鸡β-防御素Gallinacin-8基因片段的克隆与序列分析

The cDNA cloning and sequencing Analysis of chicken β-defensin(Gal-8)

本试验从鸡的肝脏中提取总RNA,根据GenBank公布的鸡β-防御素Gallinacin-8(简称gal-8)基因序列设计2对引物,运用RT-PCR技术扩增出大小为201bp的鸡β-防御素gal-8的基因片段。通过序列分析得知其由66个氨基酸组成,包括20个氨基酸残基的信号肽、5个氨基酸的原片肽及41个氨基酸的成熟肽。BLAST分析表明与GenBank中已公布的序列相比较缺失2个碱基,其同源性达到98%。将该基因克隆进pGEM-T Easy载体中,并转化大肠杆菌感受态DH5α,经筛选和测序鉴定获得了阳性重组质粒。从重组质粒中扩增出的成熟肽,大小约123bp。试验为进一步构建重组核蛋白及表达具有广谱抗微生物活性的防御素提供了实验基础。

In this study, the total cellular RNA was purified from chicken liver. According to the similarity gene sequence of birds in GenBank,two pairs of primers were designed.Then a 201bp chicken β-defensin-8（Gal-8） cDNA fragment was amplified by reverse transeriptionpoly-merase chain reaction （RT-PCR）.Analysis of the sequence data showed that the chicken β-defensin gene（the cDNA）fragment code 66 amino acids which comprised of signal peptide with 20 amino acid residues,original fragment with five amino acid and mature peptide with 41 amino acid residues. BLAST analysis showed that there was two bases difference in gene sequence of birds in GENBANK,the homogeneity of nueleotide was 99%,The gene was ligated with plasmid pGEM-T Easy,Then the recombinant plasmid was transformed into E.eoli DH5α,, and the sequence was identified by T/A clone. The mature peptide which was amplified from recombinant plasmid is 123 bp.This study provide an experimental basis for constructing recombinant nuclear protein and expressing defensins with a wide range of antimicrobial activities in the future.