飞蝗β-N-乙酰氨基葡萄糖苷酶基因的表达及酶学特性分析

【目的】β-N-乙酰氨基葡萄糖苷酶(β-N-acetylglucosaminidase,NAG)是昆虫几丁质降解过程中的重要酶类。研究旨在利用Bac-to-Bac杆状病毒表达系统获得高纯度LmNAG1蛋白并对其酶学特性进行分析,探究该酶在飞蝗(Locusta migratoria)生长发育过程中的生物学功能,为飞蝗绿色防控分子靶标研发提供理论与实践依据。【方法】根据Lm NAG1的cDNA全长序列(Gen Bank:JX888720.1)设计包含酶切位点Bam H Ⅰ、Hind Ⅲ和6×His标签的引物。采用PCR技术扩增包含开放阅读框的目标片段,双酶切后连接至pFast Bac~(TM)-Dual载体上。将重组质粒转化至大肠杆菌DH10Bac感受态细胞中,通过Tn7转座子把目的基因转座到杆状病毒基因组上,用蓝白斑结合抗生素筛选。挑取白斑用pUC/M13引物来扩增目的条带,挑选带目的基因全长的重组杆状病毒质粒(baculovirus plasmid,Bacmid)。用转染试剂将重组Bacmid转染至草地贪夜蛾(Spodoptera frugiperda)卵巢细胞系Sf9中,72 h内连续观察细胞形态,当出现感染迹象后收集细胞,离心,取上清得到P1代重组病毒粒子。用P1代病毒粒子去感染Sf9细胞,裂解并提取蛋白,Western blot检测目的蛋白是否成功表达。之后大量感染Sf9细胞,提取蛋白,利用Ni-NTA琼脂糖亲和层析柱和阴离子交换柱Q对重组蛋白进行纯化,取最纯的馏分用Bradford法测定蛋白浓度。采用4MU-GlcNAc为底物对重组目的蛋白LmNAG1的动力学参数、最适温度和最适pH进行测定。【结果】克隆得到包含1 845 bp LmNAG1全长的pFast Bac-LmNAG1重组质粒,酶切验证与目标条带一致。将重组质粒转化至DH10Bac感受态细胞中,PCR扩增挑选出纯白斑,成功将LmNAG1全长序列构建到杆状病毒基因组上。将重组Bacmid转染至Sf9细胞,72 h后在显微镜下观察可见细胞膨大,边缘不规则等感染迹象。离心收集重组病毒粒子感染新的Sf9细胞,72 h后收集细胞,Western blot检测发现在67 kD附近有明显条带,与LmNAG1的理论分子量一致,获得带6×His标签的融合蛋白。大量感染收集细胞提取蛋白,经Ni-NTA琼脂糖层析纯化,进一步透析除盐后用阴离子交换柱Q二次纯化,得到了高纯度的目的蛋白。用Bradford法测定最纯的E3组分的蛋白浓度为0.057μg·μL~(-1)。体外活性测定结果表明LmNAG1的最适pH为8.0,且在pH 6.0—8.0范围内具有较高的稳定性;LmNAG1的最适温度为40℃,在40℃以下具有很高的稳定性,当温度高于45℃时热稳定性迅速下降;采用4MU-GlcNAc为底物测得LmNAG1具有水解β-1,4糖苷键的活性,可以释放出4MU,它的动力学参数K_m值为(0.28±0.02)mmol·L~(-1),K_(cat)值为(902.88±38.15)s~(-1)。【结论】成功获得高纯度且具活性的LmNAG1蛋白,其可水解β-1,4糖苷键连接的几丁质寡糖,与已知昆虫NAG1具有相似的生物学功能,即参与几丁质的降解。

The Heterogenous Expression and Enzymatic Characteristics of β-N-acetylglucosaminidase from Locusta migratoria

【Objective】β-N-acetylglucosaminidase (NAG) is a key enzyme involved in degradation of chitin. The objective of this study is to obtain the high purified enzyme, and to analyze the enzymatic characteristics. It will be helpful for the biological function study of LmNAG1 during locust development, and will provide a theoretical and practical basis for developing molecular target of pest control. 【Method】The primers with BamH I, Hind III restriction sites and 6×His tags were designed according to the complete cDNA sequence deposited in GenBank (No. JX888720.1). The target sequence consisting of ORF was amplified by PCR, and then ligated to pFastBacTM-Dual vector after double enzyme digestion. The recombinant plasmid was transformed into E. coli DH10Bac competent cells, and the target gene was transposed to baculovirus genome through Tn7 transposon. The white clone was selected by blue-white selection combined with antibiotics screening and then the recombinant baculovirus plasmid (Bacmid) was verified by bacteria liquid PCR with the pUC/M13 primers. The recombinant Bacmid was transfected into Spodoptera frugiperda ovary cell line Sf9 using transfection reagent. The morphology of cell was observed within 72 h continuously. Once the infected phenomenon occurred, the P1 generation recombinant virions were obtained from the supernatant after cell collection and centrifugation, and then used to infect Sf9 cells again. The proteins obtained after lysing were performed western blot to examine whether the target protein was expressed or not. After that, large amounts of Sf9 cells were infected to extract proteins. The recombinant proteins were purified using Ni-NTA agarose beads and Q ion-exchange chromatography, and the protein concentration was determined according to the method of Bradford. The kinetic parameters of purified enzyme, the optimal pH and temperature were determined using the substrate, 4MU-GlcNAc. 【Result】 The recombinant plasmid pFastBac-LmNAG1 consisting of a full-length cDNA of LmNAG1 (1 845 bp) was verified by double enzyme digestion. Furthermore, it was transformed into DH10Bac competent cells, then combined into the baculovirus genome. The correct recombinant Bacmid selected by PCR was used for Sf9 cell transfection. The signs of transfection including cell enlargement and border irregularity were observed under the microscope after 72 h. The recombinant virions were collected after centrifugation, and used to infect Sf9 cells again. The infected Sf9 cells were collected for protein extraction. From western blot, an obvious band around 67 kD, which was in accordance with the molecular mass of LmNAG1. The fusion protein with 6×His tags was obtained successfully. Large amounts of protein were obtained from the infected cells and purified using Ni-NTA agarose beads followed by Q ion-exchange chromatography after dialysis to collect high purified target proteins. The top protein concentration of E3 fraction was 0.057 µg·µL^-1 determined using Bradford method. The in vitro activity detection showed that LmNAG1 exhibited the maximum activity at pH 8.0, and possessed higher stability when pH at 6.0-8.0. The optimum temperature for LmNAG1 was 40℃, the thermostability was high between 30-40℃. However, the enzyme activity was decreased rapidly when the temperature was higher than 45℃. The Km=(0.28±0.02) mmol·L^-1, Kcat= (902.88±38.15) s^-1, and the results showed that LmNAG1 could efficiently hydrolyze β-1,4 linked chitin oligosaccharide.【Conclusion】 The purified LmNAG1 was obtained in this study, and it has been shown that LmNAG1 is able to degrade β-1,4 linked chitin oligosaccharide. LmNAG1 is involved in chitin degradation, which exhibited the similar biological functions with NAG1 of other insects.