曼氏迭宫绦虫亮氨酸氨基肽酶(SmLAP)的生物信息学分析、克隆及表达

Bioinformatic analysis,cloning,and expression of leucine aminopeptidase fromSpirometra mansoni

目的利用生物信息学方法分析曼氏迭宫绦虫亮氨酸氨基肽酶(SmLAP)基因的结构和功能,并将该基因克隆至原核表达载体在大肠埃希菌中进行原核表达并纯化,为进一步研究其功能奠定基础。方法利用生物信息学相关软件,分析SmLAP基因及其编码蛋白的结构、生物学和免疫学功能特征。通过PCR扩增得到目的基因,克隆至原核表达载体pGEX-4T1中,转化感受态E.coli BL21(DE3),IPTG诱导表达,表达的重组蛋白利用GST标签层析柱纯化,采用12%SDS-PAGE分析蛋白纯度。结果曼氏迭宫绦虫LAP基因ORF finder显示核酸序列为1 662bp,编码554个氨基酸,预测在第21个氨基酸的位置出现信号肽。切除信号肽后的核酸序列为1 083bp,编码360个氨基酸,理论分子质量单位为40ku,与人LAP基因序列相似性为38%。将SmLAP和人B细胞抗原表位预测结果比对,相似度极低。构建的原核表达载体pGEX-4T1-SmLAP转化入大肠埃希菌后用IPTG进行诱导表达,目的蛋白主要以包涵体的形式存在。通过超声破碎、尿素溶解包涵体、透析、GST层析柱后获得单一组分的重组蛋白。结论构建的原核表达系统能表达SmLAP蛋白。生物信息学分析SmLAP是一个有潜在应用价值的免疫诊断分子,且主要以包涵体形式存在。

Objectives To bioinformatically characterize the structure and function of the LAP gene fromSpirometra mansoni（SmLAP）,to express the recombinant SmLAP in a prokaryotic expression system,and to purify SmLAP for further study of its functions. Methods The structural,biological,and immunological characteristics of SmLAP were analyzed using several bioinformatics programs.The SmLAP gene was amplified with PCR and inserted into the prokaryotic expression vector pGEX-4T1.Recombinant pGEX-4T1-SmLAP was transformed into E.coli for induced expression with IPTG,and the recombinant protein was purified with a GST chromatography column.Recombinant SmLAP（rSmLAP）was identified with 12% SDS-PAGE and Western blotting. Results An examination of the open reading frame（ORF）indicated that the nucleic sequence of SmLAP consisted of 1 662 bp encoding 544 amino acids,but prediction of the signal peptide indicated a likely signal peptide at amino acid 21 in the sequence.The signal peptide was cleaved,yielding a nucleic acid sequence consisting of 1 083 bp encoding 360 amino acids.The theoretical molecular mass of SmLAP was 40 ku.The sequence shared 38%similarity to LAP from Homo sapiens.Comparison of the B cell epitope in sequences of SmLAP and LAP from Homo sapiens revealed an extremely low level of similarity.In addition,rSmLAP was successfully expressed in a prokaryotic expression system.rSmLAP was ultrasonically disrupted,dissolved in urea,and dialyzed,yielding the recombinant alone. Conclusion rSmLAP was successfully expressed and purified in a prokaryotic expression system.Additionally,the results of bioinformatic analysis indicated that SmLAP might be a valuable diagnostic molecule.