犃犓犜 £⁄¥rƒ2§¤'“«‹IA›‚

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!49" !# 2021$% !"#$%&'(()&'()*JournalofNorthwestA&FUniversity Nat.Sci.Ed. Vol.49No.2 Feb.2021 +,-*./20200813 11 14 DOI 10.13207 j.cnki.jnwafu.2021.02.003 +,-*01https :// kns.cnki.net kcms detail 61.1390.S.20200812.1107.002.html ¢ 犃犓犜 £¤¥r¦2§¨©ª«¬IA- 234#20191231 56789:&'()567831872589 ); 9:;<=>?@782018YFD0900501 2019YDF0900105 ); vóCþß³h> õ 7Åÿ³h>õ¼<)( GDME20181007 ); vóCpq/qu§þT!þ"#782019KJ141 GHIJ+ %1993- ), MÕA#BQTUVWXYKbEâãy=DEmail 987947884 qq.com cdGHï#$1978- ), h%O¡WQ=D±VWXYê/bZq=DEmail liningq 126.com + C DE" FGH *IJ KL% M ? 0;N `9ê/()=Dv Äßê/=DÖpqp&'¼'t!;<2¥< vóCê/ stâãu§;<2¥<vó vµ 510380 ïÅÅÿr) ê/Em()vïÅ 201306 D E8y=D) AKT2 ScAKT2 R}*Ú*Äbainfectiousspleenandkidneynecrosisvirus ISKNV õò`yG¼)ISKNV ¯。【 þ + 犛犮犃犓犜 5åF , S -º ¦s: æ® . þß às:æ®{|¥¼¥9y ScAKT2;{| âã 4 r /0 9 ® + Ñþ¼/ . ELISA ÛÜ Ñþyp1¼/.âã23 ± WesternblotÜ ( Ñþy e Úþ +) 犃犓犜 º ¦ Éæ® . þ pCMV EGFPScAKT2 4F7* CPBÍÎT`º ¦ ScAKT2 Éæ® ÍÎ T¼ 23 ë T 4 ÛÜ 7 * p ²¼ qRTPCR Westernblot( 犛犮犃犓犜 2 mRNA ê# ±{| ê# yæ®ú 3 ( ScAKT2Éæ® ÍÎ T û É º ¦6 ½4ISKNV .¿ ScAKT2 Éæ® ÍÎ T `¼ qPCR Westernblot( ISKNV ba 5 ¬ S ISKNVMCP{|y殼 qRTPCR Ü ( 犛犮犐犚犉 犛犮犐犚犉 犛犮犕狓 犛犮犐犔 犛犮犜犚犃犉 ± 犛犮犜犚犃犉 L ² âã å mRNA ê#yæ®Þß。【 ÝçPCR jõ6`1400bp y 犃犓犜 2 ORF 786½º¦s:殢%Pn æ®-72ku y;{|6 ½ ® ScAKT2 + ÑþÑþ p 1 ® 1∶256000 ¥ 9 ¢¬ YZ Ô 10 mg mL 6½º¦ pCMVEGFPScAKT2Éæ®.þ±Éæ®ScAKT2y CPBÍÎTÉæ®ScAKT2CPBÍÎ ÷îëìÈISKNV ò¾+ëï ¹ 犛犮犐犚犉 犛犮犐犚犉 犛犮犕狓 犛犮犐犔 犛犮犜犚犃犉 ± 犛犮犜犚犃犉 L ² âã å mRNA yæ®ê#。【 ÝöScAKT2cÉï¹² âã å yæ® È ISKNV ò )ISKNV ¯ þysR9<FGH)AKT2 }*Ú*Äbabaõò IJKLMS943.127.41 NOPQRNSTM16719387 2021 02001409 Roleof 犛犻狀犻 犲狉犮犪犮犺狌犪狋狊犻AKT in roliferationofinfectious leenandkidne necrosisvirus MINGYue NIUYinjie FU Xiaozhe LIULihui LIANGHongru LINQiang LINingqiu 犓犲 犔犪犫狅狉犪狋狅狉 犉犻狊犺犲狉 犇狉狌 犇犲狏犲犾狅 犿犲狀狋 犕犻狀犻狊狋狉 狉犻犮狌犾狋狌狉犲 犌狌犪狀 犱狅狀 犘狉狅狏犻狀犮犻犪犾犓犲 犔犪犫狅狉犪狋狅狉 狌犪狋犻犮 犃狀犻犿犪犾犐犿犿狌狀犲犜犲犮犺狀狅犾狅 犵狔 犘犲犪狉犾犚犻狏犲狉犉犻狊犺犲狉犻犲狊犚犲狊犲犪狉犮犺犐狀狊狋犻狋狌狋犲 犆犺犻狀犲狊犲犃犮犪犱犲犿 犉犻狊犺犲狉 犛犮犻犲狀犮犲狊 犌狌犪狀 狕犺狅狌 犌狌犪狀 犱狅狀 510380 犆犺犻狀犪 犆狅犾犾犲 犲狅 犉犻狊犺犲狉犻犲狊犪狀犱犔犻 犲犛犮犻犲狀犮犲 犛犺犪狀 犺犪犻犗犮犲犪狀犝狀犻狏犲狉狊犻狋 犛犺犪狀 犺犪犻 201306 犆犺犻狀犪 犃犫狊狋狉犪犮狋 :【 Objective Thisstudyinvestigatedtheroleof 犛犻狀犻 犲狉犮犪犮犺狌犪狋狊犻 AKT2 ScAKT2 inthe proliferationofinfectiousspleenandkidneynecrosisvirus ISKNV Method The犛犮犃犓犜 2geneopen readingframewascloned andtheprokaryoticexpressionvectorwasconstructedforprokaryoticexpression andproteinpurification.Afterpurification polyclonalantibodieswerepreparedafterimmunizingJapanese bigearrabbitswithScAKT2recombinantprotein ELISAmethodwasusedtodetermineantibodytiter and

Transcript of 犃犓犜 £⁄¥rƒ2§¤'“«‹IA›‚

!49" !2#

2021$2%

!"#$%&'(()

(&'()*

JournalofNorthwestA&FUniversity(Nat.Sci.Ed.)Vol.49 No.2

Feb.2021

+,-*./

:20200813 11:14  DOI:10.13207/j.cnki.jnwafu.2021.02.003+,-*01

:https://kns.cnki.net/kcms/detail/61.1390.S.20200812.1107.002.html

¢犃犓犜2£¤¥r¦2§¨©ª«¬IA­�

 [234#

] 20191231

 [5678

] 9:&'()5678

(31872589);9:;<=>?@78

(2018YFD0900501,2019YDF0900105);vóCþß³h>

õ 7

(Åÿ³h>õ¼<

)(GDME20181007);vóCpq/qu§þT!þ"#78

(2019KJ141)

 [GHIJ

] + %

(1993-),M,ÕA#BQ

,TU

,VWXYKbEâã��y=D

。Email:987947884@qq.com

 [cdGH

] ï#$

(1978-),h,%O¡WQ

,=D±

,VWXYê/bZ�q=D

。Email:liningq@126.com

+ C

1,2,DE"

1,FGH

1,*IJ

1,KL%

1,M ?

1,0;N

(1`9ê/()=Dv Äßê/=DÖ

,pqp&�'¼'t!�;<2¥<

/vóCê/

stâãu§;<2¥<

,vó vµ510380;2ïÅÅÿr) ê/Em()v

,ïÅ201306)

[D E

] 【8y

】=D)AKT2(ScAKT2)R}*Ú*�Ä�ba

(infectiousspleenandkidneynecrosisvirus,

ISKNV)õò`yG¼

,�)ISKNV�¯����

。【��

】þ+犛犮犃犓犜25åF�,S-

,º¦s:æ®.þ

,ß

às:æ®�{|¥�

。¼¥�9yScAKT2;�{|âã4�r/09�®Eþ+Ñþ

,¼/.ELISA�ÛÜ

Ñþyp1

,¼/.âã23± Westernblot�Ü(Ñþye�Ú

。þ+)犃犓犜25å

,º¦Éæ®.þpCMV

EGFPScAKT2,4F7*�CPBÍÎT`º¦ScAKT2Éæ®ÍÎT

,¼23ëT4ÛÜ7*p²

,¼qRTPCR

�� Westernblot��­Ü(犛犮犃犓犜2mRNAê#±{|ê#yæ®

,ú3(ScAKT2Éæ®ÍÎTûɺ¦6

½

。4ISKNV.¿�ScAKT2Éæ®ÍÎT`

,¼qPCR�� Westernblot��­Ü(ISKNVba5¬S�

ISKNVMCP{|yæ®

,¼qRTPCR�Ü(犛犮犐犚犉3、犛犮犐犚犉7、犛犮犕狓、犛犮犐犔8、犛犮犜犚犃犉2±犛犮犜犚犃犉3L�²âã

å�mRNAê#yæ®Þß

。【Ýç

】PCRjõ6`1400bpy犃犓犜2ORF78

,6½º¦�s:殢%

,Pn

æ®-72kuy;�{|

。6½�®�ScAKT2Eþ+Ñþ

,Ñþp1®1∶256000,¥�9¢¬YZÔ�10

mg/mL。6½º¦�pCMVEGFPScAKT2Éæ®.þ±Éæ®ScAKT2yCPBÍÎT

,Éæ®ScAKT2CPBÍÎ

÷îëìÈ�ISKNVyõò

,¾+ëï¹犛犮犐犚犉3、犛犮犐犚犉7、犛犮犕狓、犛犮犐犔8、犛犮犜犚犃犉2±犛犮犜犚犃犉3L�²âãå�

mRNAyæ®ê#

。【Ýö

】ScAKT2cÉï¹�²âãå�yæ®È�ISKNVyõò

,�)ISKNV�¯���

þysR9<

[FGH

] )

;AKT2;}*Ú*�Ä�ba

;baõò

[IJKLM

] S943.127.41+9 [NOPQR

] A [NSTM

] 16719387(2021)02001409

Roleof犛犻狀犻狆犲狉犮犪犮犺狌犪狋狊犻AKT2inproliferationofinfectious

spleenandkidneynecrosisvirus

MINGYue1,2,NIUYinjie1,FUXiaozhe1,LIULihui1,

LIANGHongru1,LINQiang1,LINingqiu1

(1犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳犉犻狊犺犲狉狔犇狉狌犵犇犲狏犲犾狅狆犿犲狀狋,犕犻狀犻狊狋狉狔狅犳犃犵狉犻犮狌犾狋狌狉犲/犌狌犪狀犵犱狅狀犵犘狉狅狏犻狀犮犻犪犾犓犲狔犔犪犫狅狉犪狋狅狉狔狅犳犃狇狌犪狋犻犮

犃狀犻犿犪犾犐犿犿狌狀犲犜犲犮犺狀狅犾狅犵狔,犘犲犪狉犾犚犻狏犲狉犉犻狊犺犲狉犻犲狊犚犲狊犲犪狉犮犺犐狀狊狋犻狋狌狋犲,犆犺犻狀犲狊犲犃犮犪犱犲犿狔狅犳犉犻狊犺犲狉狔犛犮犻犲狀犮犲狊,犌狌犪狀犵狕犺狅狌,

犌狌犪狀犵犱狅狀犵510380,犆犺犻狀犪;2犆狅犾犾犲犵犲狅犳犉犻狊犺犲狉犻犲狊犪狀犱犔犻犳犲犛犮犻犲狀犮犲,犛犺犪狀犵犺犪犻犗犮犲犪狀犝狀犻狏犲狉狊犻狋狔,犛犺犪狀犵犺犪犻201306,犆犺犻狀犪)

犃犫狊狋狉犪犮狋:【Objective】Thisstudyinvestigatedtheroleof犛犻狀犻狆犲狉犮犪犮犺狌犪狋狊犻AKT2(ScAKT2)inthe

proliferationofinfectiousspleenandkidneynecrosisvirus(ISKNV).【Method】The犛犮犃犓犜2geneopen

readingframewascloned,andtheprokaryoticexpressionvectorwasconstructedforprokaryoticexpression

andproteinpurification.Afterpurification,polyclonalantibodieswerepreparedafterimmunizingJapanese

bigearrabbitswithScAKT2recombinantprotein,ELISAmethodwasusedtodetermineantibodytiter,and

IFAmethodandWesternblotmethodwereusedtodetectantibodyspecificity.The犃犓犜2genewasalso

clonedtoconstructoverexpressionvectorpCMVEGFPScAKT2transfectedintoCPBcelllinestocon

structoverexpressioncelllines.Fluorescencemicroscopewasusedtoexaminethetransfectionefficiencyof

theeukaryoticexpressionvector,andtheqRTPCRmethodandWesternblotmethodwereusedtodeter

minetheexpressionof犛犮犃犓犜2mRNAandproteinlevelstoidentifywhethertheScAKT2overexpressing

celllinewassuccessfullyconstructed.InoculatingISKNVintotheScAKT2overexpressingcellline,qPCR

methodandWesternblotmethodwereusedtodetermineISKNVviruscopynumberandISKNVMCPpro

teinexpression,respectively.TheqRTPCRmethodwasusedtoexaminethemRNAexpressionofinnate

immunefactorsincluding犛犮犐犚犉3,犛犮犐犚犉7,犛犮犕狓,犛犮犐犔8,犛犮犜犚犃犉2and犛犮犜犚犃犉3.【Result】PCRamplifi

cationobtained1400bpof犃犓犜2ORFfragmentsandsuccessfullyconstructedprokaryoticexpression

plasmidstoinducetheexpressionof72kuofrecombinantproteins.ScAKT2polyclonalantibodywassuc

cessfullypreparedwithatiterof1∶256000andaconcentrationofabout10mg/mLafterpurification.CPB

celllinespCMVEGFPScAKT2overexpressionvectorsandoverexpressionScAKT2weresuccessfully

constructed.OverexpressionofScAKT2CPBcellsextremelysignificantlyinhibitedISKNVproliferation

andsignificantlyupregulatedthemRNAexpressionlevelsofinnateimmunefactorsincluding犛犮犐犚犉3,

犛犮犐犚犉7,犛犮犕狓,犛犮犐犔8,犛犮犜犚犃犉2and犛犮犜犚犃犉3.【Conclusion】ScAKT2inhibitedtheproliferationof

ISKNVbyupregulatingtheexpressionofinnateimmunefactors.Thisstudyprovidesanewpotentialtar

getforthepreventionandcontrolofISKNV.

犓犲狔狑狅狉犱狊:犛犻狀犻狆犲狉犮犪犮犺狌犪狋狊犻;AKT2;infectiousspleenandkidneynecrosisvirus(ISKNV);viruspro

liferative

  )

(犛犻狀犻狆犲狉犮犪犮犺狌犪狋狊犻)û:9;Wy;êÂ<

]òKQ

,�*gzy³h10

。n$�

,zÕZR

Ô�]ònù)KfbÕ>

,F`}*Ú*�Ä�

ba

(infectiousspleenandkidneynecrosisvirus,

ISKNV)û=Z:9)]òy;Wbs�Í

,Fù

bÒ�

,ù�²÷®90%úï

,�)]òqG6�

>ry³h�i

[12]。å8

,FõISKNVùb��

=D�ªby�¯�*;W?@

{|Ñ� B(proteinkinaseB,PKB)5å

,�

犃犓犜,ûÍ¿sA5å

,BCbR°¶DÍÎyÎ

¢`

。AKT�AKT1、AKT2± AKT33¿(�

F`AKT2û{|Ñ�B:µyVW(�

,�*Ë

/ü3Å

(Ser/Thr)Ñ�yÙÚ

[3],ûE¿ÍÎ

É5ywE¹¯å�

[4]。*=Dæ+

,犃犓犜Éæ®

÷ÑÙFµfE¿pgå�

,¹½�²Úâãå�

yæ®�ªF>{ÑbaG¼

[56]。T�Fêba

(hepatitisBvirus,HBV)yX{|

(hepatitisBvi

rusXprotein,HBX)÷ÑÙs^FÍÎ`yAKT,

È� HBXJnyÍÎG�

,X�È�T�Fêb

ay��

[7]。RøÖòEH§ÉwPba

(porcine

reproductive and respiratory syndrome virus,

PRRSV)) * y � #

,� þ AKT � Ñ Ù

,È �

PRRSVJnyÍÎG�

,X�>{FÑbaG

¼

[8]。êIÚJêba

(vesicularstomatitisvirus,

VSV))*.

,�þAKT�Ù�

,ß�ÓïTollH

¨þ4(Tolllikereceptor4)<=>{Ñba½

¾

[9]。)*KYba

(sendaivirus,SeV)yLVc

ÉÙ� AKT,ÑÙZMù½å�3(interferon

regulatoryfactor3,IRF3)yæ®

,X�õ�βZM

Ã

(interferonβ,IFNβ)yæ®� / m Ñ b a p

g

[10]。÷Õ

,AKT÷úG�Í�Ñba9ͼ°

Ñba��y=D

8m�°)AKT2RISKNV)*É5`yG

¼Nò»O

,�8�=Dþ+�)犃犓犜2yF�,

S-&ßàs:æ®

,�®;�ScAKT2Eþ+Ñ

þ

,º¦)犃犓犜2Éæ®yÍÎT

,=DScAKT2

RISKNVõò`yG¼

,ú#�)ISKNV�¯�

�þy9<

1 �HE��

1.1 W X

1.1.1 QR

、ST

、UV;!"#$ )Ï�äÍ

Î T

(Chineseperch braincellline,CPB)[11]、

ISKNVa^

(QY20091015)[12]

�pET32a±pC

MVEGFP.þ

,ÓÏ`9ê/()=DvÄßê

/=DÖpqp&�'¼'t!�;<2¥<

/v

51!2# + %

,L

:)AKT2R}*Ú*�Ä�baõò`yG¼

óCê/stâãu§;<2¥<;b

;ríJ<

(犈.犮狅犾犻)DH5a± BL21(DE3),¤& TaKaRa(r

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1.1.2 ! % L15�]5

、�{|�

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Hanks@

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,¤

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)©ª

;Åÿst5å���¤

¢£

,¤°²_m�(u

(±¥

)*¨©ª

;Titanium

OneStepRTPCRKit¤¢£

、犜犪狇ManRealtime

PCR Master Mix ± TB Green? Premix Ex

犜犪狇TMⅡ (TliRNaseHPlus),¤°TaKaRa(r®

©ª

;iQ2¤¢£

,¤°OMEGA(Ì9

)©ª

;Ni

NTARº�S±TÉ�

(1mL),¤&m°mt°

5

(ïÅ

)»¼*¨©ª

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ÈW¢

,¤&Sigma(Ì9

)©ª

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¤¢£±PureYield(TM)¢%`�¤¢£

,¤°

Promega(Ì9

)©ª

;ProteoSpinTM

AXþ�º¥�

¤¢£

,¤Y°ZÌ[(u

(\]

)*¨©ª

;G418

(Geneticin,̂ }_Ã

)、¾FITCÍ�y23Ëѱ

DAPI*H

,¤&�`¯mt*¨©ª

(±¥

);

ISKNVMCP v þ + Ñ þ

,Ï � 2 ¥ < ; b

犈犮狅RⅠ、犛犪犾Ⅰ、犓狆狀Ⅰ�±T4DNALigase,¤&

TaKaRa(r®

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1.2.1 WXY)Z[/\]^ ¼Trizol¤¢�

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RNA^7Ý�cDNA,¼Åÿst5å���¤¢

£��)*ISKNV9yCPBÍÎDNA(d3�m

t);�

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1.2.2 犃犓犜2_`abc

(ORF))de/fg2

3UV)hi (1)犃犓犜2ORFyþ+

。_`�a

_�)Ü(y)7Ý�Ýç

,d? 犃犓犜2ut

ScAKT2F(5′CCG犌犃犃犜犜犆ATGAATGAAGTCA

GTGTTGT3′,F`bþ���犈犮狅RⅠ�Ö�<

± ScAKT2R (5′ACGC犌犜犆犌犃犆TCATTCCCG

TATGCTGGCAGAG3′,F`bþ���犛犪犾Ⅰ�

Ö�<

)。¼z;c�PrimeSTAR? HSDNAPol

ymerasejõ犃犓犜2y ORF,PCR^gÐþT�

50μL:5×PrimeSTARBuffer(Mg2+ Plus)10μL,

dNTPMixture(2.5mmol/Leach)4μL,ScAKT2F

± ScAKT2R ê 1μL,Template2μL,Prime

STAR? HSDNA Polymerase0.5μL,DEPC ê

31.5μL。^g=>

:98℃10s,55℃5s,72℃2

min,r30�pq

。PCR/t¼1.2%y�È�­

i\]9Üä

,&ÖiQ2PCR/t

(2)s:æ®.þpET32aAKT2yº¦

。¼

犈犮狅R Ⅰ ± 犛犪犾 Ⅰ x . ¦ � Ö 犃犓犜2 ORF ±

pET32a.þ

(16℃�Ö12h),¼1.2%y�È�

­i\]ÛÜ�Öp²

,ÖiQ28y=¾±6Ú

�ypET32a.þ

,4Fú3∶1yéd¼T4DNA

Ligase®.

,̂gþT�

:T4DNALigaseBuffer1

μL、8y=¾7μL、6Ú�.þ1μL± T4DNA

Ligase1μL,R16℃ô6®.Àï®.16h。4®

./t7DríJ<)¨"ÍÎ DH5α`

,�200

μL/t m

,Ée�]9

,¢�vþ+ßà<¤

PCR3(

,�PÚ<¤Üä

1.2.3 WAKT2fgjk)OP23&lm 4

º¦ys:æ®.þpET32aAKT27�ríJ<

)¨"ÍÎBL21(DE3),&.¿�fþI�g{|

h�]5

(£3iû_Ã50μg/mL)#m`

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。4Üä|�y£;

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(£3iû_Ã50

μg/mL)̀ ,°37℃jk�]�<@R600nm!y

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W,°G3s、mn4s)oJ<þ2~3min;4℃、

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。� â ã m

、9 ¹ » ß à

1∶2000,1∶4000,1∶8000,…,1∶8192000{Z

�V

,�]2μg/mLyÑsA�

,̧¼/.ELISA

��Ü(Eþ+Ñþyp1

,úâã9¹»OD4500/

âãm¹»OD4500>2(犘/犖>2)�|}Ís

。i¼

NiNTARº�S±TÉ¥�Ñþ

,¼BCA�Ü(

¥�9Ñþy¢¬YZ

,&ßàSDSPAGE3(

¸¼ Westernblot�ÛÜÑ AKT2Eþ+Ñ

þye�Ú

({|ê#

)。�þ��wµ

:i¼ RI

PAqá@��CPBÍÎÐ{|

(d3�mt);

61 ʱpË(ur))�

(&'()*

)!49"

),³BCA�Ü({|YZ9

,ßàSDSPAGE

�º&7¶�0.45μLPVDFÅï

;ú�®y0Ñ

ScAKT2Eþ+Ñþ�ÍÑ

,çÑ0IgGHRP�Ë

Ñ

,ßà WesternblotÛÜ

¸¼/.âã23�ÛÜ AKT2Eþ+Ñþ

ye�Ú

(ÍÎê#

)。��wµ

:¼ùÁy~Nf

(CPBÍÎ

,³ TritionX100���50g/L y

BSAkl9

,¼³50g/LBSA �V

(1∶50)y

ScAKT2 Ñ þ

(Í Ñ

)± ³ 50g/L BSA � V

(1∶2000)y¾*FITCÍ�yËÑ�­��

,¼

DAPI*O9§D��U@

,4Û

1.2.5 p23WAKT2QRq)ir (1)犃犓犜2

ORFy þ +

。_ ` ) 犃犓犜2 ä a

,d ? u t

AKT2F(5′CCG犌犃犃犜犜犆ATGAATGAAGTCA

GTGTTGT3′,bþ���犈犮狅R Ⅰ�Ö�<

)±

AKT2R(5′CGG犌犌犜犃犆犆TCATTCCCGTATG

CTGGCAGAG3′,bþ���犓狆狀Ⅰ�Ö�<

)。

PCRjõ犃犓犜25å

,�þ��x1.2.2(1)。

(2)Éæ®.þpCMVEGFPScAKT2yº¦

i¼犈犮狅RⅠ±犓狆狀Ⅰ4犃犓犜2ORF®.�pCMV

EGFP.þ

,º¦Éæ®.þpCMVEGFPAKT2,�

þ��x1.2.2(2)。<¤PCRÛÜ9

,Üä

(3)É æ ® AKT2 Í Î T y º ¦

。¼ Pure

Yield(TM)¢%`�¤¢£��<@`y¢%

,i

¼FuGENE? 64pCMVEGFPScAKT2.þ±

pCMVEGFPo.¢%7*� CPBÍÎ`

,�]

24h9¸¹23æ®Þß

。ª¼�ù�¢¬YZ

�1000μg/mLy G418�7*6½yCPBÍÎ

ßà®È§·

,-��O23{|����t

÷úâ(}^

(4)Éæ®AKT2ÍÎTy3(

。úâ(}^

yÉæ® AKT2ÍÎT�¤¥�

,â(}^pC

MVEGFPo.yÍÎT����

,�3�mt)

;�

,ßà7ݱ{|ê#ïy3(

。ú18S�ÿ

,¼23(¬ PCR(qRTPCR)ÛÜ 犃犓犜2m

RNAyæ®ê#

。_`GenBank`yScAKT2ä

a

(GenBankÙ

:KY984992.1),d?utF(AGC

CACAAGTTCTTCACCTCCATC)± R(CGCT

GATCTGAATCCTCCGCATC),jõ78ry�

188bp;úcDNA�æm

,18S�ÿ�

(Ö¼uty

èw d D Õ æ 1),ª ¼ TB Green? PremixEx

犜犪狇TMⅡ (TliRNaseHPlus)ÛÜ犃犓犜2mRNAy

æ®ê#

。ÐþT�20μL:2×SYBR ù ú ¢

犜犪狇TM10μL,|、̂ ut

(10μmol/L)0.5μL,ROX

�é*HⅡ0.5μL,cDNA2μL,ê6.5μL。PCR

^g5ä�

:95℃ùñÚ30s;95℃ ñÚ5s,56

℃�Ú30s,r40�pq

。¸R23dÙ

,̧¼

2-ΔΔ犆狋� ? /犃犓犜2 mRNA y è � æ ® ¬

。ú

βactin{|�ÿ�

,¼ WesternblotÛÜÉæ®

AKT2ÍÎ{|yè�æ®ê#

>1 ®¯�@PCR��Avw7°±F��

Table1 RelatedinformationoftheprimersusedinfluorescencequantitativePCR

5åÂ�

Nameofgene

utäa

(5′→3′)

Sequenceofprimer(5′→3′)

GenBank�ÝÙ

GenBankaccession

number

jõ78

ry

/bpProductsize

犛犮犐犚犉3 F:CAGATTGACAGCGGCAGGTATCC,R:GCCATTGCCACTCGCCTCTG KY646446.1 150

犛犮犐犚犉7 F:ATCCTCAGCCGGTCCTCAGTTC,R:CGAGTAGGTAGTGGAGCTGGAGAC KY646451.1 132

犛犮犐犔8 F:TGTGGTGCTCCTGGCCTTCC,R:TGATCTCAGTCTCCTCGCAGTGG JQ513375.1 167

犛犮犕狓 F:CCACTGCTGAATCCACGCTGAG,R:ACTGCTGCTGTAGGTCCTGTCC AY392097.1 80

犛犮犜犚犃犉2 F:GGTCGCAGGTGTACGGAACTTG,R:GCTGACGGTTGCTGGCTTCC KP409173.1 115

犛犮犜犚犃犉3 F:GAGACTGAACGCTCTGGAACAAGG,R:GCCAATCGGATCTCGTGGACAC KP409193.1 126

犛犮18犛 F:CATTCGTATTGTGCCGCTAGA,R:CAAATGCTTTCGCTTTGGTC AY452495.1 102

犗犚犉007 F:CGAGGCCACATCCAACATC,R:CGCCTTTAACGTGGGATATATTG NC_003494.1 83

犗犚犉007Probe FAMCACCAAACTGACCGCGGACTCGTEclipse

1.2.6 p23AKT2)CPBQRosStu@v

 (1)Éæ®AKT2�ISKNVba5¬Sy��

_`ISKNVRCPBÍÎïyõòë#

[13],¼º¦6

½yÉæ® AKT2±o.y CPBÍÎ�­)*

ISKNV()*�S

(MOI)�1),R)*9°ISKNV

yõò�#

(36,48h)��CPBÍÎyDNA,ú��

y DNA � æ m

,¼ qPCR �

[14]Ü ( ISKNV

犗犚犉0075åyba5¬S

,Ö¼y犗犚犉007ut

���ut犗犚犉007probeyèwdDÕæ1。

  (2)Éæ® AKT2�ISKNVMCP{|y�

。R)*ISKNV48h9

,��CPBÍÎyÐ{

|

,d83���

,úISKNVMCP(1∶500)vÑ

βactinÑþ

(1∶5000)(ÿ�

)�ÍÑ

,¼ Western

blotÛÜISKNVMCP{|yæ®Þß

(3)Éæ®AKT2��²âãå�R7Ýê#

ïè�殬y��

。úcDNA�æm

,18S�ÿ

71!2# + %

,L

:)AKT2R}*Ú*�Ä�baõò`yG¼

,¼qRTPCR�ÛÜ犛犮犐犚犉3、犛犮犐犚犉7、犛犮犐犔8、

犛犮犕狓、犛犮犜犚犃犉2、犛犮犜犚犃犉3L�²âãå�R7

Ýê#ïyæ®Þß

。_` GenBank`©¡yè

wäa

,̧¼Primer5.0c>d?ut

,utyèw

dDÕæ1。PCR^gþT±5äx1.2.5(4),̧ ¼

2-ΔΔ犆狋��?/ê5åmRNAyè�æ®ê#

1.3 xy²ZoK�

¸¼ GraphPadprime6.0ßàS`Ò~±�

,ª¼SPASS21.0ßàS`t�èwÚ�É

¤¥S`æu�

“#Ó0±Ís�

”,狀=3,ú“”̂

æ犘<0.05,“”̂ æ犘<0.01。

2 ÝçE�É

2.1 犛犮犃犓犜2ORFA³´7µ¶>?�·A¸¹

³PCRjõ`¯Ô1400bpy犃犓犜2ORF7

8

(�1A),Eù#ryè®

。ÜäÝçæ+

,)

犃犓犜2 y ORF � 1449 bp。s : æ ® ¢ %

pET32aScAKT2yPCRjõ6`�Ô1400bp

y78

(�1B),Üä3(6`�1449bpy78

æ+s:殢%pET32aScAKT2º¦6½

A.)犃犓犜2ORFyþ+

;B.s:殢%pET32aScAKT2yPCR3(

;M.DNAMarkerDL15000;1~4.犃犓犜2

ORFyþ+

;5,9.犃犓犜2ORF7�i�yHr

;6~8.犃犓犜2ORF7�6½yHr

A.Cloningof犃犓犜2ORF;B.PCRidentificationofprokaryoticexpressionplasmidpET32aScAKT2.M;DNAMarkerDL15000;

1-4.Cloningof犃犓犜2ORF;5,9.Samplesfailedtransfectedwith犃犓犜2ORF;6-8.Samplessuccessfullytransfectedwith犃犓犜2ORF

�1 )犃犓犜2ORFþ+�s:殢%y3(

Fig.1 Cloningof犃犓犜2ORFandidentificationofprokaryoticexpressionplasmid

2.2 ScAKT2º^+,A ¡>?7»4

³SDSPAGEÛÜ

,RPQ`ÛܯÔ72ku

(£Í�Ô20ku)y=¾

(�2A),E+ScAKT2;

�{|úAXþytçæ®

。¥�9

,;�{|V

WR`R72ku��

(�2B),÷úßà9Èâã

¤¥

A.AKT2�w{|ySDSPAGE�É

;B.AKT2�w{|y¥�

;M.{| Marker;1.PQ

;2.ï»

;3.¥�9yAKT2�w{|

A.SDSPAGEanalysisofAKT2fusionprotein;B.PurificationofAKT2fusionprotein;M.ProteinMarker;1.Precipitation;

2.Supernatant;3.ScAKT2fusionproteinafterpurification

�2 )AKT2�w{|yPnæ®�¥�

Fig.2 Induction,expressionandpurificationofScAKT2fusionprotein

2.3 pScAKT2¼³´p½A¾¿

¼¥�y;�AKT2{|âã4�r/0

,/

.ELISA�Ü(¹»âãp1÷®1∶256000(�

3A)。�¥�yScAKT2Eþ+ÑþßàSDS

PAGEÛÜ

,Ýç*�==¾

,F`Í=�Eþ+Ñ

þy;�

(Ô45ku),³Í=���

(Ô25ku),¥

81 ʱpË(ur))�

(&'()*

)!49"

�pç�Å

(�3B)。³BCA�Ü(

,¥�9yÑ þ¢¬YZÔ�10mg/mL。

A.ÑScAKT2Ñþyâãp1

;B.ÑScAKT2Ñþy¥�

;M.{| Marker;1.¥�9yÑþ

A.TheimmunetiterofantiScAKT2antibody;B.AntiScAKT2antibodypurification;M.ProteinMarker;1.Purifiedantibody

�3 ÑScAKT2Eþ+Ñþp1yÜ(E¥�

Fig.3 DeterminationofantiScAKT2polyclonalantibodytiterandpurifiedantibody

  �®yEѳ Westernblot3(

,RÔ60ku

!*1=e�Ú=¾

(�4A),æ+�®yEþ+

Ñþ¾e�Ú�­CPBÍÎy AKT2{|

。/.

âã23¤¥Ø2

,Ö�®yÑScAKT2EÑ÷�

­CPBÍÎ

,tScAKT2VWRCPBÍÎyÍÎ

¢`æ®

(�4B)。

A.WesternblotÝç

;B./.âã23Ýç

;M.{| Marker;1.ÑþECPBÍÎe�ÚÝw

A.Westernblotresult;B.Indirectfluorescentimmuneresult;M.ProteinMarker;1.SpecificbindingbetweenantibodiesandCPBcells

�4 ÑScAKT2Eþ+Ñþye�Ú�É

Fig.4 AnalysisofspecificityofantiScAKT2polyclonalantibody

2.4 ScAKT2�>?CPBÀÁÂA¸¹

³PCRjõ`¯Ô1400bpy¾*�Ö�<

犈犮狅RⅠ、犓狆狀Ⅰy犛犮犃犓犜2ORF78

(�5A);

ÜäÝçëu

,6½þ+¯1449bpyORF78

Éæ®.þpCMVEGFPScAKT2PCRjõ6`

�Ô1400bpy78

(�5B),ÜäÝçæ+

,Éæ

®.þpCMVEGFPScAKT2º¦6½

A.)犃犓犜2ORFyþ+

;B.c:殢%pCMVEGFPAKT2yPCR3(

M.DNAMarkerDL10000;1~3.犃犓犜2ORFyþ+

;5,10.7�i�yHr

;6~9.7�6½yHr

A.Cloningof犃犓犜2ORF;B.PCRidentificationofeukaryoticexpressionplasmidpCMVEGFPAKT2;M.DNAMarkerDL10000;

1-3.Cloningof犃犓犜2ORF;5,10.SamplesfailedtransfectedwithAKT2ORF;6-9.SamplessuccessfullytransfectedwithAKT2ORF

�5 )犃犓犜2ORFyþ+�c:殢%y3(

Fig.5 Cloningof犃犓犜2ORFandidentificationofeukaryoticexpressionplasmid

91!2# + %

,L

:)AKT2R}*Ú*�Ä�baõò`yG¼

  ³ÉG418EW§·��

,pCMVEGFP±pC

MVEGFPScAKT2CPByÍÎTÔ*80%yÍ

Î÷â(æ®EGFP(�6A、B)。qRTPCRÝç

æ+

,RpCMVEGFPScAKT2yCPBÍÎT`

犛犮犃犓犜2mRNAyè�殬îëìz°pCMV

EGFPCPBÍÎT

(�6C)。WesternblotÝçë

u

,R pCMVEGFPScAKT2y CPB ÍÎT`

ScAKT2{|y殬+ëz°pCMVEGFPÍ

ÎT

(� 6D)。ú ï Ý ç æ +

,) 6 ½ 6 ` �

ScAKT2Éæ®CPBÍÎT

A.â(7*pCMVEGFPyCPBÍÎ

;B.â(7*pCMVEGFPScAKT2yCPBÍÎ

;C.犛犮犃犓犜2mRNAè�殬

D.ScAKT2{|y殬

。��ïÍæuEpCMVEGFPCPB�t�îëì

(犘<0.01)

A.CPBcellsstablytransfectedwithpCMVEGFP;B.CPBcellsstablytransfectedwithpCMVEGFPScAKT2;

C.Relativeexpressionof犛犮犃犓犜2mRNA;D.TheexpressionofScAKT2protein.indicatesextremely

significantdifferencefromthepCMVEGFPCPBgroup(犘<0.01)

�6 Éæ®ScAKT2ÍÎTyº¦E3(

Fig.6 ConstructionandidentificationofoverexpressionScAKT2celllines

2.5 �>?ScAKT2CPBÀÁAp©ª}ÃK�

�­°)*ISKNV936±48h�� DNA

Hr

,¼qPCR�Ü(ISKNV£¬

。Ýç

(�7)ë

u

,pCMVEGFPScAKT2CPBÍÎ`yISKNV

£¬îëìô°pCMVEGFPCPBÍÎT

。x.

°48h2�Ð{|Hr

,¼ Westernblot�Ü(

ISKNVMCP{|yæ®Þß

,ÝçÕ�8。�8

ëu

,RÉæ®ScAKT2yCPBÍÎ`

,ISKNV

MCP{|y��

。R)*ISKNV 9�­

°36±48h��ÍÎÐ RNA Hr

,¼ RTPCR

�ÛÜ�²âãå�y7Ýê#

,Ýç

(�9)ëu

犛犮犐犚犉3、犛犮犐犚犉7、犛犮犐犔8、犛犮犕狓、犛犮犜犚犃犉2±犛犮

犜犚犃犉3ymRNAÓëìï¹

,tï¹pç�./

ÓïÚ

��ï

“”æut�îëì

(犘<0.01)

onthecolumnindicatesanextremelysignificant

difference(犘<0.01)

�7 Éæ®ScAKT2�ISKNVõòy��

Fig.7 EffectoftheproliferationofISKNVwith

overexpressionScAKT2

02 ʱpË(ur))�

(&'()*

)!49"

�8 Éæ®ScAKT2�ISKNVMCP{|y��

Fig.8 EffectofISKNVMCPproteinwith

overexpressionScAKT2

��ïÍ、æuEpCMVEGFPCPB7*�èé

t�ëì

(犘<0.05))íîëì

(犘<0.01)

,onthecolumnindicatesasignificantdifference(犘<0.05)or

anextremelysignificantdifference(犘<0.01)from

thepCMVEGFPCPBgroup

�9 Éæ®ScAKT2��²âãå�mRNAæ®y��

Fig.9 EffectofthemRNAexpressionofinnateimmune

factorswithoverexpressionScAKT2

3 � ö

AKTûE¿ÍÎÉ5ywE¹¯å�

,RÍÎ

bٱG�`���;W�O

。)*r¬=Dæ

+

,ba�*ÍÎ#/

,AKT<=�ÑÙ

,��

bayõò

。�=DÝçæ+

,) AKT2÷È�

ISKNV��õò

,��)}*Ú*�Ä�baby

�¯���þy9<

AKTG�Í�Ñba9Í

,��Ebayõò

*È�G¼

。�x�ba

(denguevirus,DENV)

±4�Ïêba

(Japaneseencephalitisvirus,JEV)

)*÷ÑÙ�þy AKT<=

,È�bay��õ

ò

[15]。©}AKTyÑÙ

,÷�z¹»�3�Hí

�ba

(theserotype3reovirus)RNAyõò

[16]。

RT�Fê

(HBV)[17]

±Ê�Fêba

(hepatitisC

virus,HCV)[18]

ýÈ)*yÞßµ

,�þAKT<=

�ÑÙ

,X��ba��

,þßÍÎbÙ

。J�ã

ba

(footandmouthdiseasevirus,FMDV)yVP1

{|÷È� AKT <=

,X�þßbay��õ

ò

[19]。�þAKTyÙ�

,÷È�í�ba71�

(enterovirus71,EV71)y)*

,�EV71y�¯�

�þy9Í

[2021]。RI¢à�ba

(bovineephem

eralfevervirus,BEFV))*9#

,�þ AKT<=

��

,ß�nùbayõòõ�

[22]。��ba

(ZIKV)yNS4A± NS4B{|÷)*Q�� ³

ZÍÎ

,nùAKT�È�

[23],ªbay��õ�

>?nù ³>m0¡

。��=DScAKT2yÑ

ba½¾

,�¤¥�®�ScAKT2Eþ+Ñþ

,/

.â ã 2 3

(IFA)± Westernblot¤ ¥ Ø 2

ScAKT2Eþ+Ñþ÷úEScAKT2{|ßàe

�Ú^g

。�¤¥ßÍCº¦�ScAKT2Éæ®

ÍÎT

,&¼qPCR± Westernblot�ÛÜ�Éæ

®ScAKT29ISKNV��yñ�Þß

,Ýçëu

ScAKT2Éæ®÷È�ISKNV yõò

,æ+R

ISKNV�*ÍÎÉ5`

,ScAKT2�ISKNVy�

�õò�*È�G¼

  ¢èst)*Hí�ba

(mammalianreovi

rus)9,�þ AKT�Ù�X�,ÑÙIFN£Ñ^

g¤>

(ISRE),&ï¹IFN £Ñ5å

(犐犛犌狊)[24]。

HrinciusL

[25]=Dæ+

,�þ)*¢)ba.

AKT÷úcÉü~ÅPn5åⅠ(retinoicacidin

duciblegeneⅠ,犚犐犌Ⅰ)ÓïÚydÙ<=�Ñ

Ù

,&þßIRF3ÑÙ±Ⅰ�ZMÃæ®

。*��

@-

,AKT3y¥¼÷È�IRF3yÙ�

,ëìóô

IFNβy/m

,&�zba.¬

;AKT3yÙ�÷Ñ

ÙIRF3,X��zIFNβy/m±õ�Ñba½

¾

[26]。�¤¥>]

,Éæ®ScAKT2yCPBÍÎ

÷cÉï¹�²âãå�犛犮犐犚犉3、犛犮犐犚犉7±犛犮

犜犚犃犉2Lyæ®�È�ISKNVyõò

,��)b

aqr'ty=>���Í�þy9<

,½��¦

D0��ISKNVE§VÇGyùb�����þ

y�ü

[u�NO

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