中国农业科学2017，Vol.50Issue(5)：849-858,10.DOI:10.3864/j.issn.0578-1752.2017.05.008
美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质
Cloning, Expression and Enzymatic Characterization of Chitin Deacetylases from Hyphantria cunea
摘要
Abstract
[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 ofH cunea.[Method] The conserved domain of chitin deacetylase 2 sequences from three kinds of lepidoptera insects Bombyx mori,Helicoverpa armigera and Choristoneurafumiferana were analyzed and chitin deacetylase gene-specific primers were designed by homologous alignment.A cDNA clone was identified to contain cDNA sequence coding for chitin deacetylase by PCR amplification.The prokaryotic recombinant expression vector pET30a-HcCDA2 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 pFastBac-HcCDA1 and pFastBac-HcCDA2 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 pH and the effects of different metal ions on enzyme activity).[Result] The full-length cDNA clone encoded chitin deacetylase 2 in H.cunea was identified (GenBank accession number:KT781841).The cDNA is 1.6 kb in length.The protein encoded by this cDNA is named HcCDA2 and the protein was shown a band on the SDS-PAGE gel with an apparent molecular weight of 61 kD.The specific antibodies reacting to HcCDA2 were obtained by immunizing rabbits.The HcCDA1 and HcCDA2 were shown as 80 kD 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 HcCDA2 almost lost its activity;HcCDA1 enzymatic reaction appropriate pH ranged from 7.0-9.0,and the optimum pH of HcCDA1 and HcCDA2 were both 8.0.Under the optimum reaction conditions,the enzyme activities of HcCDA2 protein were higher than HcCDA1 protein.Mg2+,Zn2+,Mn2+ and Ca2+ showed an inhibition on HcCDA1 and HcCDA2,an increasing Zn2+ concentration would lead to the enhancement of inhibitory effects on HcCDA1,but Mg2+ showed a trend of increasing first and then decreasing on HcCDA1,as well as an increasing Mg2+ or Ca2+ concentration showed an inhibition on HcCDA2.Co2+ and Fe2+ showed activation on HcCDA1 and HcCDA2,Fe2+ showed activation stably with increasing concentration,while Co2+ showed a trend of increasing first and then decreasing.[Conclusion] The gene HcCDA2 was cloned from H.cunea,expressed in E.coli shown as a 61 kD protein,and the HcCDA2 protein-specific polyclonal antibodies were obtained by immunizing rabbits.The active HcCDA1 and HcCDA2 proteins were obtained,and both of them showed a catalytic activity in vitro,the optimum temperature and pH were both 50℃ and 8.0,respectively.关键词
美国白蛾/几丁质脱乙酰酶/克隆表达/酶学性质Key words
Hyphantria cunea/chitin deacetylase/cloning and expression/enzymatic characterization引用本文复制引用
闫晓平,赵丹,郭巍,王伟,张雅昆,郜玉杰,赵坤莉..美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质[J].中国农业科学,2017,50(5):849-858,10.基金项目
国家自然科学基金(31471775)、国家现代农业(花生)产业技术体系(30971910)、北京市百千万人才工程项目 （31471775）
