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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| Published in | 南方农业学报 Vol. 49; no. 2; pp. 360 - 366 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
广西大学 动物科学技术学院,南宁,530004
2018
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| Subjects | |
| Online Access | Get full text |
| ISSN | 2095-1191 |
| DOI | 10.3969/j.issn.2095-1191.2018.02.24 |
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| Abstract | 【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。 |
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| AbstractList | 【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。 S828.89; [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48 h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(Sus scrofa)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2340 bp,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. |
| Abstract_FL | [Objective]The purpose of this study was to clone specificity protein 1(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 SP1 was 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-SP1 was construct-ed by plasmid pEGFP-N1 and was transfected into 293T cell.After 48 h of transfection,it was observed and taken pic-tures under inverted fluorescence microscope.[Result]The cloned Guangxi Bama mini pig SP1 gene sequence was consis-tent with wild boar reference sequence(XM_005652569.3)in GenBank(Sus scrofa),and no base mutation site was found.The coding sequence(CDS)was 2340 bp in length,coding 779 amino acids.SP1 protein of Guangxi Bama mini pig contained no transmembrane structure and signal peptide,and was not secretory protein.In its secomdary structure, there were 17.84% α-helix,21.31% extension strand,9.76% β-turn and 51.09% random coil.Green fluorescence protein has been found in 293T cells with transfected eukaryotic expression vector pEGFP-N1-SP1.[Conclusion]The encoded protein of gene SP1 CDS sequence in Guangxi Bama mini pig is mainly involved in intracellular activity,but it is not exo-crine protein.The eukaryotic expression vector pEGFP-N1-SP1 which is constructed based on gene SP1 is transfected into 293T cells and successfully expressed in it. It can be used in further research in function and interference expression of gene SP1. |
| 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 |
| AuthorAffiliation | 广西大学动物科学技术学院,南宁530004 |
| AuthorAffiliation_xml | – name: 广西大学 动物科学技术学院,南宁,530004 |
| Author_FL | CUI Yue-yue ZOU Hui LAN Gan-qiu SI Jing-lei QIU Qing-qing XIA Pan-jie ZHANG Guang-jie LI Long XIA Qin |
| Author_FL_xml | – sequence: 1 fullname: ZHANG Guang-jie – sequence: 2 fullname: CUI Yue-yue – sequence: 3 fullname: QIU Qing-qing – sequence: 4 fullname: XIA Qin – sequence: 5 fullname: LI Long – sequence: 6 fullname: SI Jing-lei – sequence: 7 fullname: XIA Pan-jie – sequence: 8 fullname: ZOU Hui – sequence: 9 fullname: LAN Gan-qiu |
| Author_xml | – sequence: 1 fullname: 张广杰;崔悦悦;邱庆庆;夏琴;李龙;司景磊;夏攀杰;邹辉;兰干球 |
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| ClassificationCodes | S828.89 |
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| Copyright | Copyright © Wanfang Data Co. Ltd. All Rights Reserved. |
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| DOI | 10.3969/j.issn.2095-1191.2018.02.24 |
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| DocumentTitleAlternate | Clone sequencing of gene SP1in Guangxi Bama mini pig and construction of its eukaryotic expression vector |
| DocumentTitle_FL | Clone sequencing of gene SP1 in Guangxi Bama mini pig and construction of its eukaryotic expression vector |
| EndPage | 366 |
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| Keywords | 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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| Notes | 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 |
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| PublicationTitle | 南方农业学报 |
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| Publisher | 广西大学 动物科学技术学院,南宁,530004 |
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| SubjectTerms | 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 |
| Title | 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 |
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