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Polyclonal Antibody Preparation and Application of Lotus japonicus Rac1 Protein

百脉根Rac1蛋白多克隆抗体的制备与应用



全 文 :书!"#$%&
,2016,36(4):0655-0660
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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f=01464000)
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xsy

z{|}uvQ~1犚狅狆,g(犚犪犮1),€‚ƒ}„…†‡ˆ‰pET28a,Š‹Œ|犚犪犮1,g~\Ž
SBL21(DE3)lS。‹Rac1‘’“”†‡•–,—˜™š›œ‹‘’,žRac1Ÿ ¡¢‰,£¤eq¢‰
¥¦Rac1k†‡Š,g#§mRac1‘’~†‡¨©。ª«¬­:(1)®¯°±˜¦²³´,µ¶·¸pET28a
Rac1„…†‡ˆ‰。(2)Rac1‘’~¹º“”†‡•–x:IPTG»¼0.1mmol/L、½¼20℃、¾¿6h,pÀ‘
’rÁÂÃtÄņ‡

œ‹~Rac1‘’®SDSPAGE¥¦,C~•Æ\Çx25kDÈÉ,ʕÆËÌ、ÍÎÏÐ
Æ
。(3)Westernblotting¬­,ž~Ÿ ¡¢‰ÑÒÓÔ]Õ¤~¢„,ÊÅÖ×Ä。(4)Øk_ŽSٔ~
ÚQŠ‹›Œ|Rac1k†‡#§~1ÛÚQ,ÜÝ1ÛÚQޑ’,WesternblottingX߬­k†‡#§m
Rac1‘’†‡à¬áÄâ㈉Õä,Må権çèk†‡ˆ‰·¸~éÅÛ。q5Zž~Rac1Ÿ ¡¢
‰ÑêÄÅÒÓ륦ìaâuvQ‰í~Rac1‘’,î€xïÎðñòRac1hTU0óŠ”ôõm~U$%
¶Ñ5ZÜöéd÷

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øùú

uvQ
;Rac1;„…†‡;Ÿ ¡¢‰
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犘狅犾狔犮犾狅狀犪犾犃狀狋犻犫狅犱狔犘狉犲狆犪狉犪狋犻狅狀犪狀犱犃狆狆犾犻犮犪狋犻狅狀狅犳
犔狅狋狌狊犼犪狆狅狀犻犮狌狊犚犪犮1犘狉狅狋犲犻狀
KEDanxia,ZHANGWei,LIXiangyong,YANGMingying,YUXiaorui,
ZHUHanxue,WANGXiaofei
(ColegeofLifeSciences&InstituteforConservationandUtilizationofAgrobioresourcesinDabieMountains,XinyangNormal
University,Xinyang,Henan464000,China)
犃犫狊狋狉犪犮狋:犚狅狆geneplaysanimportantroleintheprocessofsymbioticinteractionbetweenlegumeandrhi
zobium.A犚狅狆gene犚犪犮1wasamplifiedfromtherootcDNAofmodellegume犔狅狋狌狊犼犪狆狅狀犻犮狌狊andligated
totheprokaryoticexpressionvectorpET28a.Theengineeringbacteriumcarrying犚犪犮1genein犈.犮狅犾犻
BL21(DE3)wasobtained.TheexpressionconditionsofRac1proteinwasoptimized,andtheproteinwas
purifiedbyaffinitychromatography.Rac1proteinexpressionlevelsofoverexpressionplantweredetected
byusingthepreparedantiRac1polyclonalantibody.Theresultsshowed:(1)theprokaryoticexpression
vectorofpET28aRac1wasconstructedsuccessfulybydoubleenzymedigestionandDNAsequencing.(2)
Theoptimalexpressionconditionwasinductiontemperatureat20℃,timeat6h,andIPTGconcentration
at0.1mmol/L.Therecombinantproteinwashighefficiencyexpressedintheformofsolubleprotein.The
purifiedRac1proteinwasobtainedbyaffinitychromatographyanddetectedbySDSPAGE.Thebandsize
wasabout25kD,andthebandswereclearandsingle.(3)Westernblottinganalysisshowedthatthepoly
clonalantibodycouldspecificalyreactwiththecorrespondingantigen,andthetiterwashigh.(4)
Agrobacteriummediatedtransformationofhairyrootsmethodwasusedtoobtainthepositivehairyroots
ofRac1overexpressionplant.ThetotalproteinofpositivehairyrootswasextractedanddetectedbyWest
ernblotting.TheresultsshowedthattheexpressionofRac1proteinintheRac1overexpressionplantwas
significantlyhigherthanthatintheemptyvectorcontrol,whichprovedtheconstructionofoverexpression
vectorwaseffectivefromtranslationlevel.TheresultsshowedthatthepreparationofRac1polyclonalan
tibodycouldspecificitydetectthenativeRac1proteinin犔狅狋狌狊犼犪狆狅狀犻犮狌狊,whichwilprovideapowerful
toolforthefurtherstudyofthebiologicalfunctionofRac1inthesymbiosissignaltransductionpathway.
犓犲狔狑狅狉犱狊:犔狅狋狌狊犼犪狆狅狀犻犮犪狊;Rac1;prokaryoticexpression;polyclonalantibody
  Rop‘’ØkPûüýþgY~ÿije!P
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#$mb=>h
[10]。
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†lRop‘’!Pýþcdej)[11]。¢‰
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g
[2]。GAPphcdeqrstRop‘’~ GTP
¨T

u RopGâv"X+,L GDI€1"‹~
Ropwqrp[-x}yz,v"~ Rop{|h
GEF5",5Z}~M‘’¨©çèRop478
9"Ûý;YØkWÐ~ý;tTP€&~
(ÛU)
[1214]。
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,犚狅狆,ghO+#$
PQRSTUijklmnopLje

„…
(犕犲犱犻犮犪犵狅)†‡ÎüpL~stO+#$。hˆ
c„…
(犕犲犱犻犮犪犵狅狊犪狋犻狏犪)ƒüQRS~Qwm¥
¦}犚狅狆‰,g~†‡,î†犚狅狆,g!PTUi
jkl~ŠÎçƒ
[15]。
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(犕犲犱犻犮犪犵狅
狋狉狌狀犮犪狋狌犾犪)m~3犚狅狆,g,犕狋犚狅狆3、犕狋犚狅狆5
˜犕狋犚犪犮1kQRS“”†‡àl¬{s[16]。O
(犌犾狔犮犻狀犲犿犪狓)犚狅狆‰,g犚犪犫犃2Òӆ‡âƒ
üQRS~QÚm

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QRSªRgY~ýþ

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(犔狅狋狌狊犼犪狆狅狀犻犮狌狊)mŸX
U|}2犚狅狆,g,犔犼犚狅狆6˜犔犼犚犪犮1,gkQ
RS“”

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ªR¢CŸÿ¤£¤
[19]。
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vQm~犚狅狆,ghTUªRklmŸno§pL
¶Ñ



øâuvQm犚狅狆~5ZKL©mâŠ
—¨©

LøâåæRRop‘’?~5Z&ª«¤。
¬è­Øk·¸„…†‡ˆ‰

XUœ‹Rac1pÀ
‘’

žRac1Ÿ ¡¢‰,deq¢‰ÕRac1k
†‡Š,g#§1ÛÚQޑ’mRac1‘’~®à
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1.1 Q R
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zè­µQRSXYU$%è­µÜö

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w¬è­µb>

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1.2.2 [\]^_=`a Qƒ NCBIÄÅ»Æ
~犚犪犮1,g²Ç(GenBankȗóZ73961.1),e
PrimerPremier5.0ɖÊ;·¸犚犪犮1„…†‡
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(5’ACGCGTCGATGAGTGCT
TCCA3’)˜89Ë$ (5’GGAATTCACTCA
CAATATTGAG3’),8<’X]x犛犪犾1˜犈犮狅犚1
°±oz

rIµ~cDNAŠÎÂxsyïNPCR
z{
,PCR̤‰wx2.5μL10×PCRBufer,4μL
2.5mmol/LdNTP,1μL79ÒÓÛË$(10μmol/
L),1μL89ÒÓÛË$(10μmol/L),1μLcDNA
ŠÎÂsy
,0.25μLEx犜犪狇DNAÍI°,eddH2O
ÎωÐ}25μL。PCR̤l²x95℃яÛ
5min;94℃Û30s,55℃ÒÓ30s,72℃Ôj
1min,30^_;72℃Ôj5min。PCRÕ$deÖ
×ØÙÚ¥¦R

±ÛxÜPCRC~ÝÞ,{®¯°
±R

°±Õ$‚ƒ}†‡ˆ‰pET28a7,ŠßŽSDH5a,°±˜¦²³´1Û ¡。
1.2.3 />78bc]^XdefKg €·¸
à~pET28aRac1pÀ?áŠß†‡S§ BL21
(DE3)m;âÝÍSãâKana(50μg/mL)~LBä
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,37℃、250r/minkçåæ;kçåæ
$r1%~ƒüàŠƒß[èåæ,m,r37℃、
250r/minåæéOD600ê‡0.5~0.6¾,X]sß
IPTGé뻼x0.1、0.25˜0.5mmol/L,Ãâ
20、28˜37℃ìímåæ,2、4˜6hX]Ýî
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‰

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SDSPAGEÙÚ,õpÀˆ‰pET28ajxÕä,œ
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sß600μLPBSïðä,sddH2O´ûé1L。ï
ðä
(pH7.4)Àµx140mmol/LNaCl、2.7mmol/L
KCl、10mmol/LNa2HPO4 ˜1.8mmol/LKH2PO4。
UüeÃâý¨þI$m

ÿ!"#$&4~6
|

ú|10s,¿%1min,&Ã10min;4℃、12000r/
minÄtUü15min,7˘/()Ýî,X]
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PAGEÙÚ。
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(20mmol/LTrisHCl,50mmol/LNaCl),ßR,
{Øk9Ø»:

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‹~÷I‘’îôX;RÃ-70℃<>,že。
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PAGEÛ¾,Ð>Û7O?@Y,ÑA1cmB~
ã¿zî

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h5
JmsßKàUQL¨

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!Y\’Z[&v\3~5mL,µ\Ëjxã’
Õä

Š1|UV¢„1mg,®]¶^8ŸzUV
WX

_|WXR2\,€¢„PPQO•–R¾
/‰ÐMXþ0

ïNŠ™|s`WX

¢„¾à{
s}1.5mg。:Rú%2\s`WX1|,›¾
àP™W
。3|s`WXR,[&va\£XUË
,Westernblotting¥¦Ÿ ¡¢‰ÅÖ。ÙÚR
~‘’ÛFŠ,
,4℃bckçR,Rac1Ÿ ¡¢
‰jx΢
(1∶1000)µ½d*2h,eQkf‹$
°Dg~h¢ZIgGjx™¢(1∶2000,ijk)
µ½d*1h;¹Rue‹%nl¬C½¾¿Beyo
ECLPlus(ijk)ïN¬C,£de‹%nlµm
wn
(ProteinSimple)g—ª«。
o¢‰Åև}Lp¾

Züqrsa,
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X;â-70℃<>že。t¢‰ÅÖOQ
u

Ávws`WX1|。
1.2.6 o,pqfr6犚犪犮178>mn de
nQ_ŽSٔ~ÚQŠ‹›Œ|uvQRac1k
†‡Š,g1ÛÚQ

÷‰›!ä!xyz
[22],
€³ ´ ~ 1 Û Ú Q { 8 3~5cm,© | â ®
300mg/mL}~6~ MSV‰åæ,7,ïN1
ÛÚQ~z€åæ
,1\v1|,v3~5|,e
âRac1‘’~¥¦Xß。
h10mL‘’‚Üïðämsß1ݑ’°
S¾ƒÝ
(Roche),âý7„Ã,…ƒÝÂTRM
Xþ0

i = i e

ï ð ä
(pH 8.0)À µ x
50mmol/L TrisMES、0.5 mol/L sucrose、
1mmol/LMgCl2、10mmol/LEDTA˜5mmol/L
DTT。Ü©z€~Rac1k†‡˜ãˆ‰Š‹#§
1ÛÚQû0.1g,â†éäW~5Jm5Nµd
7564]           DEF,/:uvQRac1‘’Ÿ ¡¢‰~žP¤e
X,
‡tŠˆd‰éUüem

d‰P‚Üïðä/
‰ÐþI

⊋Œ7‹þ0

{âý7„Ã
10min;4℃、12000r/minUü20minÜ©7Ë,
sß7îïðä

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SDSPAGEÙÚ4 Westernblotting¥¦。
2 ª«PXß
2.1 kl]^_=>`a
eÊ;~Ë$z{uvQ犚犪犮1,g,z{Õ
$\Çx594bp,PÑ]ª«Îö(Ž1)。¦²ª
Ǡl

¬5Zz{Œ|~uvQ犚犪犮1,g–)
594bp,197‘,B,deh’ɖѦq‘
’XYàx 20kD ÈÉ。z{Õ$® 犛犪犾1 ˜
犈犮狅犚1¯°±,‚߆‡ˆ‰pET28a7。pÀ?
á®°±Œ|\ǒ5.3kb~ˆ‰Ýޘ594bp
ÈÉ~?ßÝÞ

Ž2)。wâpÀ‘’®éÀ‘B


deh’ɖѦpÀ‘’XYàx25kD
ÈÉ

×Rac1‘’XYà\。ïÎð~¦²ª«
ŸçèRac1,g~„…†‡ˆ‰·¸µ¶。
M.DL2000;1.PCRz{Õ$
Ž1 犚犪犮1C~ÝÞ~PCRz{
M.DL2000;1.ProductofPCRamplification
Fig.1 PCRamplificationof犚犪犮1gene
M.1kbDNAladder;1.pÀ?á°±Õ$
Ž2 pÀ?ápET28aRac1°±³´
M.1kbDNAladder;1.Productofrecombinantplasmid
digestedwith犛犪犾1犪狀犱犈犮狅犚1
Fig.2 IdentificationoftherecombinantpET28aRac1
plasmiddigestedwith犛犪犾1犪狀犱犈犮狅犚1
2.2 kl78>]^Aij
pÀ†‡ˆ‰Š‹}†‡SBL21(DE3)m,“
”R®SDSPAGEXß,h25kDÈÉél¬~†
८

\ÇPѦ~pÀ‘’XYà\Çÿ”

Ž3),ÁlpÀ‘’“”†‡µ¶。x˜Œ|¹
\ÁÂÛC~‘’Õà

Œ|pET28aRac1†‡S
§¹º“”•–

“”½¼20℃,“”¾¿6h,
IPTG»¼0.1mmol/L,SDSPAGEÙÚXßC~
‘’h\ŽSBL21(DE3)mr7ËÃt\à>
h

Ž3)。
C~‘’®6×Hisœ‹D“,Ár®-•–I
—˜>ߙšâNiNTA—×>ß.,ð*˜™1
Òәš~Б’R

€C~‘’*6

,ß»:

úÎðšX]Ýî
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ß»:R~C~‘’h25kDÈÉ,•ÆËÌÊÍ
ÎÏÐÆ

†lpÀ‘’hÄŜ‹

Ž4)。
1~5.“”†‡pÀS‰‘’;6.“”†‡ãˆ‰S‰‘’;
M.‘’marker;7.“”S§ÿ!"#$R~7Ë;
8.“”S§ÿ!"#$R~
Ž3 Rac1pÀ‘’“”†‡~SDSPAGEXß
1-5.InducedcelscontainingpET28aRac1;6.Inducedcels
containingpET28a;M.Proteinmarker;7.Solubleproteinfrom
inducedcelscontainingpET28aRac1;8.Insolubleprotein
frominducedcelscontainingpET28aRac1
Fig.3 SDSPAGEanalysisoftheexpressionof
Rac1recombinantprotein
1~3.œ‹~C~pÀ‘’;M.‘’marker;
4.,ß»:R~C~‘’
Ž4 Rac1œ‹‘’~SDSPAGEXß
13.PurifiedRac1protein;M.Proteinmarker;
4.Targetproteinafterdialyzedandconcentrated
Fig.4 SDSPAGEanalysisofpurifiedRac1
recombinantprotein
856 ! " # $ % &                   36š
2.3 9:;<=>?@Amn
€C~‘’•Æ±8

äW5NRsßR¾W
X[!Y\’Z

ž Rac1Ÿ ¡¢‰。de
Westernblotting¥¦ž¢‰~ÅÖPÒÓÛ,ª
«¬­„…†‡~÷I‘’PŸ ¡¢‰ÐC¬­
ÍΕÆ

PWX¨¢\ËÐC›é•Æœi

΢
/x1∶1000ž,Álž~Rac1Ÿ ¡¢‰
‡}QuÅÖ

ÁrñòRw½­

õpÀ†‡ˆ
‰~S‰‘’jxŸÛÕä

PŸ ¡¢‰r4W
X¨¢\˔›éÐCœ ¡•Æ

Álq¢‰÷
é×Ä~ÒÓÛ

Ž5)。
2.4 o,pqfr6犚犪犮178>mn
x˜M‘’¨©¥¦k†‡#§mRac1‘’
~†‡à

¬è­ÜÝ1ÛÚQޑ’

dež~
Rac1Ÿ ¡¢‰ïN WesternblottingÐCXß。
wŽ6Ár¢œ,1ÛÚQk†‡Üݑ’ÐC•
Æh20kDÈÉ,PѦ‘’XYà\ÇÎö,LŸ
ÛÕä

õpÀ†‡ˆ‰pET28a~S‰‘’)›é
C~•Æœi

1ÛÚQmk†‡#§ÚQPãˆ
‰p1302G #§ÿ(,Rac1‘’~†‡àl¬ 
Ä

w¦Ál

C~,g£•¤¤I}uvQQ¶,
gÀm

ÊRac1‘’Ÿ|}kà†‡。
1.õpÀ†‡ˆ‰~S‰‘’;2.“”R~C~‘’
Ž5 Rac1Ÿ ¡¢‰~ WesternblottingXß
1.InducedcelscontainingpET28a;2.Totalprotein
frominducedcelscontainingpET28aRac1
Fig.5 WesternblottinganalysisofRac1
polyclonalantibody
1.õpÀ†‡ˆ‰~S‰‘’;
2.Õä#§1ÛÚQm~Rac1‘’;
3~8.k†‡#§1ÛÚQm~Rac1‘’
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