飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性

发布时间：2018-06-12 23:08

  本文选题：飞蝗 + 几丁质脱乙酰基酶　； 参考：《中国农业科学》2017年06期

【摘要】：【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。
[Abstract]:[objective] to express chitin deacetylase 1 and 2(chitin deacetylase 1and 2 (LmCDA1 and LmCDA2) in eukaryotes of Locusta migratoriain vitro, and to determine the enzyme activity of LmCDA1 and LmCDA2. In order to further clarify the physiological function of Locust Locust LmCDA1 and LmCDA2 in chitin degradation pathway and to develop new green insecticides. [methods] using BLASTP and smart software on-line to predict LmCDA1LmCDA2a and LmCDA2b domain PCR cloning to obtain the order of LmCDA1LmCDA2a and LmCDA2b. [methods] using BLASTP and smart software on-line to predict LmCDA1LmCDA2a and LmCDA2b. The full-length sequence of LmCDA2a and LmCDA2b, The recombinant plasmids of p Fast Bac-LmCDAs were constructed and transformed into Bacmid recombinant plasmids. The recombinant plasmids were transfected into insect Sf9 cells for expression of the target protein in vitro. Western blot technique was used to detect the expression of the target protein, and the protein product was purified by Ni-NTA affinity chromatography column and anionic Q-Sepharose exchange chromatography. SDS-PAGE was used to detect the protein purity. The difference of enzyme activity between LmCDA2a and LmCDA2b was analyzed by spectrophotometric method using p-nitroacetanilide as substrate to detect the target protein. [results] BLASTP and smart software predicted that LmCDA1LmCDA2a and LmCDA2b both contained LmCDA2a and LmCDA2b, and the results of BLASTP and smart software showed that LmCDA1LmCDA2a and LmCDA2b both contained LmCDA2a and LmCDA2b. Four structural domains: N-terminal signal peptide, chitin binding rophin-Ach receptor domain low-density lipoprotein receptor class ALDLaand deacetylase catalytic domain, catalytic domain of deacetylase, catalytic domain, CDA. The chitin binding domains of the three genes all contain six conserved domains. The number and composition of amino acids of cysteine. LmCDA2a and LmCDA2b between the third cysteine and the fourth cysteine, and the sequence between the 4th and 6th cysteine (84-106aaa) were different. The results of Western blot showed that the molecular weight of LmCDA1a and LmCDA2b was about 61 KD, which was consistent with the predicted molecular weight of protein, indicating that Bacmid recombinant plasmid was successfully expressed in insect Sf9 cells. The purified proteins were detected by SDS-PAGE gel electrophoresis. The results showed that most of the proteins could be eluted by Ni-NTA affinity chromatography column, while the protein could be purified more thoroughly by Q-Sepharose exchange chromatography column. The enzyme activity of LmCDA1a and LmCDA2b were 0.268U 0.354U 渭 L ~ (-1) and 0.228U / L ~ (-1), respectively, and there were significant differences between LmCDA _ 2a and LmCDA _ 2b. [conclusion] the eukaryotic expression of LmCDA1-LmCDA2a and LmCDA2b protein in vitro were found to have the activity of chitinyldeacetylase, and the results showed that the enzyme activity of LmCDA2a and LmCDA2b were higher than that of LmCDA2b. [conclusion] the enzyme activity of LmCDA1a and LmCDA2b were determined in vitro. The enzyme activities of LmCDA2a and LmCDA2b were significantly different. It is speculated that the reasons of different phenotypes of LmCDA2a and LmCDA2b after silencing LmCDA2a and LmCDA2b in previous studies may be due to the significant differences in their enzyme activities.
【作者单位】： 山西大学应用生物学研究所;山西大学生命科学学院;
【基金】：国家自然科学青年基金(31402020) 山西省基础研究计划(2015011070) 山西省回国留学人员科研资助项目(2015-007)
【分类号】：S433.2

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