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目的克隆斑节对虾Penaeidin抗菌肽基因,构建克隆载体,测序后进行序列分析。方法从斑节对虾采集血淋巴后提取总RNA,经反转录得到cDNA,用特异性引物进行PCR扩增得到具有双酶切位点的重组Penaeidin目的基因片段。将其连接至PMD18-T载体,转化至Top10克隆菌进行克隆并测序,用生物信息学软件进行序列分析。结果测序结果表明斑节对虾Penaeidin抗菌肽基因全长225bp,编码74个氨基酸。将克隆所得的斑节对虾Penaeidin基因序列与NCBI数据库中的斑节对虾Penaeidin基因(FJ686016,AF475082)同源性为100%,与其它物种虾的Penaeidin基因同源性为48%～92%。分子进化树显示斑节对虾Penaeidin抗菌肽和印度对虾Penaeidin抗菌肽聚为一大枝,而圣保罗对虾、小褐美对虾、巴西美对虾、白滨对虾Penaeidin聚为一大枝,南方滨对虾Penaeidin则自成一枝。二级结构预测表明本次克隆得到的斑节对虾Penaeidin信号肽区为β折叠片,成熟肽主要为β折叠片和转角结构。成熟肽的N端和C端亲水性较强,N端为柔性区域。其中氨基酸序列30-40和60-70表现为潜在的T细胞抗原表位。结论成功克隆出斑节对虾Penaeidin基因,对其序列的分析结果将为后续的蛋白表达、工业化生产和理论研究奠定前期基础。
Objective To clone the Penaeidin antimicrobial peptide gene of Penaeus monodon, construct a cloning vector and sequence analysis after sequencing. Methods Total RNA was extracted from the hemolymph of Penaeus monodon, and the cDNA was obtained by reverse transcription. The recombinant Penaeidin gene fragment with double restriction enzyme digestion was amplified by specific primers. This was ligated to the PMD18-T vector, transformed into Top10 Clones, cloned and sequenced, and sequenced using bioinformatics software. Results Sequencing results showed that the Penaeidin antimicrobial peptide gene of Penaeus monodon was 225 bp in length and encoded 74 amino acids. The homology between Penaeidin gene of Penaeus monodon clone and Penaeidin gene (FJ686016, AF475082) in NCBI database was 100%, and that of Penaeidin gene was 48% -92% with that of shrimp in other species. The molecular phylogenetic tree showed that the Penaeidin antimicrobial peptide from Penaeidin and Penaeidin antimicrobial peptide from Penaeus vannamei aggregated into one branch, while Penaeidin from Penaeus vannamei, Penaeus vannamei, Penaeid shrimp and Penaeidin became a large branch, while Penaeidin from Penaeus monodon A stick. The secondary structure prediction showed that Penaeidin signal peptide region of Penaeus monodon obtained by this clone was β-sheet, and the mature peptide was mainly β-sheet and corner structure. The mature peptide N-terminal and C-terminal hydrophilic, N-terminal flexible region. In which amino acid sequences 30-40 and 60-70 appear as potential T cell epitopes. Conclusion The Penaeidin gene of Penaeus monodon was successfully cloned. The results of its sequence analysis will lay the foundation for the subsequent protein expression, industrial production and theoretical research.