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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。
OBJECTIVE: To analyze the structure and function of EgFBPA by bioinformatics methods, and to clone, express and purify the active protein by genetic engineering. Methods The bioinformatics software was used to analyze the topological structure, biology and immune function of EgFBPA. The recombinant plasmid FBPA / P-blue was digested with restriction endonucleases EcoRⅠand HandⅢ and subcloned into the expression vector pET30a. The recombinant plasmids were screened and sequenced. The recombinant plasmids with the correct sequencing were transformed into BL21 (DE3) competent cells and induced with isopropyl-β-thiogalactoside (IPTG). The recombinant proteins were purified by Ni-NTA column Recombinant protein and enzyme reaction system was established. The enzymatic activity of the recombinant protein EgFBPA was determined by the change of NADH (nicotinamide adenine dinucleotide). Results The full length of EgFBPA gene was 1 092 bp encoding a protein of 363 amino acids. The pI was 8.34 and the molecular mass unit was 39.8035ku. Subcellular localization analysis of the protein is a signal peptide-free cytoplasmic protein that belongs to a stable protein. The domain and conserved domains predicted that the activation site of FBPAI was VYLEGTLLKPN (222aa-232aa) and contained a TIMβ / α-tube structure (6aa -346aa). The secondary structure is dominated by α-helices and circular structures, with no transmembrane region and 10 types of active sites. The pET30a (+) - EgFBPA was successfully constructed by PCR, double enzyme digestion and sequencing. SDS-PAGE detection of recombinant plasmid expression product molecular quality consistent with expectations, and soluble; Bradford assay protein concentration (0.50 ± 0.02) mg / ml. Enzymatic activity detection of Ni-NTA column purified FBPA has good enzymatic activity, and there is a dose-effect relationship. Conclusion Bioinformatics prediction of EgFBPA may be a potential drug target. The recombinant plasmid Pet30a (+) - FBPA was successfully expressed in Escherichia coli BL21 and the expressed product had enzymatic activity, which laid the foundation for further study of its biological function and drug target.