530 DOI:10.13350/j.cjpb.190508 P 1ZrAlb 34k256[\ DENV 2 ]^_` BHK 21:;aJO 1, 1,[ 1,< 1, 1, 2,3,4, 1 (1. LW _ W U' X ", L 550025;2. LW _ W U 3 X ";3. LW _ M!";4. LW _ 3 ) (K M $ralb 34k2JK (DENV)* A A DENV 2# > 1) c' IJrAlb 34k2JK24h - DENV 2# BHK 21A > 2)Westernblot c' IJrAlb 34k2JK BHK 21 HepG2 HaCatA A> : V ralb 34k2 C < /4 1)' I J " \,@ _ ralb 34k2 J K # V? 0.40 ±0.023,1 _ 0.54 ± 0.029, 9 ( 犘 =0.0011) 2)Westernblot ' I J \ ralb 34k2 J K BHK 21 HepG2 HaCatA A A, BHK 21A A $;M $ C < ralb 34k2JK BHK 21 HepG2A A/4 ralb 34k2JK BHK 21 DENV * A A A>, H DENV 2cAA, W K ; W JK; A ;3 ; A> R384.1!"#$ A %& 1673 5234(2019)05 0530 04 [.2019 May;14(5):530-533,538.] 犃犲犱犲狊狊犪犾犻狏犪狉狔狊狆犲犮犻犳犻犮狉犃犾犫 34 犽 2 狆狉狅狋犲犻狀狆狉狅犿狅狋犲狊犇犈犖犞 2 犻狀犳犲犮狋犻狅狀犻狀犅犎犓 21 犮犲犾狊犻狀犲犪狉犾狔狊狋犪犵犲 XU Qian 1,TIAN Yan shen 1,ZHU Ya ni 1,ZHAO Jiu gang 1,LI Yong nian 1,WU Jia hong 2,3,4, SHANGZheng ling 1 (1. 犇犲狆犪狉狋犿犲狀狋狅犳犐犿犿狌狀狅犾狅犵狔, 犅犪狊犻犮犕犲犱犻犮犪犾犆狅犾犲犵犲, 犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔, 犌狌犻狔犪狀犵, 犌狌犻狕犺狅狌 550025, 犆犺犻狀犪 ;2. 犇犲狆犪狉狋犿犲狀狋狅犳犎狌犿犪狀犘犪狉犪狊犻狋狅犾狅犵狔, 犅犪狊犻犮犕犲犱犻犮犪犾犆狅犾犲犵犲, 犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔 ;3. 犆犺犪狉犪犮狋犲狉犻狊狋犻犮犪狀犱犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳犕狅犱犲狉狀犘犪狋犺狅犵犲狀犅犻狅犾狅犵狔犌狌犻狕犺狅狌犕犲犱犻犮犪犾犝狀犻狏犲狉狊犻狋狔 ;4. 犕犻犮狉狅 犫犻狅犾狅犵狔犪狀犱犅犻狅犮犺犲犿犻犮犪犾犘犺犪狉犿犪犮狔犈狀犵犻狀犲犲狉犻狀犵犆犲狀狋犲狉 ) 犃犫狊狋狉犪犮狋 犗犫犼犲犮狋犻狏犲 ToinvestigatetheefectsofAedesalbopictussalivaryspecificrAlb 34k2proteinonadheringva rioushostcelstopromotedenv2infection. 犕犲狋犺狅犱狊 1)TheefectsofrAlb 34k2proteinonDENV 2infectionin BHK 21celswithin24hoursweredetectedbyindirectimmunofluorescence.2)TheadhesionefectsofrAlb 34k2pro teintobhk 21,HepG2,and HaCatcels wereanalyzedby westernblotorindirectimmunofluorescence method,in whichtheblockingefectsofrabbitanti ralb 34k2antibodywerealsostudied. 犚犲狊狌犾狋狊 1)CamparedtotheDENV 2 controlgroups,theinfectionratioofbhk 21celsintherAlb 34k2proteintreatmentgroupswas0.54±0.02,whilethe infectionratiointheheated inactivatedralb 34k2proteingroupswas0.40±0.023.Theresultsshowedthattheinfection ratioofdiferentgroupswerestatisticalysignificant( 犘 =0.0011).2)Moreover,ourresultsidentifiedthattherAlb 34k2proteincouldadheretoBHK 21,HepG2andHaCatcels,inwhichtheadherionefectontheBHK21celswasin dose dependentmanner.meanwhile,theseadherionefectsonbhk 21and HepG2celwerenotcompletelyblockedby therabbitanti ralb 34k2polyclonalantibodies. 犆狅狀犮犾狌狊犻狅狀 ThisstudysuggestedthattherAlb 34k2proteinderived fromfamaleaedesalbopictussalivaryglandcouldadheretoseveralkindsofdenvtargetcelsandprovedthepotential roleoftheralb 34k2proteintopromoteDENV 2virusadsorptionandinvasionintovarioushostcels. 犓犲狔狑狅狉犱狊 Aedesalbopictus,mosquitosalivaprotein,hostcel,arbovirus,adhesion W 3c cx, b # A > [1-2] Z K D@ (DENV) * 3 b,k [3-4] Ribeiro [5] b W A 4 ' : Y (No.81260260, 81060318); La (No.[2016]09 B); L I J W [ (Y [ [2012 (4006)]) &,E mail:shangzhengling@126.com () )(1992-),-, *, b, Z 3 # ' E mail:512581441@qq.com
531, K AS34 10 3 _M JK,$?34k JK Arca [6-8] WesternblotZ Z K P W < 7@2Q34kJK,<? 34k1 34k2 X & Z 34k1JK DENV # A > [9], 34k2JKb3 # > X A K W 34k2 J K\ ] c, ( : pet28a ralb 34k2 34k2 J K ( ralb 34k2)- DENV 2 # BHK 21 A 8 9, I J K DENV * A A>,?: & P W M $34k2JK H DENV 2 W A $ *+, 1 *+ 1.1 0 ralb 34k2 J K ( : pet28a ralb 34k2, J K 6 his );V ralb 34k2 * C ralb 34k2? " ;BHK 21 HepG2 HaCatA?!" ' 9, 1.2 () AlexaFlour488(AF488) - IgG C HRP - IgG C \ ThermoGH;beta Actin C >GH; his IgG C F Abcam G H;ECL A \ Bio Rad G H; DENV 2E JK C \ GeneTexGH 2 2.1 PQ 6rAlb 34k2 DENV 2 BHK 21 -! O?5 105/ml - BHK 21 A 6,37 5% CO 2 12h,PBS 3 >: ]< :1)rAlb 34k2 J K : DENV 2(MOI= 5) O?10ng/L ralb 34k2JK;2)DENV 2- : DENV 2(MOI=5);3)@ _ J K : DENV 2 (MOI=5) O?10ng/ μ l BZZ_ ralb 34k2 J K(irAlb 34k2) PT,c A P 37 5% CO 2 c A 2h, 30min [1, 2% FBSA O [, 22h, c,pbs ; 4%* \ 7 10 min,0.1% TritonX 100 J 10 min,5% BSA "* 30 min, c,pbs ; DENV 2E JK (1 1000),"*]S 2h, PBS 3 ; IgGAF488 (1 5 000),"*& 1h,PBS DAPIT ( O?10ng/ μ l),pbs ^P \ ]! _,4 ImageJ < " 2.2 Westernblot 6rAlb 34k2 * BHK 21 HepG2" - # $ 0 ralb 34k2 % & -' R- BHK 21 HepG2 A,< ralb 34k2JK,<?4!:1)34k2JK : O?3ng/ μ l ralb 34k2JK;2) C < : O? 3ng/ μ l ralb 34k2JK V 34k2 C (1 1000)b " *] 2h A ;3) $ : O?3ng/ μ l ralb 34k2JK $V (1 1 000)"*] 2h A ;4)A - : 4A 4 2h,200g= 5 min, PBS 3,< M A JKc,B SDS PAGE ', ECL A \8IJ 2.3 Westernblot 6 4 5 ralb 34k2 * BHK 21 -# $ O ralb 34k2 JK BHK 21 A (1 10 6 /ml), J K O <?9.0 6.0 3.0 1.5 0.3ng/ μ l,4, 3D `a c 2h,200g= 5min,PBS 3, A, J K c,sds PAGE JK PVDF c,5%m bc4 # : rlb 34k2 J K * C (1 8000) beta actin C (1 2000)?, HRP IgG? (1 10000) Westernblot, ECL A \8,4 Im agej < " 2.4 PQ 6rAlb 34k2 * HaCat -#$ R HaCatA (1 10 6 /ml)c 12,500μl/,! O?10ng/ μl ralb 34k2JK,A - JK, P3QT 4 2h,PBS 3,4% * \"*715min,5%M bc"* 30min; his IgG (1 3000)"* 2h, PBS ; AF488 IgG (1 5000),"*& 1h,^P \ ]!A A 2.5 J < GrahpadPrims 7.0 <, V W E3 狋 I!, 犘 <0.01? 9 1 狉犃犾犫 34 犽 2 犇犈犖犞 2 犅犎犓 21 c' IJrAlb 34k2JK- DENV 2
532 # BHK 21A 89, A < b] < R_ 4 imagej < AF488[ /DAPI[ 4 R # V 1A? DENV 2(MOI=5)# 24h BHK 21A #,1B? # V K ralb 34k2 J K # V? 0.54 ± 0.029,@ _ J K # V? 0.40 ±0.023, DENV 2- # V? 0.44 ± 0.03 ralb 34k2JK # V DENV 2- @_ J K VW 9 ( 犘 =0.0011),4 ralb 34k2JK DENV 2# BHK 21A A 24h DENV 2# BHK 21 c' IJ B AF488/DAPI [ V 1 VWXY TU 狉犃犾犫 34 犽 2 犇犈犖犞 224 犺 犅犎犓 21(40 ) A BHK 21celinfectedbyDENV 2(MOI=5)with10ng/LrAlb 34k2proteinorheated inactiveralb 34k2protein(irAlb 34k2),respectively at24h B theratioofaf488/dapiaveragefluorescenceintensity 犉犻犵.1 犈犳犳犲犮狋狊狅犳狉犃犾犫 34 犽 2 狆狉狅狋犲犻狀狅狀狋犺犲犻狀犳犲犮狋犻狅狀狅犳犇犈犖犞 2 犻狀犅犎犓 21 犮犲犾狊狑犻狋犺犻狀 24 犺犫狔犻狀犱犻狉犲犮狋犻犿犿狌狀狅犳犾狌狅狉犲狊犮犲狀犮犲 (40 ) 2 狉犃犾犫 34 犽 2, 犅犎犓 21 犎犲狆犌 2 BV ralb 34k2 C, M I J ralb 34k2 J K, M 1IJ JK( 2A), 4 ; A I IJ ralb 34k2JK( 2B),4 JK BHK 21 HepG2 A A, A > $M $ C < A?A ralb 34k2 D M (1-4) M (5-8) ralb 34k2 ' I J B? A ralb 34k2 ' IJ 2 狉犃犾犫 34 犽 2 狉犃犾犫 34 犽 2 C, 犅犎犓 21 犎犲狆犌 2 A DetectionofrAlb 34k2proteininfirstelutingliquid(1-4)andlastelutingliquid B DetectionofrAlb 34k2incelprecipitation 犉犻犵.2 狉犃犾犫 34 犽 2 狆狉狅狋犲犻狀犮狅狌犾犱犪犱犺犲狉犲狋狅犅犎犓 21 犪狀犱犎犲狆犌 2 犮犲犾狊犪犳狋犲狉狆狉犲狋狉犲犪狋犲犱狑犻狋犺狊狆犲犮犻犳犻犮狉犪犫犫犻狋犪狀狋犻 狉犃犾犫 34 犽 2 犪狀狋犻犫狅犱狔 3 狉犃犾犫 34 犽 2, 犅犎犓 21! "? 3\ WesternblotIJ O?3.0~9.0 ng/ μ l ralb 34k2JK BHK 21A I J ralb 34k2J K \ 8 J!, J K BHK 21A A 4 狉犃犾犫 34 犽 2, 犎犪犆犪狋! 6 his ralb 34k2JK HaCat A his IgG C, '!, " @ 4 ' I J Q A $ ( Y 1 -), ralb 34k2 JK HaCatA 4 A A> 3 56# 狉犃犾犫 34 犽 2, 犅犎犓 21 犉犻犵.3 犃犱犺犲狊犻狅狀犲犳犲犮狋狊狅犳犱犻犳犳犲狉犲狀狋犮狅狀犮犲狀狋狉犪狋犻狅狀狊狅犳狉犃犾犫 34 犽 2 狆狉狅狋犲犻狀狋狅犅犎犓 21 犮犲犾狊 4 狉犃犾犫 34 犽 2, 犎犪犆犪狋 XY $%&(40 ) 犉犻犵.4 犅犻狀犱犻狀犵狊狅犳狉犃犾犫 34 犽 2 狆狉狅狋犲犻狀狅狀犎犪犆犪狋犮犲犾狊犫狔犻狀犱犻狉犲犮狋犐犿犿狌狀狅犳犾狌狅狉犲狊犮犲狀犮犲犪狊犪狔 (40 )
533 L W A X:1)" # M $ 2 C ( :HSP70/90 CD14 < DC SIGN HS ) H X WA [10-13] ;2) DENV O CA C 4,"#A 4 FcR H W, X$? C > (antibodydependentenhancing,ade) [14] DENV * 3 b # W :' K [15] Surasombatpatana [16] Z W 34k1 JK DENV # A!rAlb 34k2JK - DENV 2# 24h N O A (BHK 21) 89,A! # V DENV 2- @_ J K, ralb 34k2 J K # BHK 21A & W JK "#* #, Jin [17] A W M < LTRIN JK A c 2C β H # Conway [18-19] A W D7JK DENV c," # D7 J K CH # A A K # $, ' Western blotijralb 34k2JK,b A 4 AZ H W :(1) DENV BHK 21 A >? -,A ralb 34k2 JK- N O A (BHK 21) A > (2) DENV : # C, A # A [20], A C A [21], A (HepG2) A (HaCat)>? - I J,A ralb 34k2 J K Ac A, HaCatA A J K $ 34kJK P W M $ 4 < $JK, JK P c C \ Z 34k1 JK DENV # A, M $ W 34k1JK C >?$ DENV # Y [22-23] C D I J M $ 34k2 C < ralb 34k2 J K BHK 21 A HepG2A A,, b J K A A * <, K H DENV WA ralb 34k2JK"# c bc DENV # A 2018 WHO Q DENV -, NIH 4 W JK(v GSA) - b 2 = 3 /$ Z $ 01 [24-25], 4 ( # W JK F? A & C D MN! [1] BriantL,DespresP,ChoumetV,etal.Roleofskinimmune celsonthehostsusceptibilitytomosquito borneviruses[j].vi rology,2014,464-5(1):26-32. [2] ChavesBA,Orfano AS,NogueiraPM,etal.Coinfection with ZikaandDenguevirusesresultsinpreferentialZikatransmission byvectorbitetovertebratehost[j].jinfectdis,2018,218(4): 563-71. [3] DongF,FuY,LiX,etal.Cloning,expression,andcharacter izationofsalivaryapyrasefrom Aedesalbopictus.[J].Parasitol Res,2012,110(2):931-7. [4] VogtMB,AnismritaL,AryaRP,etal.Mosquitosalivaalone hasprofoundefectsonthehumanimmunesystem[j].plos NeglTropDis,2018,12(5):e0006439. [5] RibeiroJM,Arca B,Lombardo F,etal.Anannotatedcata logueofsalivaryglandtranscriptsintheadultfemale mosquito, 犃犲犱犲狊犪犲犵狔狆狋犻 [J].BMCGenomics,2007(8):6-33. [6] ArcaB,LombardoF,FrancischetiIM,etal.Aninsightintothe sialomeoftheadultfemalemosquito 犃犲犱犲狊犪犾犫狅狆犻犮狋狌狊 [J].Insect Biochem MolBiol,2007,37(2):107-27. [7] RibeiroJM,Martin MartinI,ArcaB,etal.Adeepinsightinto thesialomeof maleandfemale 犃犲犱犲狊犪犲犵狔狆狋犻 mosquitoes[j]. PloSOne,2016,11(3):e0151400. [8] DoucoureS,CornelieS,PatramoolS,etal.Firstscreeningof 犃 犲犱犲狊犪犾犫狅狆犻犮狋狌狊 immunogenicsalivaryproteins[j].insectmolbi ol,2013,22(4):411-23. [9] SurasombatpatanaP,PatramoolS,Luplertlop N,etal. 犃犲犱犲狊犪犲犵狔狆狋犻 salivaenhancesdenguevirusinfectionofhumankerati nocytesbysuppressinginnateimmuneresponses[j].jinvest Dermatol,2012,132(8):2103-5. [10] ValeJR,ChávezsalinasS,MedinaF,etal.Heatshockprotein 90andheatshockprotein70arecomponentsofDenguevirusre ceptorcomplexinhumancels[j].jvirol,2005,79(8):4557-67. [11] Ivory MO,BirchalJC,PiguetV,etal.Earlydenguevirusin fectioninhumanskin:acycleofinflammationandinfectivity[j]. JInvestDermatol,2015,135(7):1711-2. [12] RichterMK,daSilvaVoorhamJM,TorresPedrazadaSilva,et al.immaturedenguevirusisinfectiousinhumanimmatureden driticcelsviainteraction withthereceptor molecule DC SIGN [J].PloSOne,2014,9(6):e98785. [13] IchiyamaK,GopalaReddySB,ZhangLF,etal.Sulfatedpoly saccharide,curdlansulfate,eficientlypreventsentry/fusionand restrictsantibody dependentenhancementofdenguevirusinfec tion 犻狀狏犻狋狉狅 :apossiblecandidateforclinicalapplication[j].plos NeglTropDis,2013,7(4):e2188. (()538*)
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