《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (6): 1437-1446.doi: 10.16431/j.cnki.1671-7236.2018.06.002

• 生物技术 • 上一篇    下一篇

从江香猪γ干扰素基因克隆与序列分析

温贵兰1,2, 陈绍品2, 张升波2, 林汉卿2, 管国丹2, 王开功2, 文明2, 周碧君2, 程振涛2, 赵德刚1,3   

  1. 1. 贵州大学农业生物工程研究院, 贵州大学生命科学学院, 山地植物资源保护与种质创新省部共建教育部重点实验室, 贵阳 550025;
    2. 贵州大学动物科学学院, 预防兽医实验室, 贵阳 550025;
    3. 贵州省农业科学院, 贵阳 550006
  • 收稿日期:2017-11-29 出版日期:2018-06-20 发布日期:2018-06-15
  • 通讯作者: 赵德刚 E-mail:dgzhao@gzu.edu.cn
  • 作者简介:温贵兰(1977-),女,贵州普定人,博士,副教授,研究方向:微生物与免疫学,E-mail:524340732@qq.com
  • 基金资助:

    国家自然科学基金(31460668);贵州省科学技术基金(黔科合J字(2015)2046号);贵州大学博士基金(贵大人基合字(2013)12号);贵州省高层次创新型人才培养(黔财教[2017]号)

Cloning and Sequence Analysis of Interferon Gamma Gene in Congjiang Xiang Pig

WEN Guilan1,2, CHEN Shaopin2, ZHANG Shengbo2, LIN Hanqing2, GUAN Guodan2, WANG Kaigong2, WEN Ming2, ZHOU Bijun2, CHENG Zhentao2, ZHAO Degang1,3   

  1. 1. Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang 550025, China;
    2. College of Animal Sciences, Preventive Veterinary Laboratory, Guiyang 550025, China;
    3. Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
  • Received:2017-11-29 Online:2018-06-20 Published:2018-06-15

摘要:

试验旨在研究从江香猪γ干扰素(interferon gamma,IFN-γ)基因克隆与序列分析。试验提取贵州从江香猪肝脏组织总RNA,反转录生成cDNA,采用2对特异性引物进行巢式PCR扩增从江香猪IFN-γ(CJ-poIFN-γ)基因编码区,将其克隆至pUCm-T载体上,获得重组质粒pUCm-CJpoIFN-γ,并测序鉴定;利用NCBI、SOPMA、SignalP-4.1等在线服务器软件、DNAStar软件对CJ-poIFN-γ进行序列分析。结果表明,CJ-poIFN-γ基因编码区长501 bp,编码166个氨基酸;核苷酸序列比对结果显示,CJ-poIFN-γ与梅山猪、剑白猪、藏猪、成华猪、荣昌猪、印度猪、长白猪、内江猪同源性为99.4%~100.0%;进化树分析结果显示,CJ-poIFN-γ与梅山猪、剑白猪、藏猪、成华猪亲缘关系较近;CJ-poIFN-γ基因编码蛋白不存在跨膜结构,为分泌蛋白,前23个氨基酸为信号肽序列;CJ-poIFN-γ基因编码蛋白二级结构主要以α-螺旋(50.60%)和无规则卷曲(33.14%)为主,B细胞表位主要位于62-65、84-87、113-115、144-156和162-166位氨基酸。试验结果为进一步研究IFN-γ的生物活性、加快从江香猪品种资源的有效利用奠定基础。

关键词: 从江香猪; γ 干扰素(IFN-γ )基因; 克隆; 序列分析

Abstract:

This experiment was aimed to study the cloning and sequence analysis of interferon gamma (IFN-γ) gene in Congjiang Xiang pig.The total RNA of liver tissue in Congjiang Xiang pig was extracted and reversely transcribed into cDNA.The encoding region of Congjiang Xiang pig IFN-γ gene (CJ-poIFN-γ) was amplified using specific primers by nested PCR,ligated to pUCm-T vector,and verified by sequencing.Sequence analysis of CJ-poIFN-γ by NCBI,SOPMA,SignalP-4.1,DNAStar and so on.The results showed that the coding region of CJ-poIFN-γ gene was 501 bp,encoding 166 amino acids.The results of nucleotide sequence alignment showed that the homology of CJ-poIFN-γ among Congjiang Xiang pig, Meishan pig,Jianbai pig,Tibetan pig,Chenghua pig,Rongchang pig,Indian pig,Landrace pig and Neijiang pig was 99.4% to 100.0%.Phylogenetic tree analysis showed that CJ-poIFN-γ was closely related to Meishan pig,Jianbai pig,Tibetan pig and Chenghua pig;CJ-poIFN-γ gene coding protein was secretory protein,there was no transmembrane structure,and the first 23 amino acids were signal peptide sequences.The secondary structure of CJ-poIFN-γ gene coding protein was mainly α-helix (50.60%) and random coil (33.14%).The B cell epitopes were mainly located in 62-65,84-87,113-115,144-156 and 162-166 amino acids.The results laid a foundation for further study on biological activity of IFN-γ and speeding up the effective utilization of the resources in Congjiang Xiang pig.

Key words: Congjiang Xiang pig; IFN-γ gene; cloning; sequence analysis

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