广西巴马小型猪SP1基因克隆测序及其真核表达载体的构建

【目的】克隆广西巴马小型猪特异性蛋白1(SP1)基因,并构建其真核表达载体,为揭示其对猪生长发育的调控机理打下基础。【方法】利用RT-PCR从广西巴马小型猪背最长肌组织中扩增SP1基因,采用DNASTAR、TMHMM等分别进行基因生物信息学分析;以pEGFP-N1质粒构建真核表达载体pEGFP-N1-SP1并转染293T细胞,于转染48h后在倒置荧光显微镜下观察并拍照。【结果】克隆获得的广西巴马小型猪SP1基因与GenBank中野猪(Susscrofa)的参考序列(XM_005652569.3)一致,未发现碱基突变位点;其编码序列(CDS)全长2340bp,编码779个氨基酸。广西巴马小型猪S...

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Bibliographic Details
Published in南方农业学报 Vol. 49; no. 2; pp. 360 - 366
Main Author 张广杰;崔悦悦;邱庆庆;夏琴;李龙;司景磊;夏攀杰;邹辉;兰干球
Format Journal Article
LanguageChinese
Published 广西大学 动物科学技术学院,南宁,530004 2018
Subjects
广西巴马小型猪;SP1基因;克隆;真核表达载体;生物学信息分析
克隆
生物学信息分析
eukaryotic expression vector
clone
广西巴马小型猪
Guangxi Bama mini pig
SP1基因
gene SP1
真核表达载体
bioinformatics analysis
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Summary:【目的】克隆广西巴马小型猪特异性蛋白1(SP1)基因,并构建其真核表达载体,为揭示其对猪生长发育的调控机理打下基础。【方法】利用RT-PCR从广西巴马小型猪背最长肌组织中扩增SP1基因,采用DNASTAR、TMHMM等分别进行基因生物信息学分析;以pEGFP-N1质粒构建真核表达载体pEGFP-N1-SP1并转染293T细胞,于转染48h后在倒置荧光显微镜下观察并拍照。【结果】克隆获得的广西巴马小型猪SP1基因与GenBank中野猪(Susscrofa)的参考序列(XM_005652569.3)一致,未发现碱基突变位点;其编码序列(CDS)全长2340bp,编码779个氨基酸。广西巴马小型猪SP1蛋白不存在跨膜结构,也不存在信号肽,不属于分泌型蛋白;其二级结构中α-螺旋占17.84%,延伸链占21.31%,β-转角占9.76%,无规则卷曲占51.09%。构建的真核表达载体pEGFP-N1-SP1能成功转染至293T细胞中并表达出绿色荧光蛋白。【结论】广西巴马小型猪SP1基因CDS序列编码蛋白主要参与细胞内活动,而非外分泌型蛋白,以SP1基因构建的真核表达载体pEGFP-N1-SP1能转染293T细胞并成功表达,可直接用于后期SP1基因功能探索及干扰表达等研究。
Bibliography:45-1381/S
Guangxi Bama mini pig;gene SP1;clone;eukaryotic expression vector;bioinformatics analysis
Objective】The purpose of this study was to clone specificity protein1(SP1)gene of Guangxi Bama mini pig,construct itseukaryotic expression vector and lay a foundation for revealing its regulation mechanism to pig growth.【Method】Gene SP1was amplified from longissimus dorsi of Guangxi Bama mini pig by RT-PCR.Bioinformatics analysis on the gene was conducted using DNASTAR and TMHMM.Eukaryotic expression vector pEGFP-N1-SP1was constructed by plasmid pEGFP-N1and was transfected into293T cell.After48h of transfection,it was observed and taken pictures under inverted fluorescence microscope.【Result】The cloned Guangxi Bama mini pig SP1gene sequence was consistent with wild boar reference sequence(XM_005652569.3)in GenBank(Sus scrofa),and no base mutation site was found.The coding sequence(CDS)was2340bp in length,coding779amino acids.SP1protein of Guangxi Bama mini pig contained no transmembrane structure and signal pepti
ISSN:2095-1191
DOI:10.3969/j.issn.2095-1191.2018.02.24