细粒棘球绦虫果糖二磷酸醛缩酶的生物信息学分析及其表达与活性检测

Bioinformatic analysis,expression,purification,and assessment of the enzyme activity of Echinococcus granulosus fructose-bisphosphate aldolase

目的利用生物信息学方法分析细粒棘球绦虫果糖二磷酸醛缩酶(EgFBPA)的结构和功能特征,并利用基因工程技术进行克隆、表达、纯化,获得活性蛋白。方法利用多种生物信息学软件分析EgFBPA的拓扑结构、生物学以及免疫功能特征;用限制性内切酶EcoRⅠ和HandⅢ对重组质粒FBPA/P-blue酶切FBPA目的基因片段,亚克隆入表达载体pET30a,筛选重组克隆并测序,将测序正确的重组质粒转化入BL21(DE3)感受态细菌,用异丙基-β-硫代半乳糖苷(IPTG)诱导表达重组蛋白;用Ni-NTA柱纯化重组蛋白,并建立酶反应体系,通过NADH(烟酰胺腺嘌呤二核苷酸)的变化测定重组蛋白EgFBPA的酶催化活性。结果 EgFBPA基因全长1 092bp,编码363个氨基酸,软件分析其等电点(pI)为8.34,分子质量单位为39.8035ku。亚细胞定位分析该蛋白为无信号肽的细胞质蛋白,属于稳定蛋白;结构域和保守功能域的预测,FBPAI的激活位点为VYLEGTLLKPN(222aa-232aa),并含有TIMβ/α筒状结构(6aa-346aa),二级结构以α-螺旋和环状结构居多,无跨膜区,有10种类型的活性位点。经PCR、双酶切和测序证实pET30a(+)-EgFBPA构建成功。SDS-PAGE检测重组质粒表达产物分子质量与预期相符,且具有可溶性;Bradford法测定蛋白浓度为(0.50±0.02)mg/ml。酶活性检测Ni-NTA柱纯化后的FBPA具有良好的酶促活性,且存在量效关系。结论生物信息学预测EgFBPA可能是潜在的药物靶点。重组质粒Pet30a(+)-FBPA在大肠埃希菌BL21中成功表达,表达产物具有酶促活性,为进一步研究其生物学功能及药物靶点奠定了基础。

Objectives To bioinformatically analyze and characterize Echinococcus granulosus fructose bisphosphate aldolase （FBPA） structurally and functionally and to clone, express, and purify it and assess its bioactivity. Methods Various bioinformatics programs were used to analyze the topological, biological, and immunological characteristics of E. granulosus FBPA. EcoR I and Hind III were used to digest target fragments of FBPA. FBPA was then subcloned into the expression vector pET30a, which was subsequently screened and sequenced. The correct recombinant plasmid was transformed into BL21 （DE3） competent bacteria and expressed in the presence of isopropyl-beta-thiogalactoside （IPTG）. The recombinant protein was purified using a Ni-NTA column, and then an enzyme reaction system （nicotinamide adenine dinucleotide） was created via NADH reduction to determine the enzyme catalytic activity of the recombinant protein EgFB- PA. Results EgFBPA cDNA encoded a 336-amino acid protein and was 1 092 bp in length. Software analysis indicated that its isoelectric point （PI） was 8.34 and its molecular weight was 39. 8035 ku. Analysis of subcellular localization indicated that the protein was a stable cytoplasmic protein without signal peptides. Prediction of the domain and the conserved domain suggested an activation site at FBPAIVYLEGTLLKPN （222aa-232aa） and a TIM（β/a） barrel-like structure （6aa- 346aa）. The secondary structure consisted of an alpha-helix and cyclic structure, with 10 functional sites but no transmembrane region. Successful subcloning of pET30a （＋）-EgFBPA was confirmed with PCR, double enzyme digestion, and sequencing. The expressed recombinant plasmid was consistent with the expected molecular weight according to SDS- PAGE, and the plasmid was soluble. The protein concentration was determined to be 0. 504-0, 02 mg/ml using the Bradford method. The enzyme activity assay revealed that EgFBPA purified with a Ni-NTA column had significant enzyme activity in a dose-dependent manner. Conclusion E. granulosus FBPA may be a potential drug target. The recombinant plasmid pET30a （＋）-FBPA was successfully expressed in E. coli BL21 and purified. This work lays the foundation for further study of the biological function of E. granulosus FBPA and its targeting by drugs.