545 DOI:10.13350/j.cjpb.190511 23 VP8 56-!L ZO # 1, 2, < 3,=>> 1,? @ 1,AB 1,CDE 1 (1. WU/_ A $ WUI!, 410005;2. 9MUI! ; 3. WU E M?D ) 0 1 (Rotaviruses,RVs) - Rotarix RotaTeqVP8 J K 5 (histo blood groupantigens,hbgas) ' /" Z GenBank R Rotarix RotaTeq - VP8 JK ;,_EZ[2VS;3 Rotarix RotaTeqVP8 JK PCR,c ] < NdeI XhoI$ P PCR b9c pet 30a NdeI Xho I,B1.5% 6 b T4 c > ] c, cb DH5α#2 A PCR 7,R $2R,B NdeI XhoI7 J ; 4 9C pet 30a c H _EZ[2 BL21(DE3), IPTG G H 4,B Ni 2+ beads: 4 VP8 His J K, SDS PAGE <!IJ JK lea lex leb ley A B H1 H28M$,W!IJ VP8 JK545W M N F. *,SDS PAGE \ JK -<?26 10 3, <! \ Rotarix RotaTeqVP8 J K H15 HBGAs,W!\ `< 5W < 5W F4 - Rotarix RotaTeqVP8 JK,JK H15 HBGAs M$ ; 5;VP8 JK R373.2!"#$ A %& 1673 5234(2019)05 0545 04 [.2019 May;14(5):545-548,552.] 犆狅犿犫犻狀犻狀犵狉犲狊犲犪狉犮犺狅狀狉犲犮狅犿犫犻狀犪狀狋狉狅狋犪狏犻狉狌狊狏犪犮犮犻狀犲犞犘 8 狆狉狅狋犲犻狀犪狀犱犺犻狊狋狅 犫犾狅狅犱犵狉狅狌狆犪狀狋犻犵犲狀狊 DENGZhong hua 1,GUO Ni jun 2,XIONG Yi can 3,TAN Chao chao 1,YUE Yang hua 1,CHENJie 1, CAO You de 1 (1. 犕犲犱犻犮犪犾犔犪犫狅狉犪狋狅狉狔, 犎狌狀犪狀犘狉狅狏犻狀犮犻犪犾犘犲狅狆犾犲 ' 狊犎狅狊狆犻狋犪犾 / 犜犺犲犉犻狉狊狋犎狅狊狆犻狋犪犾犃犳犳犻犾犻犪狋犲犱狑犻狋犺犎狌狀犪狀犖狅狉犿犪犾犝狀犻狏犲狉狊犻狋狔, 犆犺犪狀犵狊犺犪, 犆犺犻狀犪 410005;2. 犕犲犱犻犮犪犾犔犪犫狅狉犪狋狅狉狔, 犎狌狀犪狀犘狉狅狏犻狀犮犻犪犾犕犪狋犲狉狀犪犾犪狀犱犆犺犻犾犱 犎犲犪犾狋犺犆犪狉犲犎狅狊狆犻狋犪犾 ;3. 犌犪狊狋狉狅犻狀狋犲狊狋犻狀犪犾犪狀犱犘犲犱犻犪狋狉犻犮犛狌狉犵犲狉狔, 犎狌狀犪狀犘狉狅狏犻狀犮犻犪犾犘犲狅狆犾犲 ' 狊犎狅狊狆犻狋犪犾 ) 犃犫狊狋狉犪犮狋 犗犫犼犲犮狋犻狏犲 ToevaluatetheimmuneefectsofVP8 proteinsoftherotavirus(rv)vaccinestrainsrotarix androtateqandfusiongenesofhisto bloodgroupantigens(hbgas). 犕犲狋犺狅犱狊 GenesequencescodingfortheVP8 proteinoftherotarixandrotateqvaccinestrainswereobtainedfromgenbank.aftersequenceoptimizationbasedonco donscommonlyusedin 犈. 犮狅犾犻,genescodingfortheVP8 proteinofrotarixandrotateqweresynthesized,andpcr wasusedtoamplifythetwogenes.ndeiandxhoirestrictionendonucleasesiteswererespectivelyintroducedupstream anddownstreamoftheprimers.thepcrproductandthevectorpet 30aweredigestedwithNdeIandXhoI.Thetar getfragmentwasrecoveredusing1.5% agarosegelelectrophoresisandligatedundertheactionoft4ligase.theligated productwastransformedintodh5αcompetentcelsandidentifiedusingpcr.thepositivebacterialsolutionwasextrac ted,andtheplasmidwasidentifiedusingndeiandxhoidoubledigestionandsequenced.thesyntheticgenesequence wasligatedtotheexpressionvectorpet 30a,introducedinto 犈. 犮狅犾犻 BL21 (DE3),andIPTG wasusedtoinduceitsex pression.a VP8 HisfusionproteinwaspurifiedusingafinitychromatographyonNi 2+ beadsandanalyzedusingsds PAGE.Thebindingpropertiesofthetargetproteintoeightoligosaccharides lea,lex,leb,ley,a,b,h1,and H2 weredetectedusinganoligosaccharidebindingassay.asalivabindingassaywasusedtoanalyzethebindingcharacteris ticsofthevp8 proteintoantigenserotypesindiferentbloodgroups. 犚犲狊狌犾狋狊 Arecombinantplasmidwassuccessful constructed,andsds PAGEindicatedthatthemolecularweightofthetargetproteinwasabout26ku,whichwascon ' :Y (No.2017JJ2151) T,E mail:youde120@aliyun.com () O(1986-),,L,b1, Z# E mail:speed1027@163.com
546 sistentwiththeexpectedmolecularweight.oligosaccharidebindingexperimentsindicatedthatvp8 proteinsfrom Ro tarixandrotateqcanbindtoh1hbgas.resultsofasalivabindingassayindicatedthatthetwocanbindtosecretedsa livaanddonotbindtonon secretedsaliva. 犆狅狀犮犾狌狊犻狅狀 TheVP8 proteinsofrvvaccinestrainsrotarixandrotateq weresuccessfulyclonedandexpressed,andbothproteinsarecharacterizedbybindingtotypeh1 HBGAs. 犓犲狔狑狅狉犱狊 Rotavirus;histo bloodgroupantigens;vp8 protein (Rotaviruses,RVs)? # $ C [1], 7 Rotarix Ro tateq ^ ) (Rotavirusdiar rhea,rvd) A a, b#, K : ), 21.5? RVs# [2] A 4 2C > A# $ Q, [3-4], X- RVs#A 2C,b? W >? [ T RV A2C., & RVs * T RV? W 5 [5-6] 2012,Huang [7] A HBGAs RV VP8 J K,K P[Ⅱ ] P [4] P [8] 5 RVs VP8 JK Leb H15 HBGAs, P [6] H15 HBGAs RVs, RVs 4 @ HBGAsE M X, P[Ⅲ ] P [9] P [14] P [25] 5 RVs VP8 A 5 HBGAs [8],P[Ⅳ] P[11] 5 RVsVP8 Ⅱ 5 X C * β N * [9] Bohm [10] A, HAL1166(P [14,Ⅲ]),K8 (P[9,Ⅲ]),RV 3 (P[6,Ⅱ]) DS 1(P [4,Ⅱ]) A 5 HBGAs, Leb H15 HBGAs! : - Rotateq Ro rarix?-," # W 4 VP8 J K, VP8 JK HBGAs M$ *+, 1 *+ #2 DH5α BL21(DE3)? G H b I;pET 30a? 9,; $ NdeI XhoI T4 DNA c : TaKaRaG H; J K Standard\ BioLabsG H;HistrapHP : \ GE Healthcare G H; 5 #? \ AmrescoGHbI;IPTG?c SangonGHb I; His C # (Horseradishperoxidase,HRP)MIgG : TransGen()GH 2 2.1-0" GenBank0 ; (RVA/Vaccine/USA/Rotarix A41CB052A/1988/ G1P1A [8] ) (GenBank:JN849113): RVA/Vac cine/usa/rotateq WI79 4/1992/G6P1A [8] (Gen Bank:GU565044.1)<- Rotarix Ro tateq VP8 ;, _ E Z [ 2 V S;3 1-,bKc ] < NdeI XhoI$ P(41), S WGH :c ;?,PCR 6 PCR 4J :95 Y$5min;94 30s,55 30s,72 1 min, 35Q,;72 10min R PCR b 9CpET 30a< NdeI XhoI,"#1. 5% 6 b T4 c > ] c, cb DH5α#2 A, - PCR 7 R $2R,B NdeI Xho I7 S WGH J PrimerName VP8 F VP8 R -1 犞犘 8 ' ^_ 犜犪犫犾犲 1 犜犺犲狆狉犻犿犲狉狊狌狊犲犱犳狅狉狋犺犲犘犆犚犪犿狆犾犻犳犻犮犪狋犻狅狀 ;(5' 3') Sequence(5' 3') GGAATTCCATATGGCCAGCCTGATCTACCGCCAGC CCGCTCGAGACGGGTATTCTGGATCGGCGGC 2.2 /K- 1 7 4 _EZ[2 BL21(DE3),b 50g/ml LBC 37 16h, c 30ml 50g/mlC LB (37,200r/min)# R# :1 100c3L 50g/mlC LB (37,200r/min) A 600?0.6CIPTG K O?0.4mmol/L,GH4 #(20,140 r/min) = 2C,PBS `Z _ J (_2s, 2s),= (12000r/min,1h),c 0.22μm #,B Ni 2+ beads: 4 VP8 HisJK, SDS PAGE < 2.3 K(R 4 JK PBS 15~20μg/ml, $,100μl/,4 #; 200μl5%O M bc,37 1h lea lex leb ley A B H1 H2 XC 2%M bc 2μg/ml, 100μl,
547 4 # 2%M bc HRP streptavidin:1 1500 100μl,37 * 1h 4: PBST 3 _ 100 μltmb (BD Biosciences),& \ 10 min;1mol/l 100μl 4, J A 450 2.4 K(R 45 W PBS :1 1000, Z10 min,5000r/min= (= 8.27cm)5min,R100μl $96,4 #, 5%M bc37 1h;100 μl20μg/ml VP8 hisjk,37 * 1h; 100μl His C (Transgene,1 1 000),37 * 1h; 100μlHRP IgG(Abgent,1 1000),37 1h 4 : PBST 3 _ 100μl TMB (BDBiosciences),& \ 10min; 1mol/L 100μl 4, J A 450 2 犞犘 8 犎犻狊 (,3 犉犻犵.2 犜犺犲狅犾犻犵狅狊犪犮犮犺犪狉犻犱犲狊犫犻狀犱犻狀犵犪狊狊犪狔狅犳犞犘 8 犎犻狊犳狌狊犻狅狀狆狉狅狋犲犻狀 3 犞犘 8 犎犻狊 (,45 IJ Q VP8 HisJK 45 W $, " @ 3 4 RotaTeq RotarixVP8 HisJK< 5W, < 5W 1 犞犘 8 犎犻狊 ( - Q2E "#_EZ[24 4,B Ni 2+ His.Bind : 4 VP8 His J K,SDS PAGE < \, J K - <?26 10 3,( 1) A A 5W AB AB5W B B 5W O O 5 W O O 5W 3 犚狅狋犪犜犲狇犞犘 8 犎犻狊 (,45 A TypeAsaliva AB TypeABsaliva B TypeBsaliva O TypeOsaliva O TypeO saliva 犉犻犵.3 犆犺犪狉犪犮狋犲狉犻狕犪狋犻狅狀狅犳狋犺犲犫犻狀犱犻狀犵狆犪狋犲狉狀犫犲狋狑犲犲狀狋犺犲犚狅狋犪犜犲狇犞犘 8 犎犻狊犳狌狊犻狅狀狆狉狅狋犲犻狀犪狀犱狋犺犲狊犪犾犻狏犪 M JK I 1 RotaTeq VP8 His J K 2 Rotarix VP8 HisJK 1 2E 犞犘 8 犎犻狊 ( b> M Proteinmarker 1 RotaTeqVP8 HisFusionProtein 2 RotarixVP8 HisFusionProtein 犉犻犵.1 犐犱犲狀狋犻犳犻犮犪狋犻狅狀狅犳狆狌狉犻犳犻犲犱犞犘 8 犎犻狊犳狌狊犻狅狀狆狉狅狋犲犻狀 2 犞犘 8 犎犻狊 (,3 IJ VP8 HisJK5 $, "@ 2 Rotarix RotaTeq VP8 JK H15, K W A A 5W AB AB5W B B 5W O O 5 W O O 5W 4 犚狅狋犪狉犻狓犞犘 8 犎犻狊 (,45 A TypeAsaliva AB TypeABsaliva B TypeBsaliva O TypeOsaliva O TypeO saliva 犉犻犵.4 犆犺犪狉犪犮狋犲狉犻狕犪狋犻狅狀狅犳狋犺犲犫犻狀犱犻狀犵狆犪狋犲狉狀犫犲狋狑犲犲狀狋犺犲犚狅狋犪狉犻狓犞犘 8 犎犻狊犳狌狊犻狅狀狆狉狅狋犲犻狀犪狀犱狋犺犲狊犪犾犻狏犪
548 L RVs LDJK# VP4JK JK VP7.,VP4 VP7JK RVs # A >A# Q, RVs<5 VP4 VP7JK, : A RVs<? 27Q G 5 35Q P 5 [11] VP4JK bjk > ] < ^? VP8 (AA 1-231) VP5 (AA 248-776) [12-13] Arias [14-15] 4 VP4 JKB#JK^? VP8 VP5Q*. 4Y #,H? VP8 J K E 4 HBGAs,4 HBGAsb RVs # A > [7] Jiang [16] \ - P 5 RVs # $ K 4 HBGAsV HBGAs ABO < 5 Lewis, 4[ 4,b W *C [17] HBGAs TU * $,K Z X C E,b ABO < 5 Lewis'QAS > ], : : c X XC45 HBGAs < 5 AS α1,2 (FUT2) XC H (O ) < Q C G H $ FUT2 H,? < 5 QC N ** < b A B > ]' H < A B LewisAS FUT3 :α 1,3 α 1,4 / X C c ' < Lewisa(Lea) Lewisx(Lex), H c Lewisb(Leb) Lewisy(Ley), $ FUT3QC Lewis A B b FUT3 ] A B LewisT GenBank - Rotateq Rotarix P [8] 5 RVs ; K VP8 ; _EZ[2-VS 3,.*pET 30a.VP8 4,"#_EZ[ 24 Rotarix RotaTeqVP8 His JK 4 His.VP8 JK< W!! WesternblotIJ His.VP8 J K His C, W!"#T '- QJK * C, His C>?,S"#c, \, * C 4 " \, Rotarix RotaTeq VP8 JK H1, K \ W!\,Rotarix RotaTeq VP8 JK A B AB O 5< 5W,< 5W 1@,< 5W H << 5W JK1@, QCXC H, b :,b RV $A* # QC < 5,,b< 5QC# RV, ; A 0 K VP8 JK4. A 4 Y,Z H K HB GAsX c, Rotarix RotaTeq VP8 JK H15 M$, 5 < 5 W A4,< 5W,? # :-' /4 D 01 5 MN! [1] LiuJ,Plats MilsJA,JumaJ,etal.Useofquantitativemolecu lardiagnosticmethodstoidentifycausesofdiarrhoeainchildren: areanalysisofthegemscase controlstudy[j].lancet,2016 (388):1291-301. [2] TateJE,Burton AH,Boschi PintoC,etal.Global,regional, andnationalestimatesofrotavirusmortalityinchildren <5years ofage,2000-2013[j].clininfectdis,2016(62):s96-105. [3] FlemingFE,Graham KL,TakadaY,etal.Determinantsofthe specificityofrotavirusinteractionswiththeα2β1integrin[j].j BiolChem,2011,286(8):6165-74. [4] GroveJ,Marsh M.Thecelbiologyofreceptor mediatedvirus entry[j].jcelbiol,2011,195(7):1071-82. [5] IsaP,AriasCF,LopezS.Roleofsialicacidsinrotavirusinfec tion[j].glycoconjj,2006,23(1-2):27-37. [6] KuhlenschmidtTB,Hanafin WP,GelbergHB,etal.Sialicacid dependenceandindependenceofgroup Arotaviruses [J].Adv Exp MedBiol,1999(473):309-17. [7] HuangP,Xia M,Tan M,etal.Spikeprotein VP8 ofhuman rotavirusrecognizeshisto bloodgroupantigensinatype specific manner[j].jvirol,2012,86 (9):4833-43. [8] Liu Y,HuangP,Tan M,etal.RotavirusVP8 :phylogeny, hostrange,andinteractionwithhisto bloodgroupantigens[j].j Virol,2012,86(18):9899-910. [9] LiuY,HuangP,JiangB,etal.Poly LacNAcasanage specific ligandforrotavirusp[11]inneonatesandinfants[j].plosone, 2013,8(11):e78113. [10] Bohm R,FlemingFE,MaggioniA,etal.Revisitingtheroleof histo bloodgroupantigensinrotavirushost celinvasion[j].nat Commun,2015(6):5907. [11] LopezS,AriasCF.Multistepentryofrotavirusintocels:a Versailesquedance[J].Trends Microbiol,2004,12(6):271-8. (()552*)
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