterization of acyl-CoA binding protein(ACBP)gene from shrimp Penaeus monodon exposed to salinity stress[J].Developmental and Comparative immunology.2013,40(1):78-82.
[8]Moretti D,Bello B D,Allavena G,et al.Calpains and cancer:Friends or enemies?[J].Archives of Biochemistry and Biophysics.2014,564:26-36.
[9]Siklos M,Benaissa M,Thatcher G R J.Cysteine proteases as therapeutic targets:does selectivity matter?A systematic review of calpain and cathepsin inhibitors[J].Acta Pharmaceutica Sinica B,.2015,5(6):506-519.
[10]Jacob U,Gaestel M,Engel K,et al.Small heat shock proteins are molecular chaperones[J].Journal of Biological Chemistry.1993,268(3):1517-1520.
[11]He Y,Huang H,Li L,et al.Changes of activated factors and activation of calpain in tilapia muscle during storage[J].Fisheries Science,2018,84(5):889-895.
[12]Carragher N O.Calpain inhibition:a therapeutic strategy targeting multiple disease states[J].Current Pharmaceutical Design,2006,12(5):615-638.
[13]Ray S K.Currently evaluated calpain and caspase inhibitors for neuroprotection in experimental brain ischemia[J].Current Medicinal Chemistry,2006,13(28):3425-3440.
[14]Liu X,Zeng J,Chen X,et al.Crystal structures of RidA,an important enzyme for the prevention of toxic side products[J].Scientific Reports,2016,6:30494.
[15]郑晖,蒋海鹰,颜亚晖.浅谈常规病理制片及免疫组化染色的质控问题[J].诊断病理学杂志,2003,10(2):119-120.
[16]龙小霞.酶消化及微波抗原修复在免疫组化中的应用[J].诊断病理学杂志,1995,2(2):115-116.