家蝇β-葡萄糖苷酶基因的克隆及重组表达

发布时间：2018-11-29 12:33

【摘要】：克隆家蝇内源性β-葡萄糖苷酶(beta-glucosidase,BG)基因并建立原核表达体系,检测其表达产物的活性,了解家蝇内源性BG酶特点,为进一步解释家蝇极强环境适应能力和发现新的种群控制措施提供分子生物学基础。以草地贪夜蛾等结构信息相对完整且研究较为透彻的6种代表性昆虫的BG酶基因序列为参照对象,利用同源克隆结合RACE-PCR的方法,从家蝇cDNA中克隆得到BG酶基因的全长cDNA序列并对其进行生物信息学分析。分别采用原核表达载体pET-28a(+)构建原核表达体系并在大肠杆菌E.coli BL21(DE3)中诱导表达BG酶基因的融合蛋白。采用七叶苷平板显色法和DNS法检测表达体系融合蛋白的酶活性,并对其性质进行初步的分析。家蝇体内克隆得到了长度为1933 bp的家蝇BG酶基因全长cDNA序列,推导其为562个氨基酸残基组成的蛋白多肽。重组原核质粒经诱导表达后,在65 kDa附近均出现特异性蛋白条带,证实重组质粒成功表达。重组表达的家蝇BG酶兼具内切β-1,4-葡聚糖酶、外切β-1,4-葡聚糖酶和BG的酶活性。家蝇BG酶是一种新的多功能纤维素酶,是昆虫纤维素酶研究的重要补充。
[Abstract]:The endogenous 尾 -glucosidase (beta-glucosidase,BG) gene was cloned and the prokaryotic expression system was established. It provides molecular biological basis for explaining the strong adaptability of housefly to environment and discovering new population control measures. The BG gene sequences of six representative insects were compared by using homologous cloning combined with RACE-PCR. The full-length cDNA sequence of BG enzyme gene was cloned from housefly cDNA and analyzed by bioinformatics. The prokaryotic expression system was constructed using prokaryotic expression vector pET-28a () and the fusion protein of BG enzyme gene was induced and expressed in Escherichia coli E.coli BL21 (DE3). The enzyme activity of fusion protein in the expression system was detected by the method of aescin plate coloration and DNS, and the properties of the fusion protein were preliminarily analyzed. The full-length cDNA sequence of housefly BG gene with length of 1933 bp was cloned in vivo, and the protein polypeptide composed of 562-amino acid residues was deduced. After induced expression of recombinant prokaryotic plasmids, specific protein bands were found near 65 kDa, which confirmed that the recombinant plasmids were successfully expressed. The recombinant housefly BG enzyme had the enzyme activities of endoglucanase, exoglucanase and BG. Housefly BG enzyme is a new multifunctional cellulase and an important supplement to insect cellulase research.
【作者单位】：贵州医科大学临床基础检验学教研室;贵州医科大学人体寄生虫学教研室;
【基金】：贵州省教育厅培育项目[(2009)0137] 国家科技支撑计划课题(2011BAC06B12) 国家自然科学基金(30960343) 国家教育部博士点专项基金[(2008)220]
【分类号】：Q963

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