Marc145细胞源LSm1基因的巢式PCR扩增及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2017.06.005

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (6): 1604-1612.doi: 10.16431/j.cnki.1671-7236.2017.06.005

Marc145细胞源LSm1基因的巢式PCR扩增及生物信息学分析

陈绍品^1,2, 林汉卿^1,2, 温贵兰^1,2, 张升波^1,2, 李昌红^1,2, 徐丽^1,2, 龚新勇³, 汪德生^1,2, 文明^1,2, 周碧君^1,2, 程振涛^1,2

1. 贵州大学动物科学学院, 贵阳 550025;
2. 贵州省动物疫病与兽医公共卫生重点实验室, 贵阳 550025;
3. 贵州大学大数据与信息工程学院, 贵阳 550025

修回日期:2017-03-27 出版日期:2017-06-20 发布日期:2017-06-28
通讯作者: 温贵兰 E-mail:524340732@qq.com
作者简介:陈绍品(1993-),男,贵州毕节人,硕士生,研究方向:兽医病毒学及免疫学,E-mail:1334177986@qq.com
基金资助:
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）

Nested PCR Amplification and Bioinformatics Analysis of LSm1 Gene from Marc145 Cells

CHEN Shao-pin^1,2, LIN Han-qing^1,2, WEN Gui-lan^1,2, ZHANG Sheng-bo^1,2, LI Chang-hong^1,2, XU Li^1,2, GONG Xin-yong³, WANG De-sheng^1,2, WEN Ming^1,2, ZHOU Bi-jun^1,2, CHENG Zhen-tao^1,2

1. College of Animal Science, Guizhou University, Guiyang 550025, China;
2. Key Laboratory of Animal Disease and Veterinary Public Health of Guizhou Province, Guiyang 550025, China;
3. College of Information and Information Engineering, Guizhou University, Guiyang 550025, China

Revised:2017-03-27 Online:2017-06-20 Published:2017-06-28

摘要/Abstract

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402 bp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。

关键词: LSm1基因; 巢式PCR; 克隆; 生物信息学分析

Abstract:

In order to analyze the character of LSm1 gene and predict its biology function in this study,LSm1 gene from the Marc145 cells was amplified and cloned by nested PCR,and the secondary structure, B cell epitopes,conserved domain, transmembrane domain and signal peptide of LSm1 protein were predicted by bioinformatics softwares. The results showed that LSm1 gene sequence with 402 bp was successfully obtained,which could encode 133 amino acids. The nucleotide sequence of LSm1 gene from Marc145 cells was high similarity with human and primates,followed by marine mammals,terrestrial wild animal and livestock,the percent identity was from 92.8% to 99.8%.The encoding amino acid sequence similarity was from 97.8% to 99.3%. The results of phylogenetic tree showed that the LSm1 gene of Marc145 cells was closely relative with human which were in the same branch,followed by primates. The alpha helix and random coil were dominant in secondary structure ,which was 45.11% and 24.06%,respectively. It was predicted that there were 4 B cell dominant epitopes and a conserved domain of Sm superfamily in the LSm1 protein, and there was no transmembrane region and signal peptide region. In conclusion,the LSm1 protein might has low transcription and expression level in the intracellular,and nested PCR was needed to achieve the target band from cDNA of cells. The method provided a reference for the amplification of LSm1 gene. At the same time,the prediction results of it's biological function would lay a foundation for studying the LSm1 artificial expression protein,the antibody and the functional mechanism of LSm1 at the cellular level.

Key words: LSm1 gene; nested PCR; cloning; bioinformatics analysis

中图分类号:

Q785

陈绍品, 林汉卿, 温贵兰, 张升波, 李昌红, 徐丽, 龚新勇, 汪德生, 文明, 周碧君, 程振涛. Marc145细胞源LSm1基因的巢式PCR扩增及生物信息学分析[J]. 《中国畜牧兽医》, 2017, 44(6): 1604-1612.

CHEN Shao-pin, LIN Han-qing, WEN Gui-lan, ZHANG Sheng-bo, LI Chang-hong, XU Li, GONG Xin-yong, WANG De-sheng, WEN Ming, ZHOU Bi-jun, CHENG Zhen-tao. Nested PCR Amplification and Bioinformatics Analysis of LSm1 Gene from Marc145 Cells[J]. , 2017, 44(6): 1604-1612.

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Marc145细胞源LSm1基因的巢式PCR扩增及生物信息学分析

Nested PCR Amplification and Bioinformatics Analysis of LSm1 Gene from Marc145 Cells

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