| 中内毒素(LPS)诱导后家蝇幼虫抗菌肽Cec Md基因的克隆、生物信息学分析及其表达载体的构建(英文) |
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| 引用本文： | 赵平森,姜宁,张爱忠.中内毒素(LPS)诱导后家蝇幼虫抗菌肽Cec Md基因的克隆、生物信息学分析及其表达载体的构建(英文)[J].中国兽医学报,2010,30(1). |
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| 作者姓名： | 赵平森 姜宁 张爱忠 |
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| 作者单位： | 黑龙江八一农垦大学动物科技学院,黑龙江,大庆,163319 |
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| 基金项目： | 黑龙江农垦局科技重点项目 |
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| 摘 要： | 从脂多糖(LPS)诱导或非诱导的家蝇幼虫体内抽提总RNA,根据家蝇Cecropin基因(GenBank:DQ 232774)设计特异性引物,应用RT-PCR技术扩增出编码CecMd开放阅读框(ORF)的cDNA序列,将其克隆入pMD18-T载体,经PCR、双酶切和测序鉴定正确后,将CecMd亚克隆基因与线性化的pET32a(+)连接,构建重组表达载体pET32a(+)/CecMd,并对此基因及其推导氨基酸序列进行系统的生物信息学分析。结果表明从脂多糖(LPS)诱导的家蝇幼虫体内成功的扩增出家蝇抗菌肽Cecropin基因,命名为CecMd,但是从未诱导的虫体内未能获得此基因;CecMd开放阅读框的cDNA全长为192 bp,编码由63个氨基酸残基组成的多肽;此多肽最可能的裂解位点在氨基酸残基A23和G24之间,提示1~23氨基酸残基组成信号肽,24~63氨基酸残基构成成熟肽;CecMd的全长肽包含4个螺旋,成熟肽(不包括信号肽)含有2个螺旋,两者均不含二硫键;CecMd与果蝇Cecropins的同源性显著高于其他昆虫,达到81.0%以上;经PCR,限制酶切和DNA测序鉴定表明,成功构建了重组表达载体pET32a(+)/CecMd。重组表达载体pET32a(+)/CecMd的构建及其CecMd基因的生物信息学分析对其生物学功能研究和高效表达工程菌的构建具有重要意义。
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| 关 键 词： | 家蝇 CecMd 基因克隆 重组表达载体 生物信息学分析 |
Cloning and bioinformic analysis of antibacterial peptide gene Cec Md from Musca domestica larvae challenged by lipopolysaccharide(LPS) and construction of its recombinant expression vector |
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| ZHAO Ping-sen,JIANG Ning,ZHANG Ai-zhong.Cloning and bioinformic analysis of antibacterial peptide gene Cec Md from Musca domestica larvae challenged by lipopolysaccharide(LPS) and construction of its recombinant expression vector[J].Chinese Journal of Veterinary Science,2010,30(1). |
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| Authors: | ZHAO Ping-sen JIANG Ning ZHANG Ai-zhong |
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| Abstract: | Total RNAs were isolated from Musca domestica larvae with or without LPS inducement and cDNA enco-ding the open reading frame (ORF) of Cec Md were amplified by RT-PCR with primers designed according to the known sequence of Musca domestica Cecropin (GenBank: DQ232774). PCR products were ligated into pMD18-T vector and then the subclones of Cec Md gene from pMD18-T/Cec Md were inserted into pET32a(+) ,which were identified by PCR,double restriction enzyme digestion and sequencing. Bioinformic analysis on fragment of Cec Md gene and its deduced amino acids were completed. Results indicated that gene of Musca domestica Cecropin gene which consisted of 192 bp and encoded 63 amino acids was successfully cloned, named Cec Md, from Musca domesti-ca larvae induced with lipopolysaccharide(LPS),but failed to obtain it from the maggots without LPS inducements. The signal peptide contained 1-23 residues in Cec Md and part of residues 24-63 constituted mature peptide. Com-plete peptide of Cec Md contained four helixes whereas the mature peptide(without signal peptide) only possessed two,both possessed no disulfide bond within them. Cec Md shared highly similarity with cecropins from Drosophila family instead of those in other insects. Results of PCR, restriction enzyme digestion and sequencing indicated that recombinant expression vector pET32a(+)/Cec Md was successfully constructed. Construction of recombinant ex-pression vector pET32a(+)/Cec Md and bioinformie analysis on Cee Md gene laid a foundation of establishing strat-egies for high-level expression and exploring biological functions of Cec Md. |
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| Keywords: | Musca domestica Cec Md gene clone recombinant expression vector Bioinformic analysis |
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