斑节对虾抗菌肽Penaeidin基因的克隆及其序列分析

Cloning and sequence analysis of antimicrobial peptide penaeidin from Penaeus monodon

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 perform cloning and sequencing of the penaeidin gene from Penaeus monodon. Methods Total RNA extracted from hemolymph of Penaeus monodon was reverse-transcribed into the cDNA. The recombinant penaeidin gene fragment was cut and cloned with PCR. The gene fragment was ligated to the PMD18-T vector for DNA sequencing. Results The result indicated that the cloned penaeidin gene contains 225 bp encoding for 74 amino acids. It showed 100% homology with those Penaeus monodon （FJ686016, AF475082）, 92% with Penaeus indicus （HM535649）, 63% with Penaeus paulensis （AY956417）, 64% with Penaeus subtilis （EF450742）, 65% with Penaeus brasiliensis （EF450745）, 52% with Penaeus schmitti （AY956420）, and 48% with Penaeus setiferus （AY039207）. Phylogenic Tree analysis showed that penaeidin from Penaeus monodon was in the same group as Penaeus indicus. Penaeus paulensis, Penaeus subtilis, Penaeus brasiliensis and Penaeus setiferus were in the same group. Penaeidin from Penaeus schmitti was an independent group. Results also showed that the differentiation of Penaeidin from Penaeus monodon and Penaeus schmitti was preceded the others, and penaeidins from Penaeus monodon and Penaeus indicus were mostly related. The mature peptide predominantly composed of beta sheets and turns, and the signal peptide was also in the beta sheet region. Both terminals were hydrophilic, and a flexible region was located in the N-terminal, in which the amino acid residues 30-40 and 60-70 were potential antigenic regions. Conclusion Penaeidin gene has been successfully cloned and the results of this study will lay the preliminary foundation for the following protein expression, industrialized production and theoretical research.