RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA

为了获得梅花鹿β-防御素-1（sika deer β-defensin-1，siBD-1）cDNA全序列，本试验以梅花鹿舌黏膜组织内提取的总RNA为模板，根据前期已获得的siBD-1 cDNA的已知部分序列设计引物，采用5'-RACE和3'-RACE技术分别扩增5'-和3'-末端序列，将此扩增产物克隆入pMD18-T载体，进行PCR、双酶切鉴定及序列测定与分析。结果表明，成功克隆出长度约为172和299 bp的siBD-1 cDNA 5'-和3'-末端序列，从而得到418 bp的siBD-1 cDNA全序列（GenBank登录号：HM588696.1），其中包含89 bp 5'-非翻译区（UTR）、192 bp的开放阅读框（ORF）、终止密码子TAA、118 bp的3'-UTR和Poly（A）16。同源性比对结果显示，siBD-1 cDNA与水牛的肠防御素（BEBD）同源性最高，为90.6%，与牛（EBD、LAP、TAP、BNBD-4）、山羊（GBD-1、GBD-2）、驯鹿（reBD-1）、绵羊（sBD-1、sBD-2）和骆驼（caBD-1）的防御素cDNA的同源性较高，分别为83.2%、83.1%、87.3%、87.0%、87.5%、87.5%、84.4%、79.9%、77.1%和70.5%；与马（hoBD-1）和猪（pBD-1）的同源性较低，为60.3%和72.4%；而与人（hBD-2）的同源性最低，为16.0%。siBD-1成熟肽由38个氨基酸残基组成，其中包含9个带正电荷的氨基酸残基。

Amplification of the Full-length cDNA of Sika β-defensin-1(siBD-1) with RACE Method

In order to obtain the full-length cDNA of sika β-defensin-1 (siBD-1),5'-RACE and 3'-RACE primers were designed according to the partial sequences of siBD-1 that obtained from this study group, cDNA ends (RACE) technology were used to amplify siBD-1 cDNA from the total RNA of tongue mucosa tissue in sika, and amplified products were cloned into pMD18-T vector and subjected to PCR, restriction endonuclease digestion and sequencing. The results indicated that the length of siBD-1 cDNA 5'- and 3'- end were about 172 and 299 bp. The full-length siBD-1 cDNA was 418 bp (GenBank accession No. HM588696.1) and includes an 5'-untranslated region (UTR) of 89 bp, a open reading frame (ORF) of 192 bp, a stop codon of TAA, 3'-UTR of 118 bp and Ploy(A)16. The sequence homology showed that siBD-1 shared the greatest identity (90.6%) with buffalo enteric β-defensin, the higher identity with cattle (EBD, LAP, TAP, BNBD-4), goats (GBD-1, GBD-2), reindeer (reBD-1), sheep (sBD-1, sBD-2) and camel (caBD-1), that were 83.2%, 83.1%, 87.3%, 87.0%, 87.5%, 87.5%, 84.4%, 79.9%, 77.1% and 70.5%,respectively,and correspondingly low identity were 60.3% and 72.4% with horse (hoBD-1), pig(pBD-1), the lowest was 16.0% with human (hBD-2). The mature peptide was consisted of 38 amino acids, and contained 9 positively charged residues.