RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA

doi:10.16431/j.cnki.1671-7236.2017.03.003

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (3): 635-643.doi: 10.16431/j.cnki.1671-7236.2017.03.003

RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA

唐娟^1,2, 金鑫², 张曼², 侯永跃¹, 李军燕¹, 田巧珍², 刘骄², 王云鹤², 杨银凤²

1. 内蒙古农牧业科学院兽医研究所, 呼和浩特 010031;
2. 内蒙古农业大学兽医学院, 呼和浩特 010018

收稿日期:2016-09-26 出版日期:2017-03-20 发布日期:2017-03-21
通讯作者: 杨银凤 E-mail:julie1963@163.com
作者简介:唐娟(1965-),女,内蒙古呼和浩特人,学士,副研究员,研究方向:中兽药的研发,E-mail:tangjuan12002@163.com;金鑫(1990-),男,内蒙古鄂尔多斯人,博士生,研究方向:动物解剖及反刍动物黏膜免疫,E-mail:jinxinnndsyxy@163.com
基金资助:
内蒙古自然科学基金（2013MS0408）

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

TANG Juan^1,2, JIN Xin², ZHANG Man², HOU Yong-yue¹, LI Jun-yan¹, TIAN Qiao-zhen², LIU Jiao², WANG Yun-he², YANG Yin-feng²

1. Academy of Agriculture and Animal Husbandry in Inner Mongolia, Hohhot 010031, China;
2. College of Veterinary Medicine, Inner Mongolia Agriculture University, Hohhot 010018, China

Received:2016-09-26 Online:2017-03-20 Published:2017-03-21

摘要/Abstract

摘要：

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。

关键词: 梅花鹿; 5'-RACE; 3'-RACE; β-防御素-1

Abstract:

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.

Key words: sika deer; 5'-RACE; 3'-RACE; β-defensin-1 (siBD-1)

中图分类号:

S865.4⁺2

唐娟, 金鑫, 张曼, 侯永跃, 李军燕, 田巧珍, 刘骄, 王云鹤, 杨银凤. RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA[J]. 《中国畜牧兽医》, 2017, 44(3): 635-643.

TANG Juan, JIN Xin, ZHANG Man, HOU Yong-yue, LI Jun-yan, TIAN Qiao-zhen, LIU Jiao, WANG Yun-he, YANG Yin-feng. Amplification of the Full-length cDNA of Sika β-defensin-1(siBD-1) with RACE Method[J]. , 2017, 44(3): 635-643.

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RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA

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

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