曼氏迭宫绦虫异柠檬酸脱氢酶的基因克隆、蛋白表达及其在裂头蚴阶段的转录水平检测分析

Gene cloning,protein expression and transcription analysis at sparganum develpment stage of isocitrate dehydrogenase from Spirometra mansoni

目的利用生物信息学方法识别和分析曼氏迭宫绦虫异柠檬酸脱氢酶(SmNAD-IDH)核苷酸序列及生物学特征;克隆该基因并诱导蛋白表达,分析其在裂头蚴阶段的转录水平。方法利用生物信息学在线分析工具对SmNAD-IDH基因的核苷酸序列进行识别,预测分析氨基酸序列的理化性质。分别以曼氏迭宫绦虫裂头蚴cDNA、孕节cDNA和成节cDNA作为模板PCR扩增目的基因,扩增产物与表达载体pET-30a重组后转化至BL21细胞中诱导蛋白表达。利用real time PCR技术分析裂头蚴阶段三羧酸循环中3个关键代谢酶的转录水平。结果BLASTx分析SmNAD-IDH基因全长1095 bp,编码蛋白由356个氨基酸组成。该基因与其他寄生虫同源基因核苷酸序列一致性>70%,与人类同源基因核苷酸序列一致性为51%。该蛋白的理论等电点为6.84,分子质量为40 ku,并且氨基酸序列中无信号肽和跨膜区,为脂溶性稳定的胞内蛋白。SmNAD-IDH在裂头蚴、成节、孕节中均有表达,在裂头蚴阶段三羧酸循环中的关键酶柠檬酸合成酶、酮戊二酸脱氢酶、SmNAD-IDH均有转录,以SmNAD-IDH的转录水平较高。结论成功重组SmNAD-IDH基因并诱导SmNAD-IDH蛋白表达,该蛋白对于曼氏迭宫绦虫,尤其是裂头蚴,是具有潜在研究价值的重要代谢酶。

Objective To identify the nucleotide sequence and biological characteristics of isocitrate dehydrogenase(SmNAD-IDH)from Spirometra mansoni by bioinformatics methods,clone the SmNAD-IDH gene,induce its protein expression and analyze its transcription level in sparganum stage.Methods The nucleotide sequence of SmNAD-IDH was identified and predicted its physicochemical properties by using bioinformatics tools.The target genes were amplified by PCR using cDNA of sparganum,mature proglottid,gravid proglottid,as templates.The amplified product was recombined with expression vector pET-30a and transformed into BL21 cells to induce protein expression.In addition,real-time PCR was used to analyze the transcription levels of three key metabolic enzymes in the tricarboxylic acid cycle(TCA)of sparganum.Results The total length of nucleotide sequence of SmNAD-IDH gene was 1095 bp,and the protein was composed of 356 amino acids by BLASTx..The identities of nucleotide sequence of SmNAD-IDH gene were more than 70%consistent with homologous genes of other parasites and 51%consistent with homologous genes of human.The theoretical isoelectric point of the protein molecule is 6.84 and the theoretical molecular weight is 40 ku.There is no signal peptide and no transmembrane region in the amino acid sequence of SmNAD-IDH.It is a stable intracellular protein molecule with lipid solubility.SmNAD-IDH is expressed in the different devolopment stages of sparganum,mature proglottid,gravid proglottid.Furthermore,the key enzymes of the tricarboxylic acid cycle,such as citrate synthase,ketoglutarate dehydrogenase and SmNAD-IDH,are transcripted in the sparganum development stage,and the transcription level of SmNAD-IDH is higher.Conclusion The SmNAD-IDH gene was successfully recombined and its protein was induced to express.The SmNAD-IDH protein is an important metabolic enzyme with potential research value for Spirometra mansoni,especially for sparganum.