飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性

【目的】体外真核表达飞蝗(Locusta migratoria)几丁质脱乙酰基酶1和2(chitin deacetylase 1and 2,LmCDA1和LmCDA2)并测定其酶活性,为进一步明确飞蝗LmCDA1和LmCDA2在几丁质降解途径中的生理功能及研发新型绿色环保杀虫剂提供依据。【方法】使用BLASTP和SMART软件在线预测LmCDA1、LmCDA2a和LmCDA2b的结构域;PCR克隆获得目的基因LmCDA1、LmCDA2a和LmCDA2b的全长序列,并分别构建p Fast Bac-LmCDAs重组质粒,转化获得Bacmid重组质粒后,转染至昆虫Sf9细胞进行目的蛋白的体外表达。采用Western blot技术对目的蛋白表达情况进行检测,并通过Ni-NTA亲和层析柱和阴离子(Q-Sepharose)交换层析柱对蛋白产物进行纯化。12%SDS-PAGE检测蛋白纯度后,采用分光光度法以对硝基乙酰苯胺为底物检测目的蛋白的酶活性,T检验法对LmCDA2a和LmCDA2b酶活力进行差异显著性分析。【结果】BLASTP和SMART软件预测结果显示LmCDA1、LmCDA2a和LmCDA2b均含有4个结构域:N-端信号肽(signal peptide)、几丁质结合域(chitin binding peritrophin-A,Ch BD)、A型低密度脂蛋白受体结构域(low-density lipoprotein receptor class A,LDLa)和脱乙酰基酶催化结构域(catalytic domain,CDA)。3个基因的几丁质结合域中均包含6个保守的半胱氨酸。LmCDA2a和LmCDA2b两个剪切子除在其第3个半胱氨酸和第4个半胱氨酸之间(67—84 aa)的氨基酸数目和组成及在第4和第6个半胱氨酸(84—106 aa)之间的序列存在差异外,其余部分完全一致。Western blot结果显示LmCDA1、LmCDA2a和LmCDA2b的蛋白分子量约为61 k D左右,与预测的蛋白分子量大小一致,表明Bacmid重组质粒在昆虫Sf9细胞中成功表达。采用12%SDS-PAGE胶电泳对各蛋白纯化组分进行检测,结果显示Ni-NTA亲和层析柱可将大部分杂蛋白洗脱,而Q-Sepharose交换层析柱可对蛋白进行更彻底地纯化。酶活检测结果显示LmCDA1、LmCDA2a和LmCDA2b的酶活力分别为0.268、0.354、0.228 U·μL~(-1),并且LmCDA2a和LmCDA2b的酶活力存在显著差异。【结论】体外真核表达LmCDA1、LmCDA2a和LmCDA2b蛋白并进行酶活测定后发现三者均具有几丁质脱乙酰基酶活力,且LmCDA2a和LmCDA2b的酶活力具有显著性差异,推测前期研究中沉默LmCDA2a和LmCDA2b后分别出现不同飞蝗表型的原因可能是由于它们酶活力存在显著性差异。

Eukaryotic Expression,Affinity Purification and Enzyme Activity of Chitin Deacetylase in Locusta migratoria

【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.