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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�
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] 20191231
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[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$�
,zÕZR
Ô�]ònù)KfbÕ>
,F`}*Ú*�Ä�
ba
(infectiousspleenandkidneynecrosisvirus,
ISKNV)û=Z:9)]òy;Wbs�Í
,Fù
bÒ�
,ù�²÷®90%úï
,�)]òqG6�
>ry³h�i
[12]。å8
,FõISKNVùb��
=D�ªby�¯�*;W?@
。
{|Ñ� B(proteinkinaseB,PKB)5å
,�
犃犓犜,ûÍ¿sA5å
,BCbR°¶DÍÎyÎ
¢`
。AKT�AKT1、AKT2± AKT33¿(�
,
F`AKT2û{|Ñ�B:µyVW(�
,�*Ë
3Å
/ü3Å
(Ser/Thr)Ñ�yÙÚ
[3],ûE¿ÍÎ
É5ywE¹¯å�
[4]。*=Dæ+
,犃犓犜Éæ®
÷ÑÙFµfE¿pgå�
,¹½�²Úâãå�
yæ®�ªF>{ÑbaG¼
[56]。T�Fêba
(hepatitisBvirus,HBV)yX{|
(hepatitisBvi
rusXprotein,HBX)÷ÑÙs^FÍÎ`yAKT,
È� HBXJnyÍÎG�
,X�È�T�Fêb
ay��
[7]。RøÖòEH§ÉwPba
(porcine
reproductive and respiratory syndrome virus,
PRRSV)) * y � #
,� þ AKT � Ñ Ù
,È �
PRRSVJnyÍÎG�
,X�>{FÑbaG
¼
[8]。êIÚJêba
(vesicularstomatitisvirus,
VSV))*.
,�þAKT�Ù�
,ß�ÓïTollH
¨þ4(Tolllikereceptor4)<=>{Ñba½
¾
[9]。)*KYba
(sendaivirus,SeV)yLVc
ÉÙ� AKT,ÑÙZMù½å�3(interferon
regulatoryfactor3,IRF3)yæ®
,X�õ�βZM
Ã
(interferonβ,IFNβ)yæ®� / m Ñ b a p
g
[10]。÷Õ
,AKT÷úG�Í�Ñba9ͼ°
Ñba��y=D
。
8m�°)AKT2RISKNV)*É5`yG
¼Nò»O
,�8�=Dþ+�)犃犓犜2yF�,
S-&ßàs:æ®
,�®;�ScAKT2Eþ+Ñ
þ
,º¦)犃犓犜2Éæ®yÍÎT
,=DScAKT2
RISKNVõò`yG¼
,ú#�)ISKNV�¯�
�þy9<
。
1 �HE��
1.1 W X
1.1.1 QR
、ST
、UV;!"#$ )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
®
)©ª
。4�r/0
,¤°æó'P]ò50
。
1.1.2 ! % L15�]5
、�{|�
、¹»±
Hanks@
,¤°Gibco(Ì9
)©ª
;Trizol¤¢
,¤
°Invitrogen(Ì9
)©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
;UzVÈW¢±UzòV
ÈW¢
,¤&Sigma(Ì9
)©ª
;FuGENE?67*
¤¢£±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®
)©ª
。
1.2 � �
1.2.1 WXY)Z[/\]^ ¼Trizol¤¢�
�CPBÍÎÐRNA(d3�mt);�
),'9�
�¼ Titanium OneStepRTPCR Kit¤ ¢ £ 4
RNA^7Ý�cDNA,¼Åÿst5å���¤¢
£��)*ISKNV9yCPBÍÎDNA(d3�m
t);�
)。
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`
,�]
12~18h9
,¢�vþ+Üä
。4Üä|�y£;
�¢%y<@.¿�@þ�]5
(£3iû_Ã50
μg/mL)̀ ,°37℃jk�]�<@R600nm!y
§3Z
(OD600)Ô�0.6.
,§DIPTGPn¢
(?
YZ1mmol/L),37℃jk�]3h;×l
(½²200
W,°G3s、mn4s)oJ<þ2~3min;4℃、
6000r/minºÃ30min,2Rï»�PQ
。��
<þPQ±ï»
,¼1×LoadingBuffer;pqá
,r
ês15min9ßàSDSPAGEÛÜ
。Ø+;�{|
RPQ`úAXþtçæ®9
,¼ProteoSpin
TMA
Xþ�º¥�¤¢£¥�PQ
(AXþ
),&cÉ
SDSPAGE�¥�y;�{|ßà3(
。
1.2.4 WAKT2ndeoV)*+ ¸R4�r
/0¹@
,�º¹»G�âãm¹»
,®¼
。ú¥�
y;�AKT2{|�Ñs
,u�vµûwâã4�
r/0
,â㢬300μg/9。ÍâÑsi¼UzV
ÈW¢ßàè�
,Ëâ
、xâ±yâÑs¼UzòV
ÈW¢ßàè�
,>9ÍWâã92ë
,°0/z¶
·! � ¹
,� º ¹ »
。� 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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22 ʱpË(ur))�
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)!49"