巴马小型猪主要细胞因子的克隆及序列分析

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.%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 DH5a, respectively. We chose positive clones and submitted them to the NCBI after sequencing the recorabinant 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.