飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性

doi:10.3864/j.issn.0578-1752.2017.06.007

Abstract

Abstract: 【Objective】 The objective of this study is to investigate the eukaryotic expression and enzyme activity of chitin deacetylase 1 and 2 (LmCDA1 and LmCDA2) in Locusta migratoria. The results will provide an experimental basis to further clarify the physiological function of LmCDA1 and LmCDA2 in chitin degradation pathway and the development of new green pesticides.【Method】The domains of LmCDA1, LmCDA2a and LmCDA2b were predicted using BLASTP and SMART softwares. The full-length sequences of LmCDA1, LmCDA2a and LmCDA2b were obtained by PCR. Recombinant plasmids of pFastBac-LmCDAs were constructed, the recombinant Bacmid plasmids were obtained by transformation, and then transfected into Sf9 insect cells to express target proteins in vitro. Target proteins were detected by Western blot technology, and then purified by Ni-NTA affinity chromatography column and anion exchange chromatography column (Q-Sepharose). The purity of proteins was detected by 12% SDS-PAGE, and the enzymes activity was detected by spectrophotometric method with p-nitroacetylaniline as substrate. The method of T test was used to analyze the difference of enzyme activity of LmCDA2a and LmCDA2b. 【Result】 The results by using BLASTP and SMART softwares showed LmCDA1, LmCDA2a and LmCDA2b contained four structural domains, that are signal peptide, chitin binding peritrophin-A (ChBD), low-density lipoprotein receptor class A (LDLa) and catalytic domain (CDA). Three genes contained six conserved cysteines in ChBD. The spacing and amino acid composition between cysteines 3 and 4 (67-84 aa) and the sequence between cysteines 4 and 6 (84-106 aa) differed between LmCDA2a and LmCDA2b, the rest were identical. Western blot result showed that the protein molecular weight of LmCDA1, LmCDA2a and LmCDA2b was about 61 kD, consistent with the prediction, which suggested that the recombinant Bacmid plasmids were expressed successfully in Sf9 insect cells. The purity of purified proteins was detected by 12% SDS-PAGE electrophoresis. Most impurities could be removed by Ni-NTA affinity chromatography, and the proteins were further purified by Q-Sepharose exchange chromatography. Enzyme activity determination showed that LmCDA1, LmCDA2a and LmCDA2b had chitin deacetylase activity of 0.268, 0.354 and 0.228 U·μL^-1, respectively, and the activity of LmCDA2a and LmCDA2b showed significant differences. 【Conclusion】Eukaryotic expression in vitro and enzyme activity determination of LmCDA1, LmCDA2a and LmCDA2b revealed that these three enzymes have chitin deacetylase activity, and LmCDA2a and LmCDA2b enzyme activity has a significant difference. It was speculated that the reasons of different phenotypes of L. migratoria when LmCDA2a and LmCDA2b were silenced might be that LmCDA2a and LmCDA2b enzyme activity had a significant difference.

Key words: Locusta migratoria, chitin deacetylase, eukaryotic expression, protein purification, activity detection

ZHAO Pan, ZHANG XueYao, LIU XiaoJian, ZHAO XiaoMing, YU RongRong, DONG Wei, MA EnBo, ZHANG JianZhen, ZHANG Min. Eukaryotic Expression, Affinity Purification and Enzyme Activity of Chitin Deacetylase in Locusta migratoria[J].Scientia Agricultura Sinica, 2017, 50(6): 1057-1066.

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