水牛（Bubalus bubalis）回肠末端表达的β-防御素cDNA的分子克隆与鉴定

Molecular cloning and characterization of beta-defensin cDNA expressed in distal ileum of buffalo (Bubalus bubalis).

作者信息

Das Dhanjit Kumar, Sharma Bhaskar, Mitra Abhijit, Kumar Ashok

机构信息

Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243 122, India.

出版信息

DNA Seq. 2005 Feb;16(1):16-20. doi: 10.1080/10425170400020399.

DOI:10.1080/10425170400020399

PMID:16040342

Abstract

Defensins play a prominent role in protection of various epithelial surfaces. In this study, we have cloned and characterized the mRNA from the distal ileum of Bubalus bubalis. Total RNA after isolation from ileal epithelium was reverse transcribed to synthesize cDNA using primers designed by taking conserved region of cattle enteric beta-defensin (EBD) mRNA, goat beta-defensin 2 (BD 2) and cattle lingual antimicrobial peptide (LAP) mRNA sequences. The PCR amplified cDNA of 254 bp was ligated to pDrive cloning vector and transformed into XL-blue strain of E coli. The sequence analysis indicated 29 nucleotide substitutions with reported cattle EBD mRNA sequence sharing 86.2% homology, 92.1% with cattle LAP, 81.6% with cattle tracheal antimicrobial peptide and 84.6% with goat BD 2. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide. Both nucleotide and amino acid sequence homology shows that the cloned sequence is closer to cattle LAP.

摘要

防御素在保护各种上皮表面中发挥着重要作用。在本研究中，我们克隆并鉴定了水牛回肠末端的mRNA。从回肠上皮分离的总RNA使用根据牛肠道β-防御素（EBD）mRNA、山羊β-防御素2（BD 2）和牛舌抗菌肽（LAP）mRNA序列的保守区域设计的引物进行逆转录以合成cDNA。PCR扩增得到的254 bp cDNA被连接到pDrive克隆载体上，并转化到大肠杆菌XL-blue菌株中。序列分析表明，与已报道的牛EBD mRNA序列有29个核苷酸替换，同源性为86.2%，与牛LAP的同源性为92.1%，与牛气管抗菌肽的同源性为81.6%，与山羊BD 2的同源性为84.6%。推导的氨基酸序列编码一个64个氨基酸的前体肽。核苷酸和氨基酸序列同源性均表明克隆的序列与牛LAP更接近。