文章摘要
钱云霞,杨孙孝,梁洪,钱伦,钱凯先.鲈PPARγ基因的克隆、组织表达及其抗体制备[J].水产学报,2010,34(8):1156~1164
PPARγ基因的克隆、组织表达及其抗体制备
Gene cloning,tissue expression and preparation of monoclonal antibody of Lateolabrax japonicus PPARγ
投稿时间:2010-05-06  修订日期:2010-06-04
DOI:10.3724/SP.J.1231.2010.06954
中文关键词:   过氧化物酶体增殖剂激活受体γ  组织表达  抗体
英文关键词: Japanese seaperch(Lateolabrax japonicus)  peroxisome proliferator activated receptor γ(PPARγ)  tissue expression  antibody
基金项目:国家自然科学基金项目(30671608);浙江省自然科学基金项目(M303345);宁波市自然科学基金项目(2006A610088)
作者单位E-mail
钱云霞 浙江大学生命科学学院 qianyunxia@nbu.edu.cn 
杨孙孝 宁波大学生命科学与生物技术学院  
梁洪 宁波大学生命科学与生物技术学院  
钱伦 宁波大学生命科学与生物技术学院  
钱凯先 浙江大学生命科学学院  
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中文摘要:
      根据GenBank上其他物种的PPARγ基因序列设计兼并引物,从鲈肝脏cDNA中扩增得到鲈PPARγ基因cDNA序列1 588 bp,分析表明该基因的开放阅读框为1 569 bp,编码522个氨基酸,理论等电点6.06,分子量59.02 ku。将鲈PPARγ氨基酸序列比对后发现与欧洲鲈同源性最高,为93.1%;与金头鲷的同源性为92.3%,与人同源性也达到为61.8%。用RT-PCR分析该基因组织表达模式,结果表明,鲈PPARγ主要分布于肝脏、鳃和脂肪组织。将鲈PPARγ开放阅读框1 569 bp序列克隆至原核表达载体pET-28a(+)构成pET-28a-PPARγ1569重组体,并转化大肠杆菌BL21(DE3),以终浓度1 mmol/L的IPTG对其进行诱导表达4 h,SDS-PAGE电泳分析表明,pET-28a-PPARγ1569菌株在66 ku 处有1条特异的蛋白带,Western-blotting 检测表明该蛋白为鲈PPARγ融合蛋白。用镍离子亲和柱纯化的鲈PPARγ融合蛋白免疫小鼠得到其多克隆抗体。用间接ELISA法检测鲈PPARγ抗体的抗体效价约为1∶16 000。实验结果为进一步研究鲈PPARγ蛋白的生物学特性及功能奠定了基础。
英文摘要:
      Peroxisome proliferator activated receptors(PPARs)are a family of ligand activated nuclear transcription factors that play pivotal roles in lipid and energy homeostasis.A cDNA of 1 688 bp encoding PPARγ was isolated from liver total RNA of Japanese Seaperch(Lateolabrax japonicus)using RT-PCR by degenerate primers based on sequence of other animals published on GenBank.The obtained 1 688 bp Seaperch PPARγ included 1 569 bp open reading frame encoding a protein of 522 amino acid residues with a theoretical pI of 6.06 and molecular weight of 59.02 ku.The blast analysis indicated that the deduced amino acid sequence of Seaperch PPARγ shared the highest identity of 93.1% with Dicentrarchus labrax,92.3% with Sparus aurata and 61.8% with Homo sapiens. RT-PCR analysis showed that the Seaperch PPARγ was ubiquitously expressed in 9 tissues tested,with the highest expression in liver,gill and lipid tissues,followed by spleen,brain,intestine and kidney,the lowest in heart and muscle.The cDNA sequence of PPARγ open reading frame was cloned into a prokaryotic expression vector pET-28a(+).The recombinant plasmid pET-28a-PPARγ1569 was transfected into Escherichia coli.BL21(DE3)pLysS and the PPARγ expression was induced at 30 ℃ by addition of 1 mmol/L isopropyl β d thiogalactopyranoside.After 4 h induction with IPTG,the expressed recombinant protein with an apparent molecular mass of 66 ku was found by SDS-PAGE and confirmed by Western-blotting using an antibody specific to 6 His tag.The expressed PPARγ protein was insoluble and present in the inclusion bodies.These inclusion bodies were solubilized by 6 mol/L guanidine hydrochloride and purified on a Ni2+ affinity column(Ni2+ His binding column).After purification,the recombinant PPARγ was used to immunize mice and the specific polyclonal antibody was obtained.Indirect ELISA(enzyme linked immunosorbent assay)was established to test the titer of the polyclonal antibody,the result was positive and the titer reached 16 000.Our research serves as a basis for further research into fish PPARγ’s biological characterization and function.
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