美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质
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摘要
【目的】研究美国白蛾(Hyphantria cunea)几丁质脱乙酰酶(chitin deacetylase,CDA)基因的酶学性质,了解其在昆虫生命过程中如何发挥功能,为美国白蛾的生物防治提供新靶标。【方法】对家蚕(Bombyxmori)、棉铃虫(Helicoverpa armigera)和云杉卷夜蛾(Choristoneura fumiferana)3种鳞翅目昆虫的几丁质脱乙酰酶2序列保守域进行分析,采用同源比对方法,设计几丁质脱乙酰酶2基因特异引物,利用PCR扩增该基因全长c DNA序列;构建原核重组表达载体p ET30a-Hc CDA2,克隆获得编码美国白蛾Hc CDA2的基因,IPTG诱导蛋白表达,SDS-PAGE电泳检测;构建重组杆状病毒表达载体p Fast Bac-Hc CDA1和p Fast Bac-Hc CDA2,脂质体转染法转染昆虫细胞Hi5,分别获得P1、P2、P3病毒,收集P3病毒上清,得到美国白蛾Hc CDA1(前期研究所得)和Hc CDA2重组蛋白,Western blot分析;重组蛋白Hc CDA1和Hc CDA2经硫酸铵粗纯化后,以对硝基乙酰苯胺为底物,测定重组几丁质脱乙酰酶Hc CDA1&2的酶活力,对其最适反应温度、最适p H以及金属离子的影响等酶学性质进行研究。【结果】获得了编码美国白蛾几丁质脱乙酰酶2基因全长c DNA序列,命名为Hc CDA2(Gen Bank登录号:KT781841),基因全长1.6 kb,该基因在大肠杆菌中成功表达61 kD目的蛋白,免疫家兔获得Hc CDA2蛋白的特异性多克隆抗体。Western blot分析结果表明,Hc CDA1和Hc CDA2在昆虫细胞(Hi5)中均成功表达约80 k D蛋白。酶学性质研究表明,昆虫细胞中分泌表达的Hc CDA1和Hc CDA2均具有催化活性,两种酶的最适反应温度均为50℃,当温度达到80℃时,Hc CDA2几乎失去酶活力;Hc CDA1酶促反应适宜的p H范围为7.0—9.0,并且Hc CDA1和Hc CDA2酶促反应的最适p H均为8.0;在最适反应条件下,Hc CDA2蛋白酶活力均高于Hc CDA1蛋白酶活力;Mg~(2+)、Zn~(2+)、Mn~(2+)和Ca~(2+)对Hc CDA1和Hc CDA2酶促反应整体呈抑制趋势,随浓度增大,Zn~(2)+对Hc CDA1抑制作用逐渐增强,而Mg~(2+)对Hc CDA1的抑制作用则呈现先升高后降低的趋势,而且Mg~(2+)和Ca~(2+)对Hc CDA2的抑制作用也是呈现先升高后降低的趋势。Co~(2+)和Fe~(2+)对Hc CDA1和Hc CDA2酶促反应有激活作用,随浓度增大,Fe~(2+)激活作用越强,而Co~(2+)激活作用则呈现先升高后降低的趋势。【结论】克隆了编码美国白蛾几丁质脱乙酰酶2基因(Hc CDA2),在原核细胞中表达61 k D目的蛋白,免疫家兔获得Hc CDA2蛋白的特异性抗体。得到具有活性的美国白蛾几丁质脱乙酰酶Hc CDA1和Hc CDA2,两者在体外均检测到催化活性,两种酶的最适反应温度均为50℃,最适pH均为8.0。
【Objective】 The objective of this paper is to study the function of the enzyme in the process of insect life, enzymatic characterization of chitin deacetylases from Hyphantria cunea and to provide a basis for understanding of its function in the insect life, and to provide new targets for the biological control strategy of H. cunea. 【Method】 The conserved domain of chitin deacetylase 2 sequences from three kinds of lepidoptera insects Bombyx mori, Helicoverpa armigera and Choristoneura fumiferana were analyzed and chitin deacetylase gene-specific primers were designed by homologous alignment. A c DNA clone was identified to contain c DNA sequence coding for chitin deacetylase by PCR amplification. The prokaryotic recombinant expression vector p ET30a-Hc CDA2 was constructed and cloned in E. coli, the protein expression was induced by IPTG and detected by SDS-PAGE electorphoresis. The recombinant baculovirus expression vector p Fast Bac-Hc CDA1 and p Fast Bac-Hc CDA2 were constructed, and liposome transfection method was used to transfect insect cells Hi5, then the P1, P2 and P3 virus were obtained, respectively. The P3 cells supernatant were collected and Western blot analysis was applied to detect the chitin deacetylase 1(from the previous study) and 2 protein expression. The recombinant proteins were crude purified with ammonium sulfate and then the enzymatic characterizations were studied with the 4'-nitroacetanilide(including the optimum temperature, the optimum p H and the effects of different metal ions on enzyme activity).【Result】 The full-length c DNA clone encoded chitin deacetylase 2 in H. cunea was identified(Gen Bank accession number: KT781841). The c DNA is 1.6 kb in length. The protein encoded by this c DNA is named Hc CDA2 and the protein was shown a band on the SDS-PAGE gel with an apparent molecular weight of 61 k D. The specific antibodies reacting to Hc CDA2 were obtained by immunizing rabbits. The Hc CDA1 and Hc CDA2 were shown as 80 k D proteins in Hi5 cell line detected by Western blot analysis. The study of enzymatic properties showed that the two enzymes which were secreting expression in insect cells possessed catalytic activity, and the optimum temperature were both 50℃, when the temperature reached 80℃, the Hc CDA2 almost lost its activity; Hc CDA1 enzymatic reaction appropriate p H ranged from 7.0-9.0, and the optimum p H of Hc CDA1 and Hc CDA2 were both 8.0. Under the optimum reaction conditions, the enzyme activities of Hc CDA2 protein were higher than Hc CDA1 protein. Mg~(2+), Zn~(2+), Mn~(2+) and Ca~(2+) showed an inhibition on Hc CDA1 and Hc CDA2, an increasing Zn~(2+) concentration would lead to the enhancement of inhibitory effects on Hc CDA1, but Mg~(2+) showed a trend of increasing first and then decreasing on Hc CDA1, as well as an increasing Mg~(2+) or Ca~(2+) concentration showed an inhibition on Hc CDA2. Co~(2+) and Fe~(2+) showed activation on Hc CDA1 and Hc CDA2, Fe~(2+) showed activation stably with increasing concentration, while Co~(2+) showed a trend of increasing first and then decreasing. 【Conclusion】 The gene Hc CDA2 was cloned from H. cunea, expressed in E. coli shown as a 61 k D protein, and the Hc CDA2 protein-specific polyclonal antibodies were obtained by immunizing rabbits. The active Hc CDA1 and Hc CDA2 proteins were obtained, and both of them showed a catalytic activity in vitro, the optimum temperature and p H were both 50℃ and 8.0, respectively.
【Objective】 The objective of this paper is to study the function of the enzyme in the process of insect life, enzymatic characterization of chitin deacetylases from Hyphantria cunea and to provide a basis for understanding of its function in the insect life, and to provide new targets for the biological control strategy of H. cunea. 【Method】 The conserved domain of chitin deacetylase 2 sequences from three kinds of lepidoptera insects Bombyx mori, Helicoverpa armigera and Choristoneura fumiferana were analyzed and chitin deacetylase gene-specific primers were designed by homologous alignment. A c DNA clone was identified to contain c DNA sequence coding for chitin deacetylase by PCR amplification. The prokaryotic recombinant expression vector p ET30a-Hc CDA2 was constructed and cloned in E. coli, the protein expression was induced by IPTG and detected by SDS-PAGE electorphoresis. The recombinant baculovirus expression vector p Fast Bac-Hc CDA1 and p Fast Bac-Hc CDA2 were constructed, and liposome transfection method was used to transfect insect cells Hi5, then the P1, P2 and P3 virus were obtained, respectively. The P3 cells supernatant were collected and Western blot analysis was applied to detect the chitin deacetylase 1(from the previous study) and 2 protein expression. The recombinant proteins were crude purified with ammonium sulfate and then the enzymatic characterizations were studied with the 4'-nitroacetanilide(including the optimum temperature, the optimum p H and the effects of different metal ions on enzyme activity).【Result】 The full-length c DNA clone encoded chitin deacetylase 2 in H. cunea was identified(Gen Bank accession number: KT781841). The c DNA is 1.6 kb in length. The protein encoded by this c DNA is named Hc CDA2 and the protein was shown a band on the SDS-PAGE gel with an apparent molecular weight of 61 k D. The specific antibodies reacting to Hc CDA2 were obtained by immunizing rabbits. The Hc CDA1 and Hc CDA2 were shown as 80 k D proteins in Hi5 cell line detected by Western blot analysis. The study of enzymatic properties showed that the two enzymes which were secreting expression in insect cells possessed catalytic activity, and the optimum temperature were both 50℃, when the temperature reached 80℃, the Hc CDA2 almost lost its activity; Hc CDA1 enzymatic reaction appropriate p H ranged from 7.0-9.0, and the optimum p H of Hc CDA1 and Hc CDA2 were both 8.0. Under the optimum reaction conditions, the enzyme activities of Hc CDA2 protein were higher than Hc CDA1 protein. Mg~(2+), Zn~(2+), Mn~(2+) and Ca~(2+) showed an inhibition on Hc CDA1 and Hc CDA2, an increasing Zn~(2+) concentration would lead to the enhancement of inhibitory effects on Hc CDA1, but Mg~(2+) showed a trend of increasing first and then decreasing on Hc CDA1, as well as an increasing Mg~(2+) or Ca~(2+) concentration showed an inhibition on Hc CDA2. Co~(2+) and Fe~(2+) showed activation on Hc CDA1 and Hc CDA2, Fe~(2+) showed activation stably with increasing concentration, while Co~(2+) showed a trend of increasing first and then decreasing. 【Conclusion】 The gene Hc CDA2 was cloned from H. cunea, expressed in E. coli shown as a 61 k D protein, and the Hc CDA2 protein-specific polyclonal antibodies were obtained by immunizing rabbits. The active Hc CDA1 and Hc CDA2 proteins were obtained, and both of them showed a catalytic activity in vitro, the optimum temperature and p H were both 50℃ and 8.0, respectively.
引文
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[4]KAUR K,DATTAJIRAO V,SHRIVASTAVA V,BHARDWAJ U.Isolation and characterization of chitosan-producing bacteria from beaches of Chennai,India.Enzyme Research,2012,2012:Article ID421683.
[5]ARAKI Y,ITO E.A pathway of chitosan formation in Mucor rouxii:enzymatic deacetylation of chitin.Biochemical and Biophysical Research Communications,1974,56(3):669-675.
[6]ARAKI Y,ITO E.A pathway of chitosan formation in Mucor rouxii.Enzymatic deacetylation of chitin.European Journal of Biochemistry,1975,55:71-78.
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[10]TOPRAK U,BALDWIN D,EELANDSON M,GILLOTT C,HOU H,COUTU C,HEGEDUS D D.A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata(Lepidoptera:Noctuidae)peritrophic matrix.Insect Molecular Biology,2008,17(5):573-585.
[11]DIXIT R,ARAKANE Y,SPECHT C A,RICHARD C,KRAMER K J,BEEMAN R W,MUTHUKRISHNAN S.Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum and three other species of insects.Insect Biochemistry and Molecular Biology,2008,38(4):440-451.
[12]ZHONG X W,WANG X H,TIAN X,Q Y XIA,XIANG Z H,ZHAO P.Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.International Journal of Molecular Sciences,2014,15:1946-1961.
[13]孙晓彤,赵丹,闫晓平,郭巍,徐大庆,李少雅,李景.甜菜夜蛾几丁质脱乙酰酶1(Se CDA1)在毕赤酵母中的表达与活性测定.中国生物防治学报,2015,31(4):586-591.SUN X T,ZHAO D,YAN X P,GUO W,XU D Q,LI S Y,LI J.Expression and enzyme activity of chitin deacetylase 1 from Spodoptera exigua in Pichia pastoris.Chinese Journal of Biological Control,2015,31(4):586-591.(in Chinese)
[14]危蓉萍,杨东航,卜莹莹,纪燕玲,于汉寿.蜜蜂螺原体几丁质脱乙酰酶基因的克隆、表达及酶学性质.南京农业大学学报,2016,39(3):417-424.WEI R P,YANG D H,BU Y Y,JI Y L,YU H S.Cloning,expression and enzymatic characterization of a chitin deacetylase gene from Spiroplasma melliferum.Journal of Nanjing Agricultural University,2016,39(3):417-424.(in Chinese)
[15]钟晓武.家蚕围食膜蛋白质组及几丁质去乙酰化酶的功能研究[D].重庆:西南大学,2012.ZHONG X W.Proteomic analysis on peritrophic membrane and functional characterization of chitin deacetylase in silkworm,Bombyx mori[D].Chongqing:Southwest University,2012.(in Chinese)
[16]ALFONSO C,NUERO O M,SANTAMARIA F,REYES F.Purification of a heat-stable chitin deacetylase from Aspergillus nidulans and its role in cell wall degradation.Current Microbiology,1995,30(1):49-54.
[17]TSIGOS I,MARTINOU A,KAFETZOPOULOS D,BOURIOTIS V.Chitin deacetylases:new,versatile tools in biotechnology.Trends in Biotechnology,2000,18(7):305-312.
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