抗棉铃虫Ⅳ型几丁质酶(HaCHT4)小鼠多克隆抗体的制备与鉴定
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摘要
目的原核表达棉铃虫Ⅳ型几丁质酶(HaCHT4)并制备其多克隆抗体。方法在大肠杆菌中表达组蛋白-HaCHT4融合蛋白(His-HaCHT4),镍柱纯化目的蛋白,经足垫加皮下注射免疫小鼠。4次免疫后,用ELISA检测抗His-HaCHT4多克隆抗体的效价,Western blot法检测其免疫特异性。结果成功纯化His-HaCHT4融合蛋白,小鼠抗His-HaCHT4的多克隆抗体的滴度高于1∶204 800。Western blot结果显示该抗体能与His-HaCHT4融合蛋白及棉铃虫体中的天然HaCHT4结合,但不能与黄粉虫的蛋白结合。结论成功原核表达和纯化了His-HaCHT4,制备了特异性的鼠多克隆抗体。
Objective The group Ⅳ chitinase from Helicoverpa armigera( HaCHT4) was expressed in prokaryotic and itspolyclonal antibody against HaCHT4 was prepared. Methods The fusion protein His-HaCHT4 was expressed in E. coli andpurified the target protein by Ni-column,and immunized the mice by footpad and subcutaneous injection. The titer ofpolyclonal antibody against His-HaCHT4 was determined by ELISA after four immunizations. The immunological specificity ofthe anti His-HaCHT4 was assayed by Western blot analysis. Results Fusion protein His-HaCHT4 was purified successfullyand the titer of mouse anti His-HaCHT4 polyclonal antibody was higher than 1∶204 800. The results of Western blot showedthat the anti His-HaCHT4 polyclonal antibody was able to bind the fusion protein His-HaCHT4,and also specifically recognizethe natural HaCHT4 in Helicoverpa armigera,but not the protein in Tenebrio molitor. Conclusion His-HaCHT4 wassuccessfully expressed in prokaryotic and purified,and its mouse anti His-HaCHT4 polyclonal antibody was prepared.
Objective The group Ⅳ chitinase from Helicoverpa armigera( HaCHT4) was expressed in prokaryotic and itspolyclonal antibody against HaCHT4 was prepared. Methods The fusion protein His-HaCHT4 was expressed in E. coli andpurified the target protein by Ni-column,and immunized the mice by footpad and subcutaneous injection. The titer ofpolyclonal antibody against His-HaCHT4 was determined by ELISA after four immunizations. The immunological specificity ofthe anti His-HaCHT4 was assayed by Western blot analysis. Results Fusion protein His-HaCHT4 was purified successfullyand the titer of mouse anti His-HaCHT4 polyclonal antibody was higher than 1∶204 800. The results of Western blot showedthat the anti His-HaCHT4 polyclonal antibody was able to bind the fusion protein His-HaCHT4,and also specifically recognizethe natural HaCHT4 in Helicoverpa armigera,but not the protein in Tenebrio molitor. Conclusion His-HaCHT4 wassuccessfully expressed in prokaryotic and purified,and its mouse anti His-HaCHT4 polyclonal antibody was prepared.
引文
[1] Chattopadhyay P,Banerjee G. Recent advancement on chemicalarsenal of Bt toxin and its application in pest management system inagricultural field[J/OL]. Biotech, 2018, 8(4):201.DOI:10. 1007/s13205-018-1223-1. Epub 2018 Mar 29.
[2] Catarino R,Ceddia G,Areal F J,et al. The impact of secondarypests on Bacillus thuringiensis(Bt)crops[J]. Plant Biotechnol J,2015,13(5):601-612.
[3]Khan F I,Rahman S,Queen A,et al. Implications of moleculardiversity of chitin and its derivatives[J]. Appl Microbiol Biotechnol,2017,101(9):3513-3536.
[4]Hegedus D,Erlandson M,Gillott C,et al. New insights into peritrophicmatrix synthesis, architecture, and function[J]. Annu RevEntomol,2009,54(54):285-302.
[5]Liu T,Chen L,Zhou Y,et al. Structure,catalysis,and inhibitionof Of Chi-h,the Lepidoptera-exclusive insect chitinase[J]. J BiolChem,2017,292(6):2080-2088.
[6]Zhao D,Liu B,Zhang Y K,et al. Structural and biochemical characteristicsof chitin-binding protein SeCBP66 from Spodoptera exigua(Hübner)[J]. Genet Mol Res,2016,15(3). DOI:10. 4238/gmr.15038789.
[7]Muthukrishnan S,Arakane Y,Yang Q,et al. Future questions ininsect chitin biology:A microreview[J/OL]. Arch Insect BiochemPhysiol,2018,98(2):e21454. DOI:10. 1002/arch. 21454.Epub 2018 Feb 25.
[8]Labroussaa F,Ionescu M,Zeilinger A R,et al. A chitinase is requiredfor Xylella fastidiosa colonization of its insect and plant hosts[J].Microbiology,2017,163(4):502-509.
[9]夏梦.亚洲玉米螟Ⅳ家族几丁质酶的克隆表达、性质表征及抑制剂研究[D].大连理工大学,2015:5-6.
[10]Pesch Y Y,Riedel D,Behr M. Drosophila chitinase 2 is expressedin chitin producing organs for cuticle formation[J]. Arthropod StructDev,2017,46(1):4-12.
[11]Suganthi M,Senthilkumar P,Arvinth S,et al. Chitinase from Pseudomonasfluorescens and its insecticidal activity against Helopeltis theivora[J].J Gen Appl Microbiol,2017,63(4):222-227.
[12] Liu S H,Li H F,Yang Y,et al. Genome-wide identification ofchitinase and chitin deacetylase gene families in the oriental fruit fly,Bactrocera dorsalis(Hendel)[J]. Comp Biochem Physiol,Part DGenomics Proteomics,2018,27:13-22.
[13]Pan Y,LüP,Wang Y,et al. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome[J/OL].J Insect Sci,2012,12:150. DOI:10. 1673/031. 012. 15001.
[14]Khajuria C,Buschman L L,Chen M S,et al. A gut-specific chitinasegene essential for regulation of chitin content of peritrophic matrix andgrowth of Ostrinia nubilalis larvae[J]. Insect Biochem Mol Bio,2010,40(8):621-629.
[15]Han J H,Lee K S,Li J,et al. Cloning and expression of a fat body-specificchitinase c DNA from the spider, Araneus ventricosus[J]. CompBiochem Physiol Part B Biochem Mol Biol, 2005, 140(3):427-435.
[16]Genta F A,Blanes L,Cristofoletti P T,et al. Purification,characterizationand molecular cloning of the major chitinase from Tenebrio molitorlarval midgut[J]. Insect Biochem Mol Biol, 2006, 36(10):789-800.
[17]Zhang D,Chen J,Yao Q,et al. Functional analysis of two chitinasegenes during the pupation and eclosion stages of the beet armywormSpodoptera exigua by RNA interference[J]. Arch Insect BiochemPhysiol,2012,79(4/5):220-234.
[18] Mamta,Reddy K R,Rajam M V. Targeting chitinase gene ofHelicoverpa armigera by host-induced RNA interference confers insectresistance in tobacco and tomato[J]. Plant Mol Biol,2016,90(3):281-292.
[19]Ganbaatar O,Cao B,Zhang Y,et al. Knockdown of Mythimna separatachitinase genes via bacterial expression and oral delivery of RNAieffectors[J/OL]. BMC Biotechnol, 2017, 17(1):9.DOI:10. 1186/s12896-017-0328-7.
[20]孟琳,田景振.动物组织蛋白质提纯方法的研究进展[J].食品与药品,2009,11(5):53-55.
[21]Liu C,Du B,Hao F,et al. Dynamic metabolic responses of brownplan thoppers towards susceptible and resistant rice plants[J]. PlantBiotechnol J,2017,15(10):1346-1357.
[22]Baia-da-silva D C,Alvarez L C S,Lizcano O V,et al. The role ofthe peritrophic matrix and red blood cell concentration in Plasmodiumvivax infection of Anopheles aquasalis[J]. Parasit Vectors,2018,11(1):148. DOI:10. 1186/s13071-018-2752-5.
[23]张婷婷,柳伟伟,高璐,等.飞蝗几丁质酶5-1抗体制备及表达特性分析[J].中国农业科学,2018,51(12):2418-2428.
[24]赵奎.蜜蜂球囊菌几丁质酶的纯化及其多克隆抗体的制备[D].福建农林大学:2008:38-39.
[25]郝婵娟,柴宝峰,王伟,等.烟草天蛾几丁质酶的表达、纯化及多克隆抗体制备[J].昆虫学报,2005,48(3):337-341.
[26]赵洁,赵文博,朱燕,等.活体电穿孔法辅助核酸免疫制备小鼠抗棉铃虫精氨酸激酶的抗体[J].细胞与分子免疫学杂志,2014,30(10):1054-1057.
[2] Catarino R,Ceddia G,Areal F J,et al. The impact of secondarypests on Bacillus thuringiensis(Bt)crops[J]. Plant Biotechnol J,2015,13(5):601-612.
[3]Khan F I,Rahman S,Queen A,et al. Implications of moleculardiversity of chitin and its derivatives[J]. Appl Microbiol Biotechnol,2017,101(9):3513-3536.
[4]Hegedus D,Erlandson M,Gillott C,et al. New insights into peritrophicmatrix synthesis, architecture, and function[J]. Annu RevEntomol,2009,54(54):285-302.
[5]Liu T,Chen L,Zhou Y,et al. Structure,catalysis,and inhibitionof Of Chi-h,the Lepidoptera-exclusive insect chitinase[J]. J BiolChem,2017,292(6):2080-2088.
[6]Zhao D,Liu B,Zhang Y K,et al. Structural and biochemical characteristicsof chitin-binding protein SeCBP66 from Spodoptera exigua(Hübner)[J]. Genet Mol Res,2016,15(3). DOI:10. 4238/gmr.15038789.
[7]Muthukrishnan S,Arakane Y,Yang Q,et al. Future questions ininsect chitin biology:A microreview[J/OL]. Arch Insect BiochemPhysiol,2018,98(2):e21454. DOI:10. 1002/arch. 21454.Epub 2018 Feb 25.
[8]Labroussaa F,Ionescu M,Zeilinger A R,et al. A chitinase is requiredfor Xylella fastidiosa colonization of its insect and plant hosts[J].Microbiology,2017,163(4):502-509.
[9]夏梦.亚洲玉米螟Ⅳ家族几丁质酶的克隆表达、性质表征及抑制剂研究[D].大连理工大学,2015:5-6.
[10]Pesch Y Y,Riedel D,Behr M. Drosophila chitinase 2 is expressedin chitin producing organs for cuticle formation[J]. Arthropod StructDev,2017,46(1):4-12.
[11]Suganthi M,Senthilkumar P,Arvinth S,et al. Chitinase from Pseudomonasfluorescens and its insecticidal activity against Helopeltis theivora[J].J Gen Appl Microbiol,2017,63(4):222-227.
[12] Liu S H,Li H F,Yang Y,et al. Genome-wide identification ofchitinase and chitin deacetylase gene families in the oriental fruit fly,Bactrocera dorsalis(Hendel)[J]. Comp Biochem Physiol,Part DGenomics Proteomics,2018,27:13-22.
[13]Pan Y,LüP,Wang Y,et al. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome[J/OL].J Insect Sci,2012,12:150. DOI:10. 1673/031. 012. 15001.
[14]Khajuria C,Buschman L L,Chen M S,et al. A gut-specific chitinasegene essential for regulation of chitin content of peritrophic matrix andgrowth of Ostrinia nubilalis larvae[J]. Insect Biochem Mol Bio,2010,40(8):621-629.
[15]Han J H,Lee K S,Li J,et al. Cloning and expression of a fat body-specificchitinase c DNA from the spider, Araneus ventricosus[J]. CompBiochem Physiol Part B Biochem Mol Biol, 2005, 140(3):427-435.
[16]Genta F A,Blanes L,Cristofoletti P T,et al. Purification,characterizationand molecular cloning of the major chitinase from Tenebrio molitorlarval midgut[J]. Insect Biochem Mol Biol, 2006, 36(10):789-800.
[17]Zhang D,Chen J,Yao Q,et al. Functional analysis of two chitinasegenes during the pupation and eclosion stages of the beet armywormSpodoptera exigua by RNA interference[J]. Arch Insect BiochemPhysiol,2012,79(4/5):220-234.
[18] Mamta,Reddy K R,Rajam M V. Targeting chitinase gene ofHelicoverpa armigera by host-induced RNA interference confers insectresistance in tobacco and tomato[J]. Plant Mol Biol,2016,90(3):281-292.
[19]Ganbaatar O,Cao B,Zhang Y,et al. Knockdown of Mythimna separatachitinase genes via bacterial expression and oral delivery of RNAieffectors[J/OL]. BMC Biotechnol, 2017, 17(1):9.DOI:10. 1186/s12896-017-0328-7.
[20]孟琳,田景振.动物组织蛋白质提纯方法的研究进展[J].食品与药品,2009,11(5):53-55.
[21]Liu C,Du B,Hao F,et al. Dynamic metabolic responses of brownplan thoppers towards susceptible and resistant rice plants[J]. PlantBiotechnol J,2017,15(10):1346-1357.
[22]Baia-da-silva D C,Alvarez L C S,Lizcano O V,et al. The role ofthe peritrophic matrix and red blood cell concentration in Plasmodiumvivax infection of Anopheles aquasalis[J]. Parasit Vectors,2018,11(1):148. DOI:10. 1186/s13071-018-2752-5.
[23]张婷婷,柳伟伟,高璐,等.飞蝗几丁质酶5-1抗体制备及表达特性分析[J].中国农业科学,2018,51(12):2418-2428.
[24]赵奎.蜜蜂球囊菌几丁质酶的纯化及其多克隆抗体的制备[D].福建农林大学:2008:38-39.
[25]郝婵娟,柴宝峰,王伟,等.烟草天蛾几丁质酶的表达、纯化及多克隆抗体制备[J].昆虫学报,2005,48(3):337-341.
[26]赵洁,赵文博,朱燕,等.活体电穿孔法辅助核酸免疫制备小鼠抗棉铃虫精氨酸激酶的抗体[J].细胞与分子免疫学杂志,2014,30(10):1054-1057.