979 DOI:10.13350/j.cjpb.180911 ] MIC10(!F F,G H,IJ,KLL,MNO,, + ( V )!", 6T 6 6 )` _6/ F 45@, 6 _ 6/ F 45@, 6!, 214064) /0FG K,-10(MIC10),.3 45:; H PCR (RT PCR) RNA H/0 cdna,q 7 cdna 8? MIC10 D X,' FG' pet28a MIC10,H LMN` BL21(DE3)K (IPTG)4 5 L M ',#$< B=T>;S FG MIC10,- FG,- E,/0 MIC10, West ernblot:;fg MIC10,- RT PCR S cdna H ^416 bp DNA, DE :; 4^8? MIC10 DX;/0 FG' pet28a MIC10H LMN` BL21(DE3)KcIPTG LM, ' F G MIC10, -; F G MIC10, - E,V W X Y 1 2000006 Westernblot:; 1 7 : % O J MIC10 : @ % C %, " MIC10^+:,- /0 OJ % FG MIC10,-,. "1,-^+:,- K ;,-10; % ; R382.5!"#$ A %& 1673 5234(2018)09 0979 05 [.2018Sep;13(9):979-983.] 犘狉犲狆犪狉犪狋犻狅狀犪狀犱犮犺犪狉犪犮狋犲狉犻狕犪狋犻狅狀狅犳狋犺犲犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻犿犻犮狉狅狀犲犿犲狆狉狅狋犲犻狀 10 ZHOU Wei,SONGLi jun,shenshuang,yin Xu ren,xu Yong liang,liu Qian,YU Chuan xin ( 犓犲狔犔犪犫狅狉犪狋狅狉狔狅狀犘犪狉犪狊犻狋犻犮犇犻狊犲犪狊犲犆狅狀狋狉狅犾犪狀犱犘狉犲狏犲狀狋犻狅狀狅犳狋犺犲犖犪狋犻狅狀犪犾犎犲犪犾狋犺犪狀犱犉犪犿犻犾狔犘犾犪狀狀犻狀犵犆狅犿犿犻狊 狊犻狅狀, 犑犻犪狀犵狊狌犘狉狅狏犻狀犮犻犪犾犓犲狔犔犪犫狅狉犪狋狅狉狔狅狀犘犪狉犪狊犻狋犲犪狀犱犞犲犮狋狅狉犆狅狀狋狉狅犾犜犲犮犺狀狅犾狅犵狔, 犑犻犪狀犵狊狌犐狀狊狋犻狋狌狋犲狅犳犘犪狉犪狊犻狋犻犮 犇犻狊犲犪狊犲狊, 犘狌犫犾犻犮犎犲犪犾狋犺犚犲狊犲犪狉犮犺犆犲狀狋犲狉, 犑犻犪狀犵狀犪狀犝狀犻狏犲狉狊犻狋狔, 犠狌狓犻 214064, 犑犻犪狀犵狊狌 ) 犃犫狊狋狉犪犮狋 犗犫犼犲犮狋犻狏犲狊 Topreparearecombinantmicronemeprotein10(MIC10)from 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 andtoana lyzeitsimmunologicalcharacteristics. 犕犲狋犺狅犱狊 ReversetranscriptionPCR (RT PCR)wasfirstusedtopreparefirst strandcdnafrom ToxoplasmatotalRNA,andthenthefirst strandcdna wasusedasatemplatetoamplifythedna fragmentencodingthemic10maturepeptidewithgene specificprimers.thisgenefragmentwasclonedintotheplasmid pet28a(+ )toconstructtherecombinantexpressionplasmidpet28a MIC10.Therecombinantexpressionplasmid pet28a MIC10wastransfectedinto 犈. 犮狅犾犻 BL21(DE3)competentcelstodevelopanengineeredbacterium.Isopropylβ D 1 thiogalactopyranoside(iptg)wasusedtoinducepositiveclonesoftheengineeredbacteriumtoexpresstherecombi nantmic10protein.thepurifiedrecombinantmic10proteinwaspreparedusingnickelchelatingafinitychromatogra phy.arabbitwasimmunizedwiththepurifiedrecombinantmic10proteintoprepareiggpolyclonalantibodiesagainst themic10protein.theimmunologicalcharacteristicsoftherecombinant MIC10protein wereanalyzedusing Western bloting. 犚犲狊狌犾狋狊 AspecificDNAbandof416bpwasamplifiedfromthefirst strandcdnaof 犜. 犵狅狀犱犻,andthere sultsofdnasequencingverifiedthatthisdnafragmentisthegeneencodingthemic10proteinof 犜. 犵狅狀犱犻.IPTGin ducedtheengineeredbacteriumcontainingtherecombinantexpressionplasmidpet28a MIC10toexpresstherecombinant MIC10protein.ThetiterofserumIgGantibodiesagainsttherecombinantprotein MIC10inarabbitimmunizedwiththe purerecombinantmic10proteinwashigherthan1:200000.thenaturalmic10proteinoftheexcretedandsecretoryan tigen(esa)of 犜. 犵狅狀犱犻 wasrecognizedbythepurifiedantibodyiggagainsttherecombinantmic10protein,whichfur therconfirmedthatmic10isanexocrineprotein. 犆狅狀犮犾狌狊犻狅狀 RecombinantMIC10proteinfrom 犜. 犵狅狀犱犻 wassuc cessfulypreparedwithnaturalimmunogenicity,andfindingshaveindicatedthatthisproteinisanexocrineprotein. ' DJ (No.BK20151120); 6 6 (No.H201635); 0 6 6 (No.Q201656) 8,E mail:chxnyu@163.com () (1990-),,!,!6! :6.E mail:852918133@qq.com
980 犓犲狔狑狅狉犱狊 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 ;micronemeprotein10;immunogenicity;immunodiagnosis K 16 " Vc P 7 Z P7 ",4 P70^ R" 1,1 ^ )% [1-2],! E 1 # $ ^ # E^ P7% K1( 6,M ( O *,' ( [3-4] Y, ), K M FK [5-6] 2 79], Y! @ [7] 3 _ _ 6/ 3!!a F 3 )% VW T UI6 &K: @ :@67, 8 VW SUI^ %,W 0XL,, #$ _ 5)5 [8] UI #3 '"^ $ 9,. ^ % %: D,-:@, A2 % :, % UI S 3 % Y [9-11],-10(MIC10)! N 6,-:@ [12], 1 K4 ^ V,C^ %!,MIC102^ V W ],6 B : @^UI] 1 S, 2 ) Y 3! D\. 38? MIC10,-:@C DX45,3' FG MIC10,- 45 :;,6a 0 ] *+, 1 *+ 1.1 XY RH! )%67,3 IG(A " ` L M N ` DH5,BL21(DE3)6 ' pet28a(+)!3 IG(A? 3 E C D P745 1.2 Q R RNA _ (TRIZOL Rea gent)t RT PCR (PhusionTM RT PCR Kit) CD NEB ;TaqDNA bb#, DNA (WizardPlus MiniprepsDNA Purification System),Q/ $ # XhoⅠ T NcoⅠ T4DNA #CD Promega ; L > A,- (SPA)=T>; T<=T>; CD 67 Q ( );HRP G EIgG C D Bethyl ; ECL (Beyo ECLPlus)CD M N O 6 7 Q ; K (NC)+, ^ Sigma N 2 2.1 MIC10 *781 ()*/0\ 2.1.1 MIC10 D : GenBank TgMIC10 & K E ( ^ XM _ 002367312.2) TgMIC10 D 7,! [ 67 Q BC 7(Prim er1 ): 5 ' TACCATGGAGCACGATTTTTTTA ACGATTA 3'; 7 (Primer2):5' TTCTC GAGCATTGATTTCCTGCGTCTTGCGAGTT 3 ' 7 5'1: Q/ $# NcoⅠT Xho Ⅰ#$ W RNA _ _ RNA,Q RT PCR B C cdna,6b^z 45 PCR PCR 8 :2X MasterMix50μl,PrimerF1 2μl,PrimerR2μl,cDNA5μl,RNase free H 2O41 μl, S :95 30s,94 30s,55 30s, 35,68 1min,68 10min,4 (A 8& K,_ PCR N710μl451%, D 2.1.2 MIC10 D PCR N 7 PCR MIC10 DX D X2 K (datp) PCR 8 2 TaqDNA b B # 0 4 5 5'1 A,.4 5 A MIC10 DNA X _ TA? pgem T 9 5 1 B,2 T4 DNA # 0,' F G pgemt MIC10 N7 H 犈. 犮狅犾犻 DH5α *B+,,H ` J (100μg/ml) LB,37 bc$ 2.1.3 O ^_ `a DNA 45 DNA, DNA c NcoⅠT XhoⅠ#$O, ` Invitrogen IE 2.2 MIC10 /0XY* Z ()/0\ 2.2.1 MIC10 pet28afg' ' Q / $ # NcoⅠ T XhoⅠ 3 MIC10 pgemt 45#$,#$N7c1% K,Q
981 PCR N7 45, # $ MIC10 X B DNA X _ c NcoⅠ XhoⅠ #$ pet28a(+) DNA : @97 1 B,2 T4DNA # 0 45,' F G ' pet28a MIC10 N7H ' " ` BL21(DE3) *B+,,H N7 ( (50μg/ml) LB,37 bc$ ^_ `a 3mlLB b c ( 50μg/ml( ),37 200r/min bc$ 61 100 $ bc` 3 ml LB bc ( 50μg/ml( ),37 200r/min b c 4h A 600 ^0.4, IPTG ^1mmol/L 45L M ', b c 4h K + `,# $ SDS PAGE :;,-' 2.2.2 MIC10,- ]' ]' 1L ` 4 5 L M ',. + `, 100mlPBS F + `, PMSF(0.1 mmol/ L), -3,JK45,610000g,4 10 min ` 7 W 7 : 45 SDS PAGE,: ; ' N 7 1 <=T > ; W 1 F G MIC10,-, " S 0,-c PBS ; G PEG20000 K45 BSA S,- ],. 4512% SDS PAGE :; 2.3 MIC10 6 7 8 9 * 4 5 _? 3 E 1,] %,MIC10,-]^1 mg,c - %,MIC10,-]T S ] R R^2 3 K 6d _ V,: VW,6 FG MIC10, - ^ %(10μg/ml), 3 ELISA S I V W X Y,% XY `a K, 1mg,- PBS 45,1 Kc P V,: VW ProteinA =T> ' =T>;, EVW PBS 1 10 K45 >;, 1mol/LTris HCl(pH=8.0)T PBS 3 IgG 45 ;\,.45,- ]T SDS PAGE :; 2.4 MIC10 ( ) : -. _ F G MIC10, - 7 : % (ESA) 1 C %(AWA)45 SDS PAGE.H+, JK6 MIC10 IgG ^,HRP G EIgG ^ 45 Westernblot,c ECL L K#$ C (GelDocXR+ C ) MIC10 _ %,- 8 1 犕犐犆 10'5 犘犆犚,ST 6 cdna ^Z, MIC10 745 PCR,c 1% U I, N7 ^400bp,_`a DX(416bp) \#( 1) c TA S B DX TA pgem T,' C F G pgemt MIC10 1 DNA 7 2 MIC10 D PCR N7 1 犕犐犆 10'5 犘犆犚 1% @A 1 ThemarkerofDNAstandardmolecularweight 2 Theprod uctofmic10amplifiedbypcr 犉犻犵.1 犃犿狆犾犻犳犻犮犪狋犻狅狀狅犳犕犐犆 10 犵犲狀犲 2 <=>@A '567 4 TA J LB ` a $ b c DNA,Q Q / &K $# XhoⅠ NcoⅠ45#$,#$N7c 1% : ;,N 6 _ TA pgem T \ #, _ ` a MIC10 D X \# X( 2) DNA E : ; 4 F G B + DNA X & KE _ Gen Bank MIC10 D& KE,' " D MIC10 C 3 <- =>?, =- #$ MIC10 DX_ #$ ' pet28a(+ ),' F G ' pet28a MIC10, N7H BL21(DE3) * B +,, bck_ `a IPTG LM,SDS PAGE :; ' N7 \3:@ ](Mr) ^ 20 10 3 FG -B,-(1FG,- 6 GJK Q, 4 ^20.6 10 3, `a 17.7 10 3 ) 1,-: @A2 +` W,^ 1, - ( 3) ]' MIC10,-,c< B=T =T>; 1 FG MIC10,-( 4)
982 1 DNA 7 2 F G pgemt MIC10 c NcoⅠ T Xho Ⅰ#$ 3 FG pgemt MIC10 2 <=> 狆犌犈犕犜 犕犐犆 10DE 4 1 ThemarkerofDNAstandard molecularweight 2 There strictiveproductsofrecombinantplasmidpgemt MIC10digestedby restrictionenzyme NcoⅠ/XhoⅠ 3 The recombinant plasmid pgemt MIC10 犉犻犵.2 犚犲狊狋狉犻犮狋犻狅狀犪狀犪犾狔狊犻狊狅犳狋犺犲狉犲犮狅犿犫犻狀犪狀狋狆犾犪狊犿犻犱狆犌犈犕犜 犕犐犆 10 1,-:@ ] 2 FG MIC10,- 4 ; < 犕犐犆 10 犛犇犛 犘犃犌犈 4 1 Proteinstandard molecularweightmarker 2 Thepurified MIC10protein 犉犻犵.4 犃狀犪犾狔狊犻狊狅犳狋犺犲狆狌狉犻犳犻犲犱狉犲犮狅犿犫犻狀犪狀狋犕犐犆 10 狆狉狅狋犲犻狀 1, - : @ ] 2 pet28a MIC10 H L M N ` BL21 3 pet28a MIC10 L M N ` BL21 ' N 7 W 4 pet28a MIC10H LMN` BL21 ` 3 < - 犕犐犆 10 犛犇犛 犘犃犌犈 4 1 Standardproteinmolecularweightmarker 2 Theprecipitate oflysate of 犈. 犮狅犾犻 BL21 containing recombinant plasmid pet28a MIC10 3 Thesupernatantoflysateof 犈. 犮狅犾犻 BL21containingre combinantplasmidpet28a MIC10 4 ThewholecelproteinofThe 犈. 犮狅犾犻 BL21transformantscontainingrecombinantplasmidpET28a MIC10 犉犻犵.3 犃狀犪犾狔狊犻狊狅犳犲狓狆狉犲狊狊犻狅狀狆狉狅犱狌犮狋狊狅犳犕犐犆 10 狆狉狅狋犲犻狀 4 < 犕犐犆 10 013O 6FG MIC10,-?3 E, 4 K V,: VW, ELISA I VWXY 1 200000 EVW ProteinA =T>;, IgG Westernblot 1 FG MIC10,- 7 : % O J MIC10,' " F G MIC10, - _ O J MIC10\# 8 1 1 %, " MIC10,- 6+: ^,-:@,2 ]Y( 5) 1,-:@ ] 2 F G MIC10, - _ MIC10 8 3 ESA _ MIC10 8 4 AWA %3 5 < 犕犐犆 10 犠犲狊狋犲狉狀犫犾狅狋 4 1 Proteinstandard molecularweightmarker 2 Recombinant MIC10proteinrecognizedbytheanti MIC10antibodyIgG 3 ESA recognizedbytheanti MIC10antibodyIgG 4 AWArecognizedby theanti MIC10antibodyIgG 犉犻犵.5 犠犲狊狋犲狉狀犫犾狅狋犻狀犵狅犳狉犲犮狅犿犫犻狀犪狀狋犕犐犆 10 Y J )%,4! # $,!TP7, :! 10%6 [13] 2 " +,$., : & > :,- _ $. [14-15] :,-(MIC) " +,$. ] :, - [16]! MIC / &'=, A,-2 $. / 0, MIC1 MIC4 MIC6 [17-18] Hof [12] <2 " +,$., : E / & ', -,
983 MIC10,W1,->A2 T7 : %(ESA) A,-:@ 4 V, A2 % :, Y, 0 a ] ^ MIC10 Y,3! 8? MIC10:@C DX,. DFG 2 +' T/0 FG MIC10 - B, - 1,-?3 EN6 XY 1 200000 6, FG MIC10,- % West ernblot:; 1 F G MIC10, -,T 7 : % (ESA) O J MIC10, -, " F G MIC10, - _ A 2 7 : % OJ MIC10,- \ DB,1FG MIC10,- 1 0^UI UI^ OJ % S Westernblot: ; F G MIC10 1 C %(AWA), -C:,'" MIC10 2 % ]%Y, A &5 MIC10:@ +:,-,DB 10^ % S U I ] 3! C K / 0 F G MIC10,1,- % T 8%,^ X % S! [1] DubeyJP.Thehistoryof 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 thefirst100years [J].JEukaryotMicrobiol,2008,55(6):467-75. [2] HeJJ,MaJ,WangJL,etal.AnalysisofmiRNA expression profilinginmousespleenafectedbyacute 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 in fection[j].infectgenetevol,2016(37):137-42. [3] DubeyJP,JonesJL. 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 infectioninhumansand animalsintheunitedstates[j].intjparasitol,2008,38(11): 1257-78. [4] `[,,,. 5 [J].V ) 2013,25(1):56-8,79. [5] HH,( [,,. K K # D ' % : ; [J]. 6 _ 6 ), 2013,8(1):12-6. [6] TorreyEF,YolkenRH.Toxoplasmaoocystsasapublichealth problem[j].trendsparasitol,2013,29(8):380-4. [7] Wang H,WangT,LuoQ,etal.Prevalenceandgenotypesof 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 inporkfrom retail meatstoresin Eastern China[J].IntJFood Microbiol,2012,157(3):393-7. [8] `,, E,. ) S! 4 [J]. 6 ),2008,21(1):28-33. [9],,,. K ROP16 ROP18 B D7&'? ' [J].)%67,2017,12(1): 57-61. [10], 4,,. K &, - 10 6 789:;[J].)%67,2017,12(9):855-9. [11],,9"F,. K MIC8CTD 0,- O [J].)%67,2014,9(3):255-7. [12] HofEF,CookSH,ShermanGD,etal. 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 : molecularcloningandcharacterizationofanovel18 kdasecretory antigen,tgmic10[j].expparasitol,2001,97(2):77-88. [13] 0, 6,!. &[J].P 7 4,2008,10(7):101-4. [14] Carruthers VB,Sibley LD.Sequentialproteinsecretionfrom threedistinctorganelesof 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 accompaniesinva sionofhumanfibroblasts[j].eurjcelbiol,1997,73(2):114-23. [15] DubremetzJF,Achbarou A,BermudesD,etal.Kineticsand paternoforganeleexocytosisduring 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 host celinteraction[j].parasitolres,1993,79(5):402-8. [16] <., - MIC3 6 7 %! [D]. :",2011. [17] Fourmaux MN,AchbarouA,Mercereau PuijalonO,etal.The MIC1micronemeproteinof 犜狅狓狅狆犾犪狊犿犪犵狅狀犱犻 containsadupli catedreceptor likedomainandbindstohostcelsurface[j].mol BiochemParasitol,1996,83(2):201-10. [18] BrechtS,CarruthersVB,FergusonDJ,etal.Thetoxoplasma micronemalprotein MIC4isanadhesincomposedofsixconserved appledomains[j].jbiolchem,2001,276(6):4119-27. 2018 05 31 Z[ 2018 07 25 檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼檼 ( 978 ) [8] ReadTD,FraserCM,HsiaRC,etal.Comparativeanalysisof 犆犺犾犪犿狔犱犻犪犫犪犮狋犲狉犻狅狆犺犪犵犲狊 revealsvariationlocalizedtoaputative receptorbindingdomain[j].microb Comp Genomics.2000,5 (4):223-31 [9] GarnerSA,EversonJS,LambdenPR,etal.Isolation,molecular characterisationandgenomesequenceofabacteriophage (Chp3) from 犆犺犾犪犿狔犱狅狆犺犻犾犪狆犲犮狅狉狌犿 [J].VirusGenes,2004,28(2): 207-14. [10],R#, $,. % `,- VP1 (7 &' B +, ' W! [J].O %, 2014,20(3):188-91. [11] RankRG,BowlinAK,CaneS,etal.Efectof 犆犺犾犪犿狔犱犻犪狆犺犪犵犲 phicpg1onthecourseofconjunctivalinfectionwith" 犆犺犾犪犿狔犱犻犪犮犪狏犻犪犲 "inguineapigs[j].infectimmun,2009,77(3):1216-21. [12] MaJY,LiuY,LiuYJ,etal.Thepresenceof 犆犺犾犪犿狔犱犻犪狆犺犪犵犲 PhiCPG1capsidprotein VP1genesandantibodiesinpatientsin fectedwith 犆犺犾犪犿狔犱犻犪狋狉犪犮犺狅犿犪狋犻狊 [J].ActaBiochim Pol,2016, 63(3):501-4. [13] (, \,& ' ;,.J % porf5, - 3 HeLa+,D [J]. ) % 6 7,2017,12(4): 311-6. [14] Hsia RC,Ting LM,BavoilPM. Microvirusof 犆犺犾犪犿狔犱犻犪狆狊犻狋犪犮犻 strain guinea piginclusionconjunctivitis:isolation and molecularcharacterization[j].microbiology,2000,146(7):1651-60. [15] LiuBL,EversonJS,FaneB,etal.Molecularcharacterizationof abacteriophage (Chp2)from 犆犺犾犪犿狔犱犻犪狆狊犻狋犪犮犻 [J].J Virol, 2000,74(8):3464-9. [16] SaitM,LivingstoneM,GrahamR,etal.Identification,sequen cingand molecularanalysisofchp4,anovelchlamydiaphageof 犆犺犾犪犿狔犱狅狆犺犻犾犪犪犫狅狉狋狌狊 belongingtothefamilymicroviridae[j].j GenVirol,2011,92(7):1733-7. 2018 05 06 Z[ 2018 07 28