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1 CHINESEJOURNALOF VIROLOGY Vol.33 No.4 June # 2#,, 2, 2, 1 3* 1*,, (1., ; 2., ; 3., ) : HEK-293-HA RT-PCR HA, pcdna3.1(+ ), pcdna3.1(+ )-HA pcdna3.1(+)-ha PLV-EF1a-EGFP(2A)Puro HEK293, HEK293-HA, (FCM) HA 15, (IFA) (WB) HA pcdna3.1(+)-ha ; 3 15 WesternblotIFA, HA HEK-293-HA, HA : ; (HA) ; :R : ; : :A : (2017) DOI: /j.cnki.bingduxuebao RNA,, [1], (A) (B) (C) (D) () [2] (InfluenzaBvirus) (Neuraminidase,NA) [3], 8 RNA,HA, RNA4, [4],, 1983 ; HA, HA1 :Victoria ( B/Victoria/02/87) HA2, HA Yamagata( B/Yamagata/16/88) [5,6],,,, [7,8], NA,,, [9] [10] HA NA : - ( : ), : ; - [11] ( : ), : :#, (1991-), ;Tel: , @qq.com; (1988-), ;Tel: , dav- idliu1988@126.com * :, (1974-), ;Tel: , xbsu92@yahoo. com;(1965-), ;Tel: , xiaofeng82@hotmail.com (Hemagglutinin,HA), HA,, pcdna3.1 (+)-HA, PLV-EF1a-EGFP(2A)Puro, DNA HEK293, HA HA,

2 4 : pcdna3.1(+) ;PLV-EF1A-EGFP(2A) 30s,41 30s,72 2min,35 ; Puro ; TG1; (Hu min 1% manembryonickidney293cels); (Ma- din-darbycaninekidneycels);victoria DNA 2 EcoRIBamHI NEB ( );PyrobestDNA T4DNA TaKaRa ( );, DNA 5 AXYGEN ( ); (BSA) (FBS) DMEM/F12 DMEM - (Pen-Strep), HA,T4 (PBS) HEPES TPCK DNA GIBCO ( ); RNA,16, RT-PCR TG1, PCR ;LipofectamineLTX Reagent, ;HA FITC HEK HRP IgG(H+L) HRP, 1d, IgG(H+L) 0.5μg/mL 3 NCBIGenBank Victoria HA (AY ), invitrogen ; (Puromycin) Invivo- gen ;HA ProSpec ;AlexaFluor647 IgG(H+L) 6 VectorNTI 4 : (infb-1f):5 - CGCGGATCCGCATTTTCTAATATCCACAAA- 3, (infb-1r):5 -CCGGAATTCAGTAGTA- ACAAGAGCATTTT-3 ; (infvb-f):5 - CGCGGATCCGCCACCATGAAGGCAATAATT- GTACTACTCATG-3 ( BamHI ), (infvb-r ):5 -CCGGAAT- TCTCAATAACGTTTCTTTGTAATGATGAC- 70% ~80%,Lipofectamine 3 ( EcoRI ), LTX Reagent 4 HA RNA, RNA,infB-1FinfB-1R, RT-PCR HA ;RT-PCR :45 30min;94 5min,94,, HA,infVB-F infvb-r,pcr HA :94 5min,94 30s,58 30s,72 2min,30 ;72 10min PCR EcoRIBamHI PCR pcdna3.1(+) 1.5μg/mL 3μg/mL, 6 6, 7 pcdna3.1(+ )-HA PLV-EF1A-EGFP(2A)Puro 10%FBS 1% Pen-StrepDMEM 10cm HEK293 HEK293 pcdna3.1(+)-ha PLV-EF1A- EGFP(2A)Puro HEK293 (pcdna3.1 MDCK (+)-HA PLV-EF1A-EGFP(2A)-Puro=3 1), RNA 1.5μg/mL,

496 33 2d,, 1870bp (, 0.5μg/mL 1),,,, 8 HA 8.1 (Flowcytometry,FCM) 75%,1% 1h,PBS3,1 100 HA 1h, PBS 3, 1 100 AlexaFluor647 IgG(H+L) 1h, PBS 3, HA Figure1 HAgeneproductidentifiedbyelectrophoresis 8.

1(+)-ha BamHI 15,, EcoRI, 5400bp,37 1830bp ;,,PBST 3, PCR 1830bp 1%BSA 37 1h;1 100 (2), HA,37 1h, PBST 3, 1 100 FITC GenBank HA IgG(H+L),, 37 1h,PBST 3, 8.

3 d,, 1870bp (, 0.5μg/mL 1),,,, 8 HA 8.1 (Flowcytometry,FCM) 75%,1% 1h,PBS3,1 100 HA 1h, PBS 3, AlexaFluor647 IgG(H+L) 1h, PBS 3, HA Figure1 HAgeneproductidentifiedbyelectrophoresis 8.2 (Indirectimmunofluorescene assay,ifa) 2 pcdna3.1(+)-ha BamHI 15,, EcoRI, 5400bp, bp ;,,PBST 3, PCR 1830bp 1%BSA 37 1h;1 100 (2), HA,37 1h, PBST 3, FITC GenBank HA IgG(H+L),, 37 1h,PBST 3, 8.3 (Westernblot,WB) 15, 2 loadingbufer,100 10min, 12%SDS-PAGE,,4 5%,0.1% TBST 3, HA, 2h,0.1%TBST 3, HRP IgG(H+L), 1h,0.1%TBST 3, 1 1 ECL,ChemStudio 1 HA RT-PCR M:DNA markertrans2kplus;1,2:ha RT-PCR M:DNA markertrans2kplus;1,2:rt-pcr productofhagene 1 HA RT-PCR M:DNA markerdl2000;1,2: PCR ; 3: PCR ;4,5: ; 6: ;M:DNA markertrans5k M:DNA markerdl2000;1,2:pcrproductof pcdna3.1(+)-ha;3:pcrproductofpcdna3.1(+); 4,5:pcDNA3.1(+)-HAdoubledigestion;6:pcDNA3.1(+) plasmid;m:dna markertrans5k PCR 1% digestionofrecombinantplasmidpcdna3.1(+)-ha 2 pcdna3.1(+)-ha PCR Figure2 PCRanalysesanddoublerestriction-enzyme

4 4 : HA,HEK FCM 0.5μg/mL; 15 HEK293-HA (3d),7d, HEK293, 1.5μg/mL, 3 HEK293-HA, T, P<0.05, HEK239HEK293-HA, 15 (3, HEK293-HA-1~15 4) 3 FITC APC Figure3 PercentageofAPCfluorescence-positiveand MFIinFITC-positivecelpopulations 4 HEK239HEK293-HA t Figure4 Analyses(Student st-test)of HEK293andHEK293-HAcels, [12], [13],,B 25%,, [14], HA1 [15],HA, 4.2 IFA HA, ;HA,, HEK293-HA, [16] HEK293 (5) (Streptomyces 4.3 Westernblot alboniger), Westernblot, 15 HEK293-HA 75kD pac N-,HEK293 (6) (PAC),

NormalHEK293cels Westernblot HEK293-HA HA Figure6 ExpressionofHAproteinby Westernbloting,, HA, pcdna3.1 (+)-HA PLV-EF1a-EGFP(2A)Puro HEK293,,IFA, HA, HA HA,, ; HA, HA [17] : ;, [1]PaleseP,Shaw M L.

5 498 33, pac HEK293,,, HA PLV-EF1a-EGFP(2A)Puro pcdna3.1(+)-ha 5 A. HEK293-HA ;B.HEK293 A.RecombinantHEK293-HAcels;B.NormalHEK293cels HEK293-HA HA ( 40) Figure5 IdentificationofexpressionofHAproteinbyimmunofluorescenceassay( 40magnification) M:Proteinmarker;1.HEK293-HA ;2.HEK293 6 M:Proteinmarker;1:HEK293-HAcels; 2.NormalHEK293cels Westernblot HEK293-HA HA Figure6 ExpressionofHAproteinby Westernbloting,, HA, pcdna3.1 (+)-HA PLV-EF1a-EGFP(2A)Puro HEK293,,IFA, HA, HA HA,, ; HA, HA [17] : ;, [1]PaleseP,Shaw M L.Orthomyxoviridae:Theviruses,, andtheirreplication[j].viro,5ed,2007, [19] [2]SongH,QiJ,KhedriZ,DiazS,Yu H,ChenX,VarkiA, HA ShiY,GaoGF.Anopenreceptor-bindingcavityofhe-, magglutinin-esterase-fusionglycoproteinfrom newly-i- dentifiedinfluenzadvirus:basisforitsbroadceltrop-, ism[j/ol].plospath,2016,12(1):e , [3],,,,,.B

6 4 : 499 [J].,,,. S-03,2016,32(6): [4],. [J].,2011,(3): ,2013,34(10): laysia, [j/ol].plos One,2015,10(8): e [5]Y O Xiang,K T Ng,T Y Lam,Y KPang,K GChan,NS Hanafi,A Kamarulzaman,K K Tee.Epidemiological andevolutionarydynamicsofinfluenzabvirusesin Ma- [6]EuropeanCentreforDiseasePreventionandControl.In- fluenza virus characterisation september 2015 [R]. Stockholm:ECDC,2015, [7]RotaP A,WalisT R,Harmon M W,RotaJS,Kendal A P,NeromeK.Cocirculationoftwodistinctevolution- arylineagesofinfluenzatypebvirussince1983[j].vi- rology,1990,175(1): [8]Shaw M W,XuX,LiY,NormandS,UekiR T,Kunimo- pearanceandglobalspreadofvariantsofinfluenzab/ 2002seasons[J].Virology,2002,303(1):1-8. [9],. [M]. manr,chen W H,WinokurP,BelsheR,GrahamIL,,1997,5-42. NoahDL,GuoK,HilH.Safetyandimmunogenicity [10]D D,RJ,FG. [M].,2012, [11],.,,,, [19]NicholsonK G.Influenzaandvaccinedevelopment:a HA1 [J]. continuedbatle[j].expertrevvaccines,2009,8(4):,2010,(10): [12],,,,,,, [J]. [13],,,,,,,, HA [J].,2011,15(8): [15],,,,,. HA1 [J].,2008,18(3): [16]UlmerJB.InfluenzaDNAvaccines[J].Vaccine,2002, 20(16): tog Y,HalH,KlimovA,CoxNJ,SubbaraoK.Reap- victoria/2/87lineagevirusesinthe and2001- [14]DushofJ,PlotkinJB,ViboudC,EarnDJ,Simonsen L.Mortalityduetoinfluenzainthe UnitedStates-an annualized regression approach using multiple-cause mortalitydata[j].amjepidemiol,2006,163(2):181- [17]PalacheB.New vaccineapproachesforseasonaland pandemicinfluenza[j].vaccine,2008,26(49): [18]BradyRC,TreanorJJ,AtmarRL,KeitelW A,Edel- ofasubvirioninactivatedinfluenza A/H5N1 vaccine with or withoutaluminum hydroxideamong healthy elderlyadults[j].vaccine,2009,27(37):

7 DevelopmentofaStableCelLineExpressingtheHemagglutininProteinof InfluenzaBVirus HUANG Wenbo 1,LIU Zhenwei 2,ZHOU Rong 2,LIXiao 2, ZENGJun 1,LIXiaofeng 3*,SU Xiaobo 1* (1.Departmentof MedicalGeneticsand CelBiology,GMU-GIBH JointSchoolof LifeSciences, Guangzhou Medical University,Guangzhou511436,China;2.GuangzhouInstituteof Respiratory Disease, State Key Laboratoryof Respiratory Disease,Guangzhou Medical University,Guangzhou510230,China; 3.Guangdong Hecin Scientific,Inc,Guangzhou510663,China) [KH-*2D] Abstract:Wewishedtodevelopastablecellinethatcouldexpresshemagglutinin(HA)intheinfluenzaB virus(ibv).the HA geneintheful-lengthibv wasgeneratedbyamplificationofthereversetran- scriptase-polymerasechainreactionfromtheibv.thehagenewasligatedwithpcdna3.1(+)tocon- structtheeukaryoticexpressionplasmidpcdna3.1(+)-ha,whichwasco-transfectedintohek293cels withaplv-ef1a-egfp(2a)puroplasmidbyaliposome-mediated method.therecombinantcelline HEK293-HA wasobtainedafterscreeningwithpuromycinandexpressionofhaproteinwasdetermined byflowcytometry.recombinantcelsweresubculturedfor15passagesandtestedforstabilityofexpres- sionofha proteinbyimmunofluorescenceassaysand Westernbloting.TherecombinantplasmidpcD- NA3.1(+)-HA wasidentifiedcorrectlybyecoriandbamhiendonucleasedigestionandsequencingana- lyses.threepositivecelcloneswithhighexpressionofha proteinwereobtained.stableexpressionof HAproteinwasshownbyimmunofluorescenceassaysand Westernblotinginrecombinantcelsaftercul- turefor15passages.a HEK293-HAcellineforstableexpressionoftheHAproteinoftheIBV wases- tablished:thiscouldprovidethefoundationforfurtherstudyofthehaproteinoftheibv. Keywords:InfluenzaBvirus;Hemagglutinin(HA)protein;Celline Funding:Thepresent work wassupportedbytwo Guangzhou Scienceand Technology Programs:(i)The majorprojectsof Health CareCol- laborativeinnovation (projectnumber ;specialized passiveimmunizationagainstsevereviralinfectionand developmentof an adenovirus monoclonalantibody);(i)projectnumber (Establishmentand applicationof ananimal modelofthehumanadeno- virusandinfluenzavirus). *Corresponding author:su

:TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposomet

:TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposomet 2 ( )208 58 JournalofNanchangUniversity(MedicalSciences)208,Vol.58No. TGF-β bfgf,, 2 (., 330006; 2., 5040) : -β (TGF-β ) (bfgf) (BMSCs), SD BMSCs Percol PCR TGF-β bfgf, pcdna3.(+), 3 BMSCs RT-PCR Westernblot