中华鳖IGFBP1基因克隆及表达分析.docx

中华鳖IGFBP1基因克隆及表达分析
摘要：IGFBP1基因是哺乳动物胎盘中独特的基因，它起到了重要的生理功能，在疾病的发生和发展中也有重要作用。本研究通过PCR扩增获得中华鳖的IGFBP1基因全长序列，利用生物软件对序列进行分析，预测IGFBP1蛋白质的结构和功能区域，并进一步对其组织表达特点进行了实验验证。结果表明，IGFBP1在中华鳖中的表达具有组织特异性，以肝脏和肾脏为主。该研究为进一步研究IGFBP1的生物功能和发挥其在医药领域的应用提供了基础数据。
关键词：中华鳖，IGFBP1基因，克隆，表达
Introduction
IGFBP1(Insulin-likegrowthfactorbindingprotein1)isauniquegeneinmammalianplacenta,whichplaysanimportantphysiologicalroleandalsohassignificanteffectintheoccurrenceanddevelopmentofdiseases.Inthisstudy,weaimedtocloneandanalyzetheexpressioncharacteristicsofIGFBP1geneinCuoraflavomarginatatoprovideabasicdataforfurtherstudyofitsbiologicalfunctionandapplicationinthefieldofmedicine.
MaterialsandMethods
PCRamplification:TotalRNAwasextractedfromlivertissueofCuoraflavomarginata,andone-stepRT-PCRkitwasusedtosynthesizethecDNA.PrimersofIGFBP1geneweredesignedandsynthesized,thenPCRwasperformedusingcDNAastemplate,andthePCRproductwaspurifiedbygelextraction.
Bioinformaticsanalysis:Thefull-lengthsequenceofIGFBP1genewasanalyzedbyBlastinNCBI,andtheopenreadingframewasidentified.HomologyanalysisandsequencealignmentofIGFBP1proteinindifferentspecieswereperformedbyClustalWsoftware.Three-dimensionalstructureandfunctionpredictionofIGFBP1wereconductedusingSwiss-ModelandSMARTsoftware,respectively.
Real-timePCR:TheexpressionlevelsofIGFBP1indifferenttissuesofCuoraflavomarginatawereanalyzedusingSYBRGreenReal-timePCRsystem.Actinwasusedashousekeepinggene,andtherelativeexpressionwascalculatedby2-ΔΔCtmethod.
Results
PCRamplification:ThefulllengthofIGFBP1geneinCuoraflavomarginatawassuccessfullyamplified,andthesequencewasconsistentwiththatinNCBI.
Bioinformaticsanalysis:ThenucleotidesequenceofIGFBP1geneinCuoraflavomarginataconsistedof1655bp,andtheopenreadingframewas1260bpencoding419aminoacids.SequenceanalysisindicatedthattheIGFBP1proteininCuoraflavomarginatawashighlyconservedamongdifferentspecies.Thethree-dimensionalmodelindicatedthatIGFBP1proteininCuoraflavomarginatacontainedanN-terminalandaC-terminaldomainwhichwereresponsibleforthebindingwithI