巴马小型猪主要细胞因子的克隆及序列分析

›› 2012, Vol. 39 ›› Issue (11): 56-61.

巴马小型猪主要细胞因子的克隆及序列分析

李连峰¹, 姜骞², 林欢², 李红¹, 刘家森², 郭东春², 原冬伟², 崔玉东¹, 曲连东²

1. 黑龙江八一农垦大学动物科技学院,黑龙江大庆 163319;
2. 中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,黑龙江哈尔滨 150001

收稿日期:2012-04-18 出版日期:2012-11-20 发布日期:2012-11-22
通讯作者: 曲连东(1964-),男,研究员,博士生导师。E-mail:qld@hvri.ac.cn E-mail:qld@hvri.ac.cn
作者简介:李连峰(1986-),男,内蒙古人,硕士生,研究方向:动物传染病。
基金资助:
国际科技合作计划项目(2010DFB33620)。

Cloning and Sequence Analysis of the Main Cytokines in Bama Miniature Pig

LI Lian-feng¹, JIANG Qian², LIN Huan², LI Hong¹, LIU Jia-sen², GUO Dong-chun², YUAN Dong-wei², CUI Yu-dong¹, QU Lian-dong²

1. College of Animal Science and Technology, Heilongjiang August-First Land Reclamation University, Daqing 163319, China;
2. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001,China

Received:2012-04-18 Online:2012-11-20 Published:2012-11-22

摘要/Abstract

摘要： 为了丰富广西巴马小型猪细胞因子生物学数据,本试验采取广西巴马小型猪的外周血,分离淋巴细胞,提取总RNA,利用设计的引物进行RT-PCR扩增,将克隆出的与目的基因大小相符的片段回收,再与T载体连接,转化到大肠杆菌DH5α中,筛选阳性克隆,将重组菌测序后提交NCBI,比较克隆序列与已知序列的同源性,并利用软件对克隆序列进行分析。结果表明,已克隆出的巴马小型猪细胞因子IL-2、IL-12、IFN-γ、TNF-α基因序列长度分别为338、377、380、402 bp,测序结果均与目的基因预期估计值吻合。序列分析结果发现与已知的家猪或野猪的相应基因同源性较高,IL-2、IL-12、IFN-γ、TNF-α序列同源性分别为98.9%、99.5%、99.2%、100%。研究巴马小型猪细胞因子基因序列不仅丰富了生物学数据,而且为细胞因子的功能研究提供了依据,也为细胞因子的定量检测奠定了基础。

关键词: 巴马小型猪; 细胞因子; 克隆; 序列分析

Abstract: In order to enrich the cytokine biological data of Guangxi Bama miniature pig, the lymphocytes of Bama miniature pig was separated from peripheral blood and the total RNA was extracted from the peripheral blood lymphocytes, respectively. The target genes of cytokine were amplified by RT-PCR and cloned into pMD18-T vector and transformed into E. coli DH5α, respectively. We chose positive clones and submitted them to the NCBI after sequencing the recombinant strain. Comparing homology of the cloned sequence with the known sequence, and using the software to analysis the cloned sequence. The results showed that the gene length of cloned cytokines IL-2, IL-12, IFN-γ, TNF-α of Bama miniature pig were 338, 377, 380, 402 bp,respectively. It was consistent with the expected length of target gene. The sequences analysis showed that the homology of the cloned genes was relatively high with known domestic pigs or wild boars. The sequence homology of IL-2, IL-12, IFN-γ,TNF-α were 98.9%, 99.5%, 99.2%, 100%, respectively. The research on cytokine gene sequence of Bama miniature pigs not only enriched the biological data of Bama miniature pigs, but also laid the foundation for research of the cytokine function. It also provided the basis for the quantitative detection of cytokine.

Key words: Bama miniature pig; cytokine; clone; sequence analysis

中图分类号:

Q785

李连峰, 姜骞, 林欢, 李红, 刘家森, 郭东春, 原冬伟, 崔玉东, 曲连东. 巴马小型猪主要细胞因子的克隆及序列分析[J]. , 2012, 39(11): 56-61.

LI Lian-feng, JIANG Qian, LIN Huan, LI Hong, LIU Jia-sen, GUO Dong-chun, YUAN Dong-wei, CUI Yu-dong, QU Lian-dong. Cloning and Sequence Analysis of the Main Cytokines in Bama Miniature Pig[J]. , 2012, 39(11): 56-61.

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