猪BST-2全基因克隆、真核表达载体构建及组织表达谱分析
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- 英文论文题名：Gene clone,eukaryotic expression vector and tissue expression profile analysis of porcine BST-2 gene
- 论文作者：孔宁 ; 吴永光 ; 孟琼 ; 王忠泽 ; 童武 ; 郑浩 ; 李国新 ; 单同领 ; 周恩民 ; 童光志
- 英文论文作者：KONG Ning ; WU Yong-guang ; MENG Qiong ; WANG Zhong-ze ; ZUO Ye-wen ; TONG Wu ; ZHENG Hao ; LI Guoxin ; SHAN Tong-ling ; ZHOU En-min ; TONG Guang-zhi ; Department of Preventive Veterinary Medicine ; College of Veterinary Medicine ; Northwest A&F University ; Department of Swine Infectious Disease ; Shanghai Veterinary Research Institute ; Chinese Academy of Agricultural Sciences ; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses
- 年：2016
- 作者机构：西北农林科技大学动物医学院;中国农业科学院上海兽医研究所;江苏高校动物重要疫病与人兽共患病防控协同创新中心;
- 论文关键词：猪BST-2基因 ; 蛋白结构与功能 ; 组织表达谱
- 英文论文关键词：pig BST-2 gene ; structure and function of protein ; tissue expression profile
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S828
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：8
- 文件大小：2116k
- 原文格式：D
- 会议级别：全国
摘要
为研究猪BST-2基因的生物学功能,用特异性的引物扩增猪BST-2基因,并利用生物信息学软件对猪BST-2基因及氨基酸进行分子特性分析,同时进行了猪BST-2蛋白的真核表达及组织表达谱分析。结果表明:猪BST-2基因全长851 bp,其中5’-UTR为23 bp,3’-UTR为294 bp,CDS区为534 bp,编码177个氨基酸,猪源BST-2蛋白氨基酸序列与大猩猩、仓鼠、家鼠、驴、猫、牛、猕猴、绵羊、人BST-2蛋白氨基酸序列同源性分别为46.1%,41.7%,39.5%,35.4%,42.0%,40.5%,44.4%,38.7%,46.8%。含有两个跨膜结构(27–49 aa和154–176 aa),两个潜在的糖基化位点,14个潜在的磷酸化位点,包含磷酸激酶ATM、CKⅡ、PKA、PKC的结合位点。构建真核质粒并转染发现猪BST-2蛋白能够在Vero细胞内正确表达。半定量PCR检测发现BST-2基因在所有组织中都有表达,尤其是在免疫组织及器官(淋巴结、胸腺、扁桃体、脾)、大肠、小肠中的表达量较高。本研究为今后进一步分析验证猪BST-2蛋白的抗病毒机制奠定了基础。
In order to study the biological function of pig BST-2 gene,the BST-2 gene was amplified with specific primers from porcine kidney tissue,and molecular characterization of BST-2 nuclectide and amino acid sequence were analyzed with bioinformatics tools and online server.Then the prokaryotic expression and tissue expression profile analysis was carried out.The results showed that the full length of pig BST-2 gene was 851 bp and contained 23 bp of 5'-UTR,294 bp of 3'-UTR and 534 bp of CDS and the gene encoded 177 aa.Sequence analysis showed 46.1 % identity with gorilla gorilla,41.7 % with cricetulus griseus,39.5 % with mus musculus,35.4 % with equus asinus,42.0 % with felis catus,40.5 % with bos mutus,44.4 % with macaca mulatta,38.7 %with ovis aries,46.8 % with homo sapiens.BST-2 protein contain 2 transmembrane structure(27 – 49 aa and 154 –176 aa),2 glycosylation sites and 14 potential phosphorylation sites including ATM、CKⅡ、PKA、PKC binding sites.The pig BST-2 protein was expressed in Vero cells after translated the recombinant plasmid FLAG-BST-2.Semi-quantitative PCR results showed that BST-2 gene expressed in all the tissues,especially in lymph nodes,thymus,tonsils,spleen,large intestine and small intestine.This study provide a foundation for further studying the antiviral mechanism of pig BST-2 protein.
In order to study the biological function of pig BST-2 gene,the BST-2 gene was amplified with specific primers from porcine kidney tissue,and molecular characterization of BST-2 nuclectide and amino acid sequence were analyzed with bioinformatics tools and online server.Then the prokaryotic expression and tissue expression profile analysis was carried out.The results showed that the full length of pig BST-2 gene was 851 bp and contained 23 bp of 5'-UTR,294 bp of 3'-UTR and 534 bp of CDS and the gene encoded 177 aa.Sequence analysis showed 46.1 % identity with gorilla gorilla,41.7 % with cricetulus griseus,39.5 % with mus musculus,35.4 % with equus asinus,42.0 % with felis catus,40.5 % with bos mutus,44.4 % with macaca mulatta,38.7 %with ovis aries,46.8 % with homo sapiens.BST-2 protein contain 2 transmembrane structure(27 – 49 aa and 154 –176 aa),2 glycosylation sites and 14 potential phosphorylation sites including ATM、CKⅡ、PKA、PKC binding sites.The pig BST-2 protein was expressed in Vero cells after translated the recombinant plasmid FLAG-BST-2.Semi-quantitative PCR results showed that BST-2 gene expressed in all the tissues,especially in lymph nodes,thymus,tonsils,spleen,large intestine and small intestine.This study provide a foundation for further studying the antiviral mechanism of pig BST-2 protein.
引文
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[2]Goto T,Kennel SJ,Abe M,et al.A novel membrane antigen selectively expressed on terminally differentiated human B cells[J].Blood,1994,84:1922-1930.
[3]Rollason R,Korolchuk V,Hamilton C,et al.Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif[J].J Cell Sci,2007,120:3850-3858.
[4]Ozaki S,Kosaka M,Wakatsuki S,et al.Immunotherapy of multiple myeloma with a monoclonal antibody directed against a plasma cell-specific antigen,HM1.24[J].Blood,1997,90:3179-3186.
[5]Neil SJ,Zang T,Bieniasz PD Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu[J].Nature,2008,451:425-430.
[6]Zhang F,Landford WN,Ng M,et al.SIV Nef proteins recruit the AP-2 complex to antagonize Tetherin and facilitate virion release[J].PLo S Pathog,2011,7:e1002039.
[7]Sakuma T,Noda T,Urata S,et al.Inhibition of Lassa and Marburg virus production by tetherin[J].J Virol,2009,83:2382-2385.
[8]Kaletsky RL,Francica JR,Agrawal-Gamse C,et al.Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein[J].Proc Natl Acad Sci U S A,2009,106:2886-2891.
[9]Yan R,Zhao X,Cai D,et al.The Interferon-Inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion[J].J Virol,2015,89:9200-9212.
[10]Lv M,Zhang B,Shi Y,et al.Identification of BST-2/tetherin-induced hepatitis B virus restriction and hepatocyte-specific BST-2 inactivation[J].Sci Rep,2015,5:11736.
[11]Galao RP,Le Tortorec A,Pickering S,et al.Innate sensing of HIV-1 assembly by Tetherin induces NFkappa B-dependent proinflammatory responses[J].Cell Host Microbe,2012,12:633-644.
[12]Tokarev A,Suarez M,Kwan W,et al.Stimulation of NF-kappa B activity by the HIV restriction factor BST2[J].J Virol,2013,87:2046-2057.
[13]Bond AE,Row PE,Dudley E Post-translation modification of proteins;methodologies and applications in plant sciences[J].Phytochemistry,2011,72:975-996.
[14]Cobos Jimenez V,Booiman T,de Taeye SW,et al.Differential expression of HIV-1 interfering factors in monocyte-derived macrophages stimulated with polarizing cytokines or interferons[J].Sci Rep,2012,2:763.
[15]Liu MQ,Zhou DJ,Wang X,et al.IFN-lambda3 inhibits HIV infection of macrophages through the JAK-STAT pathway[J].PLo S One,2012,7:e35902.
[16]Amet T,Byrd D,Hu N,et al.BST-2 expression in human hepatocytes is inducible by all three types of interferons and restricts production of hepatitis C virus[J].Curr Mol Med,2014,14:349-360.
[17]Neil SJ,Eastman SW,Jouvenet N,et al.HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane[J].PLo S Pathog,2006,2:e39.