一个植物半胱氨酸蛋白酶多克隆抗体的制备及其应用
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摘要
半胱氨酸蛋白酶(cysteine proteinase,Cys P)是一类重要的蛋白酶家族,广泛参与植物多种生理过程。为了分析植物Cys P的特性,本实验首先通过RT-PCR技术从本氏烟中扩增获得了一个编码Cys P的基因(NbCys P)序列并连接至p EASYTM-T5 Zero载体,测序验证后亚克隆至原核表达载体p GEX-6P1,命名为p GEXNb Cys P;将其导入大肠埃希菌BL21plys S中诱导表达;重组表达的Nb Cys P融合蛋白经过亲和层析纯化,免疫兔子制备多克隆抗体,Western印迹分析显示,该抗体能和重组Nb Cys P蛋白发生强烈的免疫学反应且条带单一,表明所获得的Cys P抗体具有良好的特异性。上述结果为进一步鉴定植物Cys P的功能特性奠定了基础。
Cysteine proteases( Cys Ps) are a family of important proteases that are involved in a wide range of plant physiological processes. To characterize such proteinase,a gene encoding Cys P was amplified by RT-PCR from Nicotiana benthamiana plants and ligated into p EASYTM-T5 Zero vector. The insertion was sequenced and then subcloned into the prokaryotic expression plasmid p GEX-6 P1,named as p GEX-Nb Cys P. The plasmid was transformed into Escherichia coli BL21 plys S for inducible expression. The recombinant Nb Cys P fusion protein was purified with affinity chromatography and used for producing polyclonal antibody by immunizing rabbits. In immunoblotting assays,the polyclonal antibody could react strongly with the recombinant Nb Cys P protein as the presence of a single band,indicating that the antibody was specific against the proteinase. These results laid a foundation for further characterization of its function.
Cysteine proteases( Cys Ps) are a family of important proteases that are involved in a wide range of plant physiological processes. To characterize such proteinase,a gene encoding Cys P was amplified by RT-PCR from Nicotiana benthamiana plants and ligated into p EASYTM-T5 Zero vector. The insertion was sequenced and then subcloned into the prokaryotic expression plasmid p GEX-6 P1,named as p GEX-Nb Cys P. The plasmid was transformed into Escherichia coli BL21 plys S for inducible expression. The recombinant Nb Cys P fusion protein was purified with affinity chromatography and used for producing polyclonal antibody by immunizing rabbits. In immunoblotting assays,the polyclonal antibody could react strongly with the recombinant Nb Cys P protein as the presence of a single band,indicating that the antibody was specific against the proteinase. These results laid a foundation for further characterization of its function.
引文
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[14]BARZIV A,LEVY Y,CITOVSKY V,et al.The Tomato yellow leaf curl virus(TYLCV)V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1[J].Biochemical&Biophysical Research Communications,2015,460(3):525-529.
[15]BERNOUX M,TIMMERS T A,BRIERE C,et al.RD19,an Arabidopsis cysteine protease required for RRS1-R-Mediated resistance,is relocalized to the nucleus by the Ralstonia solanacearum Pop P2 effector[J].Plant Cell,2008,20(8):2252-2264.
[16]羊健,张恒木,陈剑平,等.水稻黑条矮缩病毒p8蛋白的原核表达、抗血清制备及其特性[J].植物保护学报,2007,34(3):252-256.YANG J,ZHANG,H M,CHEN J P,et al.Prekaryotic expression antiserum preparation and some properties of p8 protein of Rice black-streaked dwarf Fijivirus[J].Acta Phytophylacica Sinica,2007,34(3):252-256.(in Chinese with English abstract)
[17]RICHAU K H,KASCHANI F,VERDOES M,et al.Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics[J].Plant Physiology,2012,158(4):1583-1599.
[18]GE Y,CAI Y M,BONNEAU L,et al.Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis[J].Cell Death&Differentiation,2016,23(9):1493-1501.
[19]GILORY E M,HEIN I,HOOM R V D,et al.Involvement of cathepsin B in the plant disease resistance hypersensitive response[J].Plant Journal,2007,52(1):1-13.
[20]KOIZUMI M,YAMAGUCHIi-SHINOZAKI K,TSUJI H,et al.Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana[J].Gene,1993,129(2):175-182.
[21]BOZKURT TO,SCHORNACK S,WIN J,et al.Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(51):20832-20837.
[2]ZHANG X M,WANG Y,LV X M,et al.Nt CP56,a new cysteine protease in Nicotiana tabacum L.involved in pollen grain development[J].Journal of Experimental Botany,2009,60(6):1569-1577.
[3]CHEN G H,HUANG L T,YAP M N,et al.Molecular characterization of a senescence-associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet potato[J].Plant&Cell Physiology,2002,43(9):984-991.
[4]ABE K,EMORI Y,KONDO H,et al.Molecular cloning of a cysteine proteinase inhibitor of rice(oryzacystatin).Homology with animal cystatins and transient expression in the ripening process of rice seeds[J].Journal of Biological Chemistry,1987,262(35):16793-16797.
[5]TRIPATHI S K,SINGH A P,SANE A P,et al.Transcriptional activation of a 37 k Da ethylene responsive cysteine protease gene,Rb CP1,is associated with protein degradation during petal abscission in rose[J].Journal of Experimental Botany,2009,60(7):2035-2044.
[6]WIEDERABDERS B.Structure-function relationships in class CA1 cysteine peptidase propeptides[J].Acta Biochimica Polonica,2003,50(3):691-713.
[7]AVILA E L,ZOUHAR J,CARTER D G,et al.Tools to study plant organelle biogenesis.point mutation lines with disrupted vacuoles and high-speed confocal screening of green fluorescent protein-tagged organelles[J].Plant Physiology,2003,133(4):1673-1676.
[8]MATARASSO N,SCHUSTER S,AVNI A.A novel plant cysteine protease has a dual function as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase gene expression[J].Plant Cell,2005,17(4):1205-1216.
[9]SHINDO T,MISASVILLAMIL J C,HORGER A C,et al.A role in immunity for Arabidopsis cysteine protease RD21,the ortholog of the tomato immune protease C14[J].PLo S One,2012,7(1):e29317.
[10]KASCHANI F,SHABAB M,BOKURT T,et al.An effectortargeted protease contributes to defense against phytophthora infestans and is under diversifying selection in natural hosts[J].Plant Physiology,2011,154(1):1794-1804.
[11]LOZANOTORRES JL,WILBERS RH,GAWRONSKI P,et al.Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(25):10119-10135.
[12]SONG J,WIN J,TIAN M Y,et al.Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(5):1654-1659.
[13]ILYAS M,HORGER A,BOZKURT T,et al.Functional divergence of two secreted immune proteases of tomato[J].Current Biology,2015,25(17):2300-2306.
[14]BARZIV A,LEVY Y,CITOVSKY V,et al.The Tomato yellow leaf curl virus(TYLCV)V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1[J].Biochemical&Biophysical Research Communications,2015,460(3):525-529.
[15]BERNOUX M,TIMMERS T A,BRIERE C,et al.RD19,an Arabidopsis cysteine protease required for RRS1-R-Mediated resistance,is relocalized to the nucleus by the Ralstonia solanacearum Pop P2 effector[J].Plant Cell,2008,20(8):2252-2264.
[16]羊健,张恒木,陈剑平,等.水稻黑条矮缩病毒p8蛋白的原核表达、抗血清制备及其特性[J].植物保护学报,2007,34(3):252-256.YANG J,ZHANG,H M,CHEN J P,et al.Prekaryotic expression antiserum preparation and some properties of p8 protein of Rice black-streaked dwarf Fijivirus[J].Acta Phytophylacica Sinica,2007,34(3):252-256.(in Chinese with English abstract)
[17]RICHAU K H,KASCHANI F,VERDOES M,et al.Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics[J].Plant Physiology,2012,158(4):1583-1599.
[18]GE Y,CAI Y M,BONNEAU L,et al.Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis[J].Cell Death&Differentiation,2016,23(9):1493-1501.
[19]GILORY E M,HEIN I,HOOM R V D,et al.Involvement of cathepsin B in the plant disease resistance hypersensitive response[J].Plant Journal,2007,52(1):1-13.
[20]KOIZUMI M,YAMAGUCHIi-SHINOZAKI K,TSUJI H,et al.Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana[J].Gene,1993,129(2):175-182.
[21]BOZKURT TO,SCHORNACK S,WIN J,et al.Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(51):20832-20837.