Marc145细胞源LSm1基因的巢式PCR扩增及生物信息学分析

发布时间：2018-04-24 01:24

  本文选题：LSm基因 + 巢式PCR　； 参考：《中国畜牧兽医》2017年06期

【摘要】：为分析LSm1基因的特点,预测其编码蛋白的生物学功能,试验利用巢式PCR方法对Marc145细胞源LSm1基因进行扩增、克隆及序列测定,应用生物信息学方法对Marc145细胞源LSm1基因进行序列分析,并对其编码蛋白进行二级结构、B细胞表位、保守结构域、跨膜结构域和信号肽预测。结果显示,经过两轮的PCR扩增,成功获得402bp的Marc145细胞源LSm1基因,编码133个氨基酸。Marc145细胞源LSm1基因核苷酸序列与人类、灵长类同源性较高,其次是海洋哺乳动物类,最后是陆地野生动物及家畜,同源性为92.8%~99.8%,而其编码的氨基酸虽没有此规律,但氨基酸序列同源性均较高,为97.8%~99.3%。系统进化树结果显示,Marc145细胞源LSm1基因与人类LSm1基因亲缘关系较近,处在同一分支,其次是灵长类。LSm1蛋白二级结构中α-螺旋和无规则卷曲所占比例较大,分别为45.11%和24.06%。预测此蛋白存在4个B细胞优势抗原表位,具有Sm超家族保守结构域,无跨膜区域,无信号肽区域。结果表明,LSm1蛋白在细胞内转录及表达水平可能较低,细胞cDNA需通过巢式PCR扩增两轮才能出现目的条带,本试验方法为LSm1基因的扩增提供了参考。同时,LSm1生物学功能预测结果为获得LSm1人工表达蛋白与针对LSm1蛋白的特异性抗体制备,以及在细胞水平研究LSm1的功能机制及其在病毒复制中的作用奠定基础。
[Abstract]:In order to analyze the characteristics of LSm1 gene and predict the biological function of its encoded protein, the LSm1 gene derived from Marc145 cells was amplified, cloned and sequenced by nested PCR method. Marc145 cell-derived LSm1 gene was sequenced by bioinformatics, and its encoded protein was predicted by secondary structure B cell epitope, conserved domain, transmembrane domain and signal peptide. The results showed that after two rounds of PCR amplification, the Marc145 cell-derived LSm1 gene of 402bp was successfully obtained. The nucleotide sequence of the LSm1 gene encoding 133 amino acids. Marc145 cells was highly homologous to human and primates, followed by marine mammals. Finally, wild animals and domestic animals on land, the homology was 92.8% and 99.8%, while the amino acids encoded by them had no such rule, but the amino acid sequence homology was 97.8% (99.3%), and the amino acid sequence was 97.8% (99.3%). Phylogenetic tree results showed that LSm1 gene derived from Marc145 cells was closely related to human LSm1 gene and was in the same branch, followed by 伪 -helix and irregular curl in the secondary structure of primate .LSm1 protein, which were 45.11% and 24.06%, respectively. It was predicted that the protein had four B cell epitopes with Sm superfamily conserved domain, no transmembrane domain and no signal peptide region. The results showed that the transcriptional and expression level of LSm1 protein in cells may be low, and the target bands of cDNA could be obtained by nested PCR amplification. This method provides a reference for the amplification of LSm1 gene. At the same time, the prediction of the biological function of LSm1 lays a foundation for the preparation of LSm1 artificially expressed protein and the specific antibody against LSm1 protein, and for the study of the functional mechanism of LSm1 and its role in virus replication at the cellular level.
【作者单位】： 贵州大学动物科学学院;贵州省动物疫病与兽医公共卫生重点实验室;贵州大学大数据与信息工程学院;
【基金】：国家自然科学基金:P-body在PRRSV复制和持续感染过程中的作用机制研究(国科金计项[2014]44) 贵州省科学技术基金:NSP2亚类在PRRSV复制过程中的作用机制研究(黔科合J字[2015]2046号)
【分类号】：Q78;Q811.4
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本文编号：1794519