牛气管黏膜抗菌肽成熟肽基因的克隆及序列分析  

Cloning and Sequencing of Matural Peptide of Bovine Tracheal Antimicrobial Peptide

作　　者：王金涛[1] 程广东[1] 徐世文[1] 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030

出　　处：《生物技术通讯》2006年第4期550-552,共3页Letters in Biotechnology

摘　　要：目的:获得牛气管黏膜抗菌肽(bTAP)成熟肽的基因序列,为后续的研究工作奠定基础。方法:从新屠宰的黄牛气管黏膜中提取总RNA,反转录获得cDNA,以此cDNA为模板进行PCR扩增目的片段,并将其克隆至pMD18-T载体中,经鉴定随机挑选1个阳性重组子进行测序,将测序结果与已报道的序列进行比较,并做NCBIBlast比对。结果:PCR扩增出bTAP成熟肽基因,核苷酸序列测定验证了其正确性;NCBIBlast比对表明,与bTAP成熟肽基因同源性较高的分别是牛β-防御素11、牛β-防御素12、牛β-防御素402、牛β-防御素403、绵羊β-防御素1、绵羊β-防御素2、山羊β-防御素1及山羊β-防御素2,核苷酸序列同源性分别为78.07%、78.95%、80.70%、83.33%、83.33%、80.70%、81.58%和81.58%,氨基酸序列同源性分别为68.42%、65.79%、68.42%、76.32%、71.05%、63.16%、63.16%和68.42%。结论:成功克隆了bTAP成熟肽的基因序列,NCBIBlas比对表明bTAP与防御素可能来自一个共同的祖系基因。Objective： To obtain the matural peptide gene of bovine tracheal antimicrobial peptide（bTAP） and establish the foundation for future research. Methods： Total RNA was isolated from tracheal mucosa of new butchered cattle, cDNA was obtained by RT-PCR reaction and this cDNA was used as the template in PCR amplification of targeted fragment. The targeted fragment was inserted into pMD18-T Vector. One positive recombinant was selected and sequenced, and was compared with the reported sequence and NCBI Blast. Results： The matural peptide gene of bTAP was successfully amplified by PCR. NCBI Blast results showed that bTAP has higher homology with bovine β-defensinll, 12, 402 and 403, ovis aries β-defensin 1 and 2, goat β-defensin 1 and 2. The nucleotide sequence homology were 78.07%, 78.95%, 80.70%, 83.33%, 83.33%, 80.70%, 81.58% and 81.58% respectively; and the amino acids sequence homology were 68.42%, 65.79%, 68.42%, 76.32%, 71.05%, 63.16%, 63.16% and 68.42% respectively. Conclusion： The matural peptide gene of bTAP was successfully amplified, bTAP and defensins may come from the common ancestor gene.

关 键 词：牛气管黏膜抗菌肽 克隆 序列分析 NCBI BLAST 

分 类 号：Q785[生物学—分子生物学]