简介：目的:设计合成新型2-喹诺酮类Polo样激酶1(Plk1)抑制剂。方法:以Plk1抑制剂ON01910为先导化合物,利用生物电子等排原理设计一系列2-喹诺酮类衍生物,用Autodock软件将该类化合物与Plk1进行分子对接和虚拟筛选,计算结合自由能;以取代的氯(溴)苄为起始原料,先后经巯基乙酸取代、双氧水氧化、与(对甲氧基)苯胺酰化,再经环合、水解制得目标化合物。结果:设计的化合物大多数与Plk1的结合自由能均比ON01910的低,结合强度高、稳定性好;合成了16个2-喹诺酮类衍生物,产物结构经1H-NMR确证。结论:所得化合物中有15个为新化合物,化合物的结构设计科学合理,虚拟筛选结果良好,为后续实体筛选和化合物结构优化提供了理论依据和参考。

简介：目的:利用慢病毒载体建立稳定表达猪载脂蛋白BmRNA编辑酶催化多肽样蛋白3F(pAPOBEC3F,简称pA3F)的PK15细胞系。方法:以实验室前期构建的pBPLV-flag-pA3F质粒为模板,PCR扩增pA3F基因,克隆到pLenti-Puro-3Flag载体构建成pLenti-Puro-pA3F-3Flag质粒,Western印迹鉴定其在HEK293T细胞中的表达;将pLenti-Puro-pA3F-3Flag质粒与包装载体共转染HEK293T细胞,包装成Lenti-pA3F慢病毒并测定病毒滴度;将Lenti-pA3F慢病毒感染PK15细胞,通过嘌呤霉素压力筛选并结合有限稀释法,筛选稳定表达pA3F的细胞单克隆株,最终Western印迹检测细胞单克隆株中pA3F的表达。结果:菌落PCR和序列测定表明pLenti-Puro-pA3F-3Flag质粒构建正确,Western印迹显示该质粒可在HEK293T细胞中表达pA3F蛋白;包装了Lenti-pA3F慢病毒,滴度为1.32×10^8TU/mL,慢病毒感染PK15细胞后可检测到pA3F蛋白的表达;经Western印迹鉴定,筛选得到的单克隆细胞可稳定表达pA3F蛋白。结论:建立了稳定表达pA3F的PK15细胞单克隆株,为进一步深入研究猪内源性反转录病毒在pA3F作用下的免疫逃逸机制奠定了基础。

简介：【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77d,但差异不显著;除1龄幼虫体重增加(0.0773mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。

简介：Themechanismsofmagnetoreceptionhavebeenproposedasthemagnetitebased,thechemicalradical-pairandbiocompassmodel,inwhichmagnetiteparticles,thecryptochrome(Cry)oriron-sulfurclusterassembly1(IscA1)maybeinvolved.However,littleisknownabouttheassociationamongthemolecules.HereweinvestigatedthemolecularcharacterizationandthemRNAexpressionofIscA1indifferentdevelopmentalstages,tissuesandmagneticfieldsinthemigratorybrownplanthopper(BPH),Nilaparvatalugens.NlIscA1containsanopenreadingframeof390bp,encodingaminoacidsof129,withthepredictedmolecularweightof14.0kDaandtheisoelectricpointof9.10.Well-conservedFe-Sclusterbindingsiteswereobservedinthepredictedprotein.PhylogeneticanalysisdemonstratedNlIscA1tobeclusteredintotheinsect'sIscA1.NlIscA1showedup-regulatedmRNAexpressionduringtheperiodofmigration.ThemRNAexpressionofNlIscA1couldbedetectedinallthethreetissuesofhead,thoraxandabdomen,withthehighestexpressionlevelintheabdomen.ForthemacropterousmigratoryNilaparvatalugens,mRNAexpressionofNlIscA1andN.lugenscryptochromel(Nlcry1)wereup-regulatedunderthemagneticfieldsof5Gaussand10Gaussinstrength(vs.localgeomagneticfield),whileN.lugenscryptochrome2(Nlcry2)remainedstable.Forthebrachyterousnon-migratoryNilaparvatalugens,nosignificantchangeswerefoundinmRNAexpressionofNlIscA1,Nlcry1andNlcry2amongdifferentmagneticfields.ThesefindingspreliminarilyrevealthattheexpressionofNlIscA1andNlcry1exhibitedcoordinatedresponsestothemagneticfield.Itsuggestssomepotentialassociationsamongtheputativemagneto-sensitivemoleculesofcryptochromeandiron-sulfurclusterassembly.

简介：目的:构建人角质细胞生长因子2(KGF-2)基因真核表达载体,探索KGF-2的上皮细胞迁移功能。方法:以人乳腺cDNA文库为模板,PCR扩增KGF-2基因片段,将其插入pXJ-40-myc,经双酶切和测序验证后,将重组质粒转染HEK-293T细胞,采用Western印迹检测重组蛋白;转染人肠上皮细胞FHC,采用细胞划痕实验检测细胞迁移能力。结果:PCR扩增获得627bp的DNA产物,插入pXJ-40-myc载体,双酶切及测序结果证明重组质粒含有目的序列;转染HEK-293T细胞,Western印迹检测到相对分子质量约23×10^3的目的蛋白;细胞划痕实验显示,转染Myc-KGF-2的FHC细胞较未转染或转染空载体的细胞迁移能力强。结论:构建了人KGF-2基因真核表达载体,并验证了其促进细胞迁移的功能。

简介：Theinsectcuticleplaysimportantrolesinnumerousphysiologicalfunctionstoprotectthebodyfrominvasionofpathogens,physicalinjuryanddehydration.Inthisreport,weconductedacomprehensivegenome-widesearchforgenesencodingproteinswithperitrophinA-type(ChtBD2)chitin-bindingdomain(CBD)inthesilkworm,Bombyxmori.Oneofthesegenes,whichencodesthecuticleproteinBmCBP1,wasadditionallycloned,anditsexpressionandlocationduringtheprocessofdevelopmentandmoltinginB.moriwereinvestigated.Intotal,46protein-codinggeneswereidentifiedinthesilkwormgenome,includingthoseencoding15cuticleproteinsanalogoustoperitrophinswithoneCBD(CPAP1s),ninecuticleproteinsanalogoustoperitrophinswiththreeCBD(CPAP3s),15peritrophicmembraneproteins(PMPs),fourchitinases,andthreechitindeacetylases,whichcontainedatleastoneChtBD2domain.MicroarrayanalysisindicatedthatCPAP-encodinggeneswerewidelyexpressedinvarioustissues,whereasPMPgeneswerehighlyexpressedinthemidgut.QuantitativepolymerasechainreactionandwesternblottingshowedthatthecuticleproteinBmCBP1washighlyexpressedintheepidermisandhead,particularlyduringmoltingandmetamorphosis.Animmunofluorescencestudyrevealedthatchitinco-localizedwithBmCBP1attheepidermalsurfaceduringmolting.Additionally,BmCBP1wasnotablyup-regulatedby20-hydroxyecdysonetreatment.Theseresultsprovideagenome-levelviewofthechitin-bindingproteininsilkwormandsuggestthatBmCBP1participatesintheformationofthenewcuticleduringmolting.

简介：目的:利用昆虫细胞表达系统真核表达并纯化小电导钙激活钾离子通道蛋白1(KCNN1)。方法:以基因重组方法构建杆状病毒穿梭质粒reBacmid-KCNN1,将其转染至杆状病毒/Sf9细胞表达系统表达目的蛋白,并用Western印迹鉴定KCNN1的表达水平;用Ni-IDA-SepharoseCL-6B亲和层析柱纯化裂解细胞上清中的KCNN1,并用Western印迹鉴定纯化结果。结果:KCNN1在Sf9细胞中高效表达,通过亲和层析获得了纯化的KCNN1。结论:膜蛋白KCCN1在昆虫细胞Sf9中的表达与纯化,为深入研究其分子生物学功能提供了材料,也为全长膜蛋白的体外表达提供了一套可借鉴的实验方法。

简介：Pheromone-bindingproteins(PBPs)arethoughttobindandtransportsexpheromonesontotheolfactoryreceptorsonthedendritemembraneofolfactoryneurons,andthusplayavitalroleinsexpheromoneperception.However,thefunctionofPBPshasrarelybeendemonstratedinvivo.Inthisstudy,twoPBPs(PBP1andPBP3)ofChilosuppressalis,oneofthemostnotoriouspyralidpests,wereinvivofunctionallycharacterizedusinginsectswiththePBPgeneknockedoutbytheCRISPR/Cas9system.First,throughdirectinjectionofPBP-singleguideRNA(sgRNA)/Cas9messengerRNAintonewlylaideggs,ahighrateoftarget-geneediting(checkedwithpolledeggs)wasinducedat24hafterinjection,21.3%forPBPl-sgRNAinjectedeggsand19.5%forPBP3-sgRNAinjectedeggs.Second,byanin-crossingstrategy,insectswithmutantPBP1orPBP3(bothwithaprematurestopcodon)werescreenedandhomozygousmutantswereobtainedintheG3generation.Third,themutantinsectsweremeasuredforelectroantennogram(EAG)responsetofemalesexpheromones.Asaresult,bothPBPmutantmalesdisplayedsignificantreductioninEAGresponse,andthisreductioninPBP1mutantswashigherthanthatinPBP3mutants,indicatingamoreimportantroleofPBP1.Finally,therelativeimportanceoftwoPBPsandthepossibleofftargeteffectinducedbysgRNA-injectionarediscussed.Takentogether,ourstudyprovidesadeeperinsightintothefunctionofandinteractionbetweendifferentPBPgenesinsexpheromoneperceptionofC.suppressalis,aswellasavaluablereferenceinmethodologyforgenefunctionalstudyinothergenesandothermothspecies.