摘要
Objective:Toachieveanoptimizedmethodforsolubleexpressionofhumancarboxylesterase1(hCE-1)inescherichiacoilandpurificationbyNi2+-NTAagaroseaffinitychromatography,togetimprovedproteinyieldandpurityforfurtherdevelopmentofhepatocellularcarcinoma(HCC)diagnosisELISAkits.Methods:ThebestantigenepitopesofhCE1werepredictedbycomparingsecondarystructure,flexibleregions,hydrophilicity,antigenicindexsurfaceprobabilityofresidues.Afterwards,pET-42a(+)withaHis-tagandaGST-tagwasappliedtoformrecombinantplasmidpET-42a(+)/hCE1,whichfacilitatedpurificationwhenusingNi2+-NTAagaroseaffinitychromatography.ProteinqualitywasmeasuredbySDS-PAGEandBCAproteinassay.Western-blotidentificationwasalsoperformedtoensurethecorrectexpressionofhCE1protein.Results:Theresiduesfrom500to567nearC-terminalofhCE1proteinwereconsideredthebestepitopeswhichexhibitedhighhydrophilicityandhighsurfaceprobabilityandrelativelyflexiblesecondarystructureandlowhomologycomparedwithhCE2andhCE3.His-hCE1500-567fusionproteinwasachievedbyIPTG-inductedexpressionwithanexpectedmassof42kDa.Afterpurification,thefinalproductwasspeciallyidentified,whichreachedover95%purityandmorethan10mg/Lofmicrobialculture.InWesternblot,thepurifiedfusionproteinwasrecognizedbyanti-hCE1monoclonalantibody,alongwithprevioussequencingvalidation,whichdemonstratedthecorrectpreparationofsolublehCE1protein.Conclusion:ThisisanefficaciousandaffordablestrategytogeneratefusionhCE1ofhighqualityinEcoli,whichfacilitatespreparationofhCE1monoclonalantibodyandfurtherHCCdiagnosisresearch.
出版日期
2014年01月11日（中国期刊网平台首次上网日期，不代表论文的发表时间）
