邢唷> LNIJK欹 鳵bjbj駘駘<剬剬 xx8 !6wcvevevevevevev$鈞剓T塿Q111塿4趘 譕譕譕1cv譕1cv譕譕R飊蟩0!刺v鉶Ov鷙<6wp貃MH|<蟩貃L$|$蟩譕[,!(塿塿1O6w1111H|x : RP[uirf[瀃寶II 藛}v(崉vh埦0瘇S蔛纇Km 瀃寶NY恘鶺郪(W'Y爛Fg虄-N剉駤黐h埦 0瀃寶顅剉0 惽,g瀃寶哊銐Y恘鶺郪(W烻8h苸迉-N駤黐h埦徟`礠0 0瀃寶烻t0 \Y恘鶺郪KQ問(WO(u+T glac蚫\OP[剉/T≧P[剉h埦弣廠O-N vQ(WE. coli-Nh埦0枡HQ(W N燫駤黐BR剉ag鯪 N鵚{Q縖;N虄 lacI鶺郪h埦弰vu剉;朞愃唥vLacINlac蚫\OP[觺T 蜰 N齹蹚LY恘鶺郪剉h埦6qTT鵚{Q鶺-N燫eQ駤黐irIPTG dk鰁LacI;朞愃唥v豐刧1Y;m N齹Nlac蚫\OP[觺T Y恘鶺郪h埦0 0瀃寶闚hV0Pg檈N諎BR0 瀃寶闚hV厤繯錧\O餝0諎0Gd奮0粂胈:gI{0 虄*h孴(崚|虄*h'Y爛Fg虄BL21蛻膥(崚|pET-30a-X鶺郪XNpET-30a蛻膥0 ;N亯諎BR 50mg/ml aS }M10ml (u0.22m鋘渷菑鋘d柼 Rb10齆 .峏[嶯-20!0 1M IPTG\2.4g IPTG秐嶯10mL ddH2O -N (u0.22m鋘渷菑鋘d柼 蟢齆1ml .峏[嶯-20!0 LB瞞SO鵚{Q鶺 饊藛}v鑰tryptone 10g u懲ku懲k弫yeast extract 5g Nacl 10g Gd≧筟hV魐髞秐(崒[hQ秐銐,(uNaOH(1M)寕侾H髞7.0 燫eQ籗粂P[4l髞;`SO飝:N1L 121!n韕mp虄20R煍0 0瀃寶ek0 1蛓P[瞞剉6RY c諷LB鶹SOs^g N剉'Y爛Fg虄BL21pET-30a-X US虄= 蛓嶯+T5laS }剉5ml LB瞞SO鵚{Q鶺剉諎-N 37! 200rpm/ca凒W{Q菑Y0(鑜a諷虄*h亯(W厤繯錧\O餝 N蚫\O N歔鑜a鄀虄). 2l彞c鵚{Q 諷50l虄瞞R+Rl彞c0R$N/e+T g5laS }剉5ml LB瞞SO鵚{Q鶺諎-N1%蛓蠎 37!0200rpml彞c鵚{Q0 3. IPTG駤黐h埦 S_鵚{Q2.5\鰁 OvQOD600f:y@w5u髄剉MR縧MOn S_c:yBR0R緩軶鰛昢钀鰁 sS颯\Pbk5u髄 200V 45min鎉骃 0 瀃寶Pg檈 瀃寶N-N剉駤黐/*g駤黐h埦弰v'Y爛Fg虄BL21pET-X 瀃寶闚hV: 俉魐g5u髄闚 ;N亯諎BR 藛}v(峂arker 30 Acr/Bis N飌p扂秐瞞 29.2g Acr + 0.8g Bis baPeQ蘏竸4l20ml 錘籹僼襤bad蘠肙蹚秐銐0籹僼襤d≧E^ N亯*Y'Y錘MQ秐瞞昢钀剉蘏N飌p扂nmw峷^ibce0Rzzl-N0孾hQ秐銐剉秐瞞擽n坥鄀rf0卂秐銐孾hQTe垹R蘏竸4l髞100 mL PeQ鄀r偦s僼諎BR鰐-N 4!癚眥颯軴X[10*Ng0 1.5 M Tris-HClpH 8.8 20ml \3.63 g Tris眡秐嶯10ml籗粂P[4l-N (u衯x宲HeY(u (uKNMR37!{弡忳mS秐瞞髞SDShQ秐0 0瀃寶ek0 1. 7h罷6RY 諷鶴-20!軴X[剉BL21pET- cry9ea 虄SO塴續 燫eQ80l ddH2O蛻琡塴續 燫eQ20l 5X N7h睶瞞鱩S 竘4ltm10R煍 12000r/min 4!粂胈5R煍 N7h鰁諷 Nn钀R 癚 N>en0鑜aFU罷藛}vMarker_N梺N5X N7h睶瞞鱩S竘4ltm 2. 10R粂鰛6RY 秅}Y鰛gO(u0.75mm剉籹僼g M憂10R粂鰛6R鰛(u4ml M5ml 10R粂鰛5ml 籗粂P[4l 1.9 ml 30Acr + Bis 1.7 ml 1.5 M Tris-Hcl(pH 8.8) 1.3 ml 10SDS 50 ul 10AP 50 ul TEMED 3 ul 鑜agT燫AP孴TEMED0 鱩ST燫eQ$N籹僼9Y-N v^\胈(W鰛b N燫eQ1cm竸彊4l 37!40R煍 I{鰛陙6q軶ZT>P渆P鶴竸彊4l(W鰛b N燫eQ竸彊4l饄:N4l\ vQ顅剉/f軴c鰛b梥^te孴2朾kzzl蹚eQ q_蚑軶鰛 0 3. 5Sm)鰛6RY6R鰛(u4ml M5ml 5Sm)鰛M懝e5ml  籗粂P[4l 3.5 ml 30Acr-Bis 0.825 ml 0.5M Tris-Hcl(PH6.8) 0.625 ml 10SDS 50 ul 10AP 50 ul TEMED 5 ul 鱩ST燫eQ0R籹僼g-N v^襝eQ0.75mm砲P[ 37!40R煍軶鶹T\胈詁鶴砲P[0 4. N7h 鍌/f10T[鰛蟢T[ N7h蠎15ul 15T[鰛蟢T[ N10ul 鵞gq N7h蠎:NYt剉1/2SO飝0 軴翄苸迉uir蠎N魜 藛}v N7h蠎剉 愰b亯9hncwQSO臽礠砆歔 鵞嶯l毌e甆輨觛r 俌済7h罷:N虄SO藛}v7h罷 g髞\50ag錘 N剉ag&^亯>f鶴eg 梺 N7h蠎(W15 g鎉骃sS颯 俌済藛}vag&^\嶯6ag 俌藛}vMarkerb瘇S6柕kTg剉藛}v7h罷 N7h蠎颯螿\髞2gsS颯>f鶴npf剉ag&^0 5.5u髄 \籹僼g軶鰛>eeQ5u髄齣-N v^(W遼-N燫eQ1X5u髄睶瞞0 燫7h諷@b6RY7h罷 藛}vMaker 5 ulR+R N7h0 緥歔5u髄 z廭200V 90min 瀃寶 N 藛}v(嵗hKm Al毌e甆輨R250觛r傉l Coomassie Bright Blue R250 Staining 0烻t0 l毌e甆輨 /fN蛓藛}v(嵱g檈 ;N亯 gR-250孴G-250$N蛓{|媁0l毌e甆輨颯錘孴藛}v絸-N眡'`(l鶺x憢k鶺b硞櫃蟚(l鶺x憢k鶺Arg Trp Tyr His Phe 觺T0l毌e甆輨R250Y(u嶯ZN飌p扂軶鰛5u髄T藛}v(峚g&^剉觛rl毌e甆輨G250/f^蒪穇弝穇(Bradford)"(0248:<FNRZ\^fj  $ , 6 D L 蹼犭蹼淄酌朊墜白哀灖暟裝寖寉儗yoyf儗yf宖hxh<}]CJhxh錮6丆Jhxh\=6丆Jhxh錮CJhxh\=CJhxhCvCJhxh w[CJhxhCJhxh訿CJhxh*5丆Jhxh箉5丆Jhxh+?5丆Jhxh訿5丆Jhxh?>5丆Jhxh靌5丆Jhxh齗U5丆J(24\j , n ~  & Fd,G$H$gd;$d,88G$H$XDdYDda$gd勦dhG$H$WD`勦gd勦d,G$H$WD`勦gd;$d,88G$H$XDdYDda$gd; $d,G$H$a$gd;L N T Z ^ v z |    6 8 X h j p r 黜澉澉澉澉澉阡餮染鵲憚殦姎姎亃qzqdqhxh訟CJOJQJhxh訟CJ h訟CJo(hxh;CJhxhxCJ h%XoCJo(hxhVCJhxhCvCJhxhCJhxhz6KCJhxh訿5丆Jhxh訿CJhxh/CJhxhCv6丆Jhxh<}]CJhxh\=6丆Jhxh\=CJ&   * , 4 h j l n ~ B F   黝麋鬏黝星罱傳礄儲傳搭岊儁僷gpgp` h= CJo(hxh繳 CJhxh8KHCJhxh訿5丆Jhxh訿CJhxhVCJOJQJhxh CJhxh&zCJhxh鑕yCJhxh;CJhxhV5丆Jhxh- CJhxh- CJOJQJhxhlCJH* h繷CJo(hxhVCJhxhlCJ%~ J :jY勦d,G$H$WD`勦gd;R勦d,E苺SG$H$WD`勦gdl$d,88G$H$XDdYDda$gd;剉d,G$H$VD,^剉gd d,G$H$gd;勑d,G$H$WD,`勑gd;    " $ & * , : < @ B ^ `    8:<NRT黝孱孱孱茴茴項祁堿愁☆☆橆橆彙釹喅|r|rhxh5丆Jhxh5丆JhxhCJhxh;ICJhxhhCJhxhCJhxh辬CJhxhCJhxh8KH6丆JhxhlCJhxh8KHCJOJQJhxh蒳CJhxh繳 CJhxh8KHCJhxh= CJ,:<Thzk$勦dhG$VDWD^`勦a$gdx$勦dhG$WD`勦a$gdx$匎d,G$^`匎a$gd^$d,G$XD2a$gd^ $d,G$H$a$gd舙刋d,G$H$WD`刋gdx d,G$H$gd勦d,G$H$WD`勦gd d,G$H$gd T\`nt $@X^`fhjnr黟麈鬏髟髟黟鬏魘朗鞣癌⒎鞣鞣暦媱{蕅gqhxh舙5丆Jhxh^5丆Jhx5丆Jo( h欽5丆JhxhxCJo(hxh8KHCJOJQJ h 3CJo( hv4CJo( hqCJo(hxh8KHCJhxh;I5丆Jhxh5丆JhxhhCJhxhCJhxhCJH* hCJo(hxhCJ%RTXbrv鏃羞漣漣列P擼枺嚕~tjaWKWhxh*P5丆JQJhxh*P5丆Jhxh,txCJhxh;5丆Jhxh,tx5丆Jh'5丆Jo(hxh^5丅*OJphhxhxB*OJphhxh^B*OJph hxh^0JB*H*OJphhxh^0JB*OJphhxh^B*OJ\乸hhxh^5丅*OJ\乸hhxh>5丅*OJ\乸hXrtv8tt d,G$H$gd恴 d,G$H$gd  d,G$H$gd d,G$H$gd;$d,88G$H$XDdYDda$gd,tx$d,G$XD2a$gd^&筱筘縣呢幌嗇徊牟ツ材矖墆墆塲aTahxhCJmHsHhxhCJhxhCJQJmHsHhxh5丆JQJmHsHhxh5丆JmHsHhxh5丆JmHsHhxh;CJOJQJhxh;CJhxh鷞CJhxh;CJQJhxh*PCJhxh*PCJQJhxh;5丆JQJmHsHhxh;5丆JQJ&,4HLPVhv|~蹯蹯蹯蹯蹯嘖焓禐禐囀祎aVMVMVMVhxh盢CJhxh盢CJQJ$hxh*P5丅*OJmHphsH$hxh;5丅*OJmHphsH,hxh盢5丅*KHOJQJmHphsH,hxhl5丅*KHOJQJmHphsH(hxh;5丅*KHOJmHphsH(hxh盢5丅*KHOJmHphsHhxhCJOJQJhxhCJhxhCJQJ *02468>@FHNdrt黛黛鬟溜聯聯晘iS@%hxh鷞B*KHOJmHphsH+hxh 5丅*KHOJmHo(phsH)h 5丅*KHOJQJmHo(phsH,hxh鷞5丅*KHOJQJmHphsH(hxh鷞5丅*KHOJmHphsH,hxh恴5丅*KHOJQJmHphsH(hxh恴5丅*KHOJmHphsHhxhCJhxh盢CJOJQJhxh盢CJQJhxh盢CJ"&(4hjrt曜曜耆宮泭w`O=O0hxh B*OJph#h; h獸D5丅*KHOJo(ph h; h鷞5丅*KHOJph,hxh鷞5丅*KHOJQJmHphsH$h; h鷞5丅*KHOJQJph h; hT5丅*KHOJph h; h*P5丅*KHOJphhxh,txB*OJphhxh恴B*OJQJphhxh鷞B*OJQJph%hxh鷞B*KHOJmHphsH)hxh鷞B*KHOJQJmHphsHtvx HJLRZhj翮厥繰緗冂冂噞rlrerZrZlrS h恴CJo(hxhCJQJ h; CJo( h; CJhxh恴CJhxh恴5丆JKHhxh恴5丆JKHQJhxh5丆JKHhxh B*OJphhxh鷞5丅*OJphhxh鷞B*QJphh獸DB*OJQJo(phhxh鷞B*OJQJphhxh鷞B*OJphh獸DB*OJo(phR@^t&@z|勔d,G$H$^勔gd沀w d,G$H$gd楿凾刪d,G$H$VDWD^凾`刪gd楿 d,G$H$gdL~C d,G$H$gd 勑d,G$H$WD,`勑gd勔勦d,G$H$VDd^勔`勦gd%勔d,G$H$VDd^勔gd恴  .2>@BDNRX^dj苡裼苡苡苡毆毆ofh腒h楿CJh腒h楿5丆JKHh腒h楿CJo(h腒h CJh腒hL~CCJQJh腒h CJh腒hL~CCJh腒hL~C5丆JKHQJh腒hL~C5丆JKHh腒h楿5丆JKHo(hxh恴CJhxh恴CJQJhxh%CJo( h; CJo( h%CJo(%   &.068>@RVhjlrx~ $&.8:>@黝麇坼襄棋誨誨誨黝麇襄棋誨插讒銦槽插棋誨黝髹悉檷勏h腒h楿CJh腒h楿CJQJo(h腒h楿CJQJh腒hL~C5丆JKHo(h腒hCJh腒hL~CCJQJh腒h CJh腒h楿5丆JKHh腒h楿CJo(h腒hL~CCJh腒h CJh腒hGDCJ3@BDFHRX\^lrvxz|~黟縻靨麓搪特槑槄ymd[dNdEhxhM CJhxh8KHCJOJQJhxh剗`CJhxh8KHCJhxh8KH5丆JKHhxh跙c5丆JKHhxh跙cCJhxh^5丆Jhxh^CJh1h沀w5丆JhL~CCJQJo(h1hL~C5丆JOJQJh1hL~C5丆Jh1hL~C5丆JQJhxhL~CCJQJhxhL~CCJOJQJ h1CJo(hxhL~CCJ| 0 Z!!"b""8#j#勑d,G$H$WD,`勑gd 勑d,G$H$WD,`勑gd; d,G$H$gd; d,G$H$gd騄W d,G$H$gd>$d,88G$H$XDdYDda$gd  & Fd,G$H$gd跙c$d,88G$H$XDdYDda$gd跙c "&*@BDFPfpx|蹼賡猩新共Σ孤澛剮剮攳攢敳攜峱yg`gp峺 hO(CJo(hxh/CJhxh3CJ h3CJo(hxh镮CJOJQJ h/CJo(hxh镮CJhxh{CJh%h%CJH*o( h%CJo(hxhCJ h{CJo( h 3CJo(hxh跙cCJhxh剗`CJhxh8KHCJhxh6丆JhxhM 6丆J%      & ( 0 < D ~ !!麟哂哂拭矯椽∈樖嬍媬q媎媬W婮hxh沀wCJmHsHhxhluCJmHsHhxh::CCJmHsHhxh訬CJmHsHhxh镮CJmHsHhxh8KHCJmHsHhxhluCJhxh CJhxh8KHCJo(hxh?)CJ h?)CJ h?)CJo(hxh8KHCJhxh8KH5丆JKHhxhlu5丆JKHhxh跙c5丆JKHhxhCJ!!B!H!L!P!T!f!!!!"" "X""""""6#8#>#@#J#N#P#Z#^#b#h#j#篋囅炭蠖董ざ潖劧l`x`漐漄`hxh?)CJ h?)CJhxh8KH5丆JKHhxh>5丆JKHhxhlu5丆JKHh?)CJOJQJo(h?)h?)CJOJQJo( h?)CJo(hxh跙cCJhxhluCJhxh8KHCJhxh訬CJmHsHhxhluCJmHsHhxh::CCJmHsHhxh CJmHsHhxh8KHCJmHsHj#t#z#|############2$6$8$>$F$J$X$^$$$$$$$ %%%%`%b%d%f%n%%%%%%%%%%&&黝珙髭髭挖坎訁訁挖坎誆誆ネ誑諭駿諭タペ駿鄺滅巸zhxh?)CJh?)CJOJQJo(h?)h?)CJOJQJo(hxhGDCJh?)hluCJmHsHh?)h訬CJmHsHh?)h?)CJmHo(sHh?)h::CCJmHsHh?)h8KHCJmHsH h?)CJo(hxhluCJhxh8KHCJ0j###L$$%p%%&&&&'''} d,G$H$gd鯨` 勦d,G$H$WD`勦gd% d,G$H$gd $d,88G$H$XDdYDda$gd;剶d,G$H$WD`剶gd>勑d,G$H$WD,`勑gd; d,G$H$gd; d,G$H$gd騄W &"&$&&&*&6&<&>&@&D&\&h&p&z&&&&&&''''''''箬諼露堵炍炍稐巺帡~ocWNEhxh;CJhxh8KHCJhxh8KH5丆JKHhxh燞5丆JKHhxh%B*CJo(ph h CJo(hxh>CJhxh恴CJ h%CJo(h'hz&5丆Jo(h'h 5丆Jo(h'h%5丆Jo(h'hO(5丆Jo(h'h鸗5丆Jo(hxh,tx5丆JKHo(h?)5丆JKHo(hxh,tx5丆JKH''(8(N(P(l(((((4d篹靍頴鴈勦dhG$H$WD`勦gd觭$$勜dh1$G$H$WD`勜a$gd觭$ d,G$H$gd; $d,G$H$a$gd沀w d,G$H$gd; & Fd,G$H$gdU( & Fd,G$H$gd;'((("(0(6(8(:(>(L(N(P(\(d(h(j(l(n(((((((((((黝孱揞障掌戰票屁諟悳悇渼zm_Shxh+f5丆JKHhxh+f5丆JKHQJhxhx5丆J\乷(h+?5丆J\乷(hxh>5丆J\hxh+?5丆J\hxh沀w5丆J\hxh舙CJhxh+f5丆J\hxh>CJhxh沀wCJ h欽CJhxh+?CJ hU(CJo(hxh;CJhxh8KHCJhxh镮CJ(((()$)F)v)|)))))))d"d2d4d>dFdJdLdPdZdbd謉辒鈊鋎鑔頳鰀e坋竐篹疸疸鷦損境境境本慳殟|k|k|殟|k|k|殟歗hxh觭$OJQJ^J!hxh+fB*OJQJ^Jph!hxh+fB*OJQJ^Jphhxh+fOJQJ^Jhxh+fOJ^Jhxh+f5丆JKHUhxh+fCJKHhxh+fCJKHQJhxh+fCJhxh+fB*CJQJphhxh+fB*CJph333hxh+fB*CJQJph333$藛}v(崥[蠎諎BR剉;N亯bR (ueg蹚L埶唥v(峉mKm歔0 l毌e甆輨G250-N剉G1\/fGreen OP~ ~輨r N藛}v(崉v觺T蚐擽ASR艔 /f8^(u剉藛}v觛檈 觺TT觛檈豐:N輨r 8^(ueg\O:N藛}v(+T蠎剉歔蠎Km歔0l毌e甆輨R250-N剉R鉔h圧ed OP 輨r N藛}v(嵱~T}6q詋儚ba FO/f觛檈颯錘z愝Q鰛 觛鰛He済}Y 觛rT:N輨r NN鰛剉觺T颯錘珗m1 N籗 @b錘颯錘(ueg鵞5u髄ag&^觛r0G250_N颯觛鰛 FO觛r偳 zbaN觛rT虁of貧 1乺傪V緰0 觛剉upOe颯錘緩0RgNO颯纇鶴0.1 g藛}v 0諎BR0 觛r偛m500ml 225ml 2u噾 + 225ml 4l + 50ml 癚YNx + 1.25 g R250 鋘竳菑鋘颯Y!kO(u 觛r(W2\鰁錘 Nb菑Y 1乺偛m1000ml 450ml 2u噾 + 450ml 4l + 100ml 癚YNx 0瀃寶ek0觛r倢T1乺5u髄觺_gT \鰛g蜰5u髄齣-N諷鶴 \胈蜰籹僼g N諷鶴軶鰛 >eeQl毌e甆輨觛r偛m-N 1乺侴d奮觛r偊~1\鰁0\軶鰛l廵Q鍿N鵚{Q縱 燫eQ1乺偛m 1乺侴d奮1乺$N!k ,{N!k1\鰁 ,{孨!k0.5\鰁 sS颯聣遊藛}vag&^0 B藛}v(岯g誰 Western Blotting 0瀃寶烻t0 Western blotting枡HQ/f亯\5u髄TR粂剉藛}v蜰軶鰛-Nl忹y0RPVDF渷 N ,g瀃寶O(u5u髄pS鶑nl徴l0購蛓筫誰/f(u gT[剉QX檈孴醠玪\軶鰛孴NC/PVDF渷9Yb Nf籰 b_秗 TxmeQ$N*Ns^L5u乬-N魰剉睶瞞-N蹚L5u髄 愰b怱_剉5u髄筫T1\颯錘O藛}v((W5u:W汻剉\O(u N粂_軶鰛觺T0RPVDF渷 N0 l忹yTPVDF渷1\饄:NN*NpS鶑blot (u嶯鵞藛}v(崉v蹚Nek纇Km0pS鶑枡HQ(u藛}v秐瞞俌1%剉BSAb1乿Y墊秐瞞 Yt錘\頃NC/PVDF渷(,g瀃寶(uPVDF渷) NiRYO剉弖4l觺TMO筽 T(u@b亯xvz剉藛}v(崉v梑SON梑 Yt pS鶑-N闟 g卂xvz剉藛}v(NN梑yr_觺Tb_b梑烻梑SO YTir vQ僛剉藛}v( N齹NN梑觺T 購7hnmd柣S*g觺T剉N梑T pS鶑-N闟 g卂xvz剉藛}v(崉vMOn N觺T@wN梑0Yt菑剉pS鶑蹚Nek(u怱_h皨剉孨梑Yt 孨梑/fcN梑剉梑SO 俌N梑/f蜰TQ-N穬梍剉 R孨梑1\/f梑TQIgG剉梑SO0YtT &^ gh皨剉孨梑NN梑觺Tb_b梑SO YTir颯錘c:yN梑剉MOn sS/f卂xvz剉藛}v(崉vMOn0顅MR g觺TT蛓h皨ir剉梑SOyr歔IgG剉梑SO孨梑 颯錘魐-峱N g8^(u剉N蛓/fv戅弰v孨梑 pS鶑(uv戅弻N梑YtT 峇(u怱_剉昢ir秐瞞Yt S_v懍PS昢irub g湗r倓vir鰁 1\Ou颯翂剉:S&^ c:y@b亯xvz剉藛}v(峂On0(Wv戅彈bSO-NO(u剉v8^/f眡'`鱴x憊AP 0眡'`鱴x憊戯S錘\鄀r倓v昢ir5-磏-4-/l2T赥鱴x懶vBCIP lS:N輨r倓vir0 0;N亯諎BR0 1 ( 渷l忹y瞞Transfer Buffer  1000 ml 10 ( 5u髄睶瞞Electrode Buffer 100ml 2u噾 200ml 燫籗粂P[4l髞 1000ml. 10 ( TBS睶瞞500 ml Tris 眡30.25g NaCl 43.35g (uHCl.宲H 7.5. m渷睶瞞TBST 1 ( TBS 0.1%剉Tween-20 \頃瞞Blocking solution 1% BSA 100ml 1 ( TBS 100ml . BSA1g Store at 4! 纇Km瞞Detection Buffer 00.1M Tris-HClpH 9.5 0.1M NaCl 50mM MgCl2 030ml 1M Tris-HClpH 9.5 3ml NaCl0.175g MgCl20.1425g 籗粂P[4le報30ml >fr偛mColor-substrate solution 10 ml NBT solution0.3 mg/ml (u70% 孨2u鶺2up扂+30% H2O秐銐 Store at 4!  66 (l BCIP solution0.15 mg/ml (u100% 孨2u鶺2up扂秐銐 Store at 4! 33 (l 燫(W10ml纇Km瞞detection buffer -N 0瀃寶ek0 藛}v(峫彍 1軶鰛(W渷l忹y睶瞞-Ns^a10min 2jR}YT:\鳾剉PVDF渷 HQ(W2u噾-Nxm5 min l忬仠乴忹y睶瞞xm10min 3jR}YT:\鳾剉鋘竳2 _瀀歋鋘竳b4 _歋鋘竳b6 _剠鋘竳 (u渷l忹y睶瞞xmn 4(Wl彍侁N剉3杹gs^g N9hnc N-Nz槒^ZP}Yl弍S Nf籰 鑜a N齹 gl醠 5艌}Y4杹gs^g T N塠hQ講 6 NT悇v5u髄闚_薡l弍S 鑜a5u乬迯ck鵞ck 嶚[ 100V 45min N歔亯癚 N蹚L0 7sQ頃5u髄闚 Sb_塠hQ講孴4杹g5u乬 諷鶴PVDF渷  \頃蔛Bg 8l弍ST剉PVDF渷 (u渷l忹y睶瞞om10min 6qTn嶯20 ml \頃瞞(1% BSA)-N )n baGd≧45-60min b4!菑Y 9T\頃瞞1% BSA -N燫eQ20 ulN梑 烪is梑SO 鴙S_嶯\His梑SOz蕬1000 PegO(u )n baGd≧60min b4!菑Y 10TBSTm渷4 x 5min 6qTTBSm渷1 x 5min 11峇\渷l忹y蹚20 ml \頃瞞(1% BSA)-N 燫eQ1ul孨梑梑 烮gG 鴙S_嶯\孨梑z蕬20000 PegO(u baGd≧45-60min 12TBSTm渷4 x 5min TBSm渷1 x 5min >fr偼S擽 13.燫eQ10mlS+T>fr倳^irNBT-66l0BCIP-33l 剉纇Km瞞>fr偛m )n 怚Q Y梟 聣遊湗r偼S擽 14酧鱏緩0R亯BlT n4lom 葉bk蚐擽 怚Q0~fr^0 瀃寶踁 藛}v(崉v瘇S-M曬g睳孴B\恎 0瀃寶顅剉0 f[`N惽彶N孴B\恎誰瘇S顅剉藛}v0 0瀃寶烻t0 鶺郪崌T1\/f\$N*NbY*N_>e麐xFh cN歔z槒^迯(WNw 崌T桘婩h秅剉h埦彠Nir/fN*NBg孴藛}v0(WE. coli剉 pETh埦忹|邁-Nh埦廚飠+T gHis-tag膥(lx憁QZSO 剉崌Tv椝唥v 6qT(u睳孴B\恎剉筫誰蹚L埊~S0 YZ膥(lx扆NY蛓菑!n褢^\孴菑!n褢^\瘒Tir觺T 郪dk&^磃2梽v6 X His-tag剉藛}v(嶟觺T嶯鶹SNi2+h 蜰 \&^ ghis-tag膥(lxh~{剉崌T藛}vNvQ僛藛}v:SR_eg0膥(lx憢k鶺剉擭CQ猅U痵/f藛}vNNi粂P[\O(u剉sQ.0S_b霳(u貧Sm剉猅Uimidazole 秐瞞m1亜v鰁疧 猅U縊N崌T藛}vhis-tag剉猅U痵込塏觺T g葉\崌T藛}vm1 Neg0 0瀃寶闚hV0Pg檈N諎BR0 瀃寶闚hV zzltmGd奮0厤繯錧\O餝0NO)n粂胈:g05u髄闚0厤餢4x巟闚I{0 瀃寶Pg檈 eg陙 瀃寶N @b6e茤剉E. coli BL21/pET-cry9ea虄SO塴續 ;N亯諎BR d朚Rb棡[寶@bM憂SDS-PAGEo兞TY 貜桵6R 10mg/ml秐虄v:(u鄀虄4lM6Rb10mg/ml剉秐虄v懚n瞞 R艌b\齆v^軴X[嶯-20!0 2M NaCl 50 ml 觺T睶瞞0.1M Tris-HClpH 8.0 50ml 1.5M 猅U M5ml, R艌b1ml\齆v^軴X[嶯-20!0 0瀃寶ek0 1. 諷鶴-20!軴X[剉E. coli BL21/pET-X虄SO塴續eg陙1.5ml虄瞞 燫eQ1/10SO飝剉觺T睶瞞0.1M Tris-HCl pH8.0 150l 蛻琡苸迉塴續 燫eQ1.5 l剉秐虄v秐虄v(WpH=8-9購*N凈V匭詋儚}Y裇%c\O(u 髞葉Sm:N100g/ml 癚 N>en30R煍 2. 厤餢鈒Yt50!k厤餢5襶/魰10襶 5R煍  3. 12000r/min 4 !粂胈15R煍 諷 Nn100 l 4. 諷beads 30-50l 5. 燫eQ500l籗粂P[mp虄4l Y楏楓mThV鱩S20R-1\鰁 Y!k粂胈bc4l 髞pH7.0. 6. cM 粂胈籗4l 燫eQ1Mkxx慚500l Y楏楓mThV鱩S20R-1\鰁 2!k粂胈bckxx慚睶瞞 髞pH2~3. 7. 500l籗粂P[mp虄4l Y楏楓mThV鱩S20R-1\鰁 Y!k粂胈bc4l 髞pH7.0. 8. 粂胈 燫eQ500l 10mM Tris-HClpH8.0 秐瞞s^a坆eads Y楏楓mThV鱩S30R0 9.粂胈 200l+T0.5M/lS爺剉10 mM Tris-HClpH8.0 秐瞞s^a坆eads Y楏楓mThV鱩S30R0 11. T7h罷 Nn-N燫eQ33l 2M剉/lS爺 髞/lS爺葉Sm:N0.5M0 12. 諷beads 10l 燫eQ7h罷-N 4! Y楏楓mThV鱩S1\鰁0 13. 粂胈 _ Nn (u+T0.5M/lS爺剉10mM Tris-HClpH8.0 秐瞞mbeads 蟢!k500l m2!k0 14. 燫eQ+T15mM猅U00.5M/lS爺剉10mMTris-HClpH8.0 秐瞞(uMRM憂 20L mbeads,Y楏楓mThV鱩S1\鰁 粂胈諷 Nn0 15. 燫eQ+T60mM猅U00.5M/lS爺剉10mMTris-HClpH8.0 秐瞞(uMRM憂 20L mbeads,Y楏楓mThV鱩S1\鰁 粂胈諷 Nn0 "16. 燫eQ+T300mM猅U00.5M/lS爺剉10mMTris-HClpH8.0 秐瞞(uMRM憂 20Lmbeads Y楏楓mThV鱩S1\鰁 粂胈諷 Nn0 17. \錘 N Nek@b諷 Nn鱩T(WNw0 18. SDS-PAGE5u髄纇Km0     篹豦鋏阤靍餰鬳鰁鴈ffff$f*f8f:fNfPf榝爁癴秄苀蘤趂鄁餱騠鬴鴉黤g襦裼腔鉗墏墏墏墺湧拤倝倝yoe[oyhxh15丆Jhxh!5丆Jhxh>5丆Jhxh CJ h搟CJo(hxh+?CJh搟h搟CJo(h搟5丆Jo(hxh+?5丆Jhxh+?5丆J\hxh15丆J\hxh>5丆J\hxh+fOJQJ^J hxh+fOJQJ^JnHtHhxh+fOJ^JnHtH"鴈榝餱騠趃頶h"h$h\imm}}g$d,88G$H$XDdYDda$gd! $dhG$H$a$gd> d,G$H$gd!$d,88G$H$XDdYDda$gd>$剦剋d,88G$H$WD待XDdYDd^剦`剋鷄$gdx d,G$H$gd;剉d,G$H$WD1`剉gdx>)d,G$H$VD1WD奎^>`)鴊dx g g\gdghgjglgpgtgxg坓抔爂篻膅蝕謌豨趃躦辡鄃阦頶餲hhhhh h"h黝遘瘴茴蓬寮盞瞵坯拡~垝垝rfr\h!5丆J\乷(hxh!5丆J\hxh>5丆J\hxh舙5丆Jhxh 5丆Jhxh+?5丆Jhx5丆Jo( h/sV5丆Jhxh1CJ h躉\CJo(hxhtCJhxh訬CJ h= CJo( h%CJo(hxh訫qCJhxhC}CJhxh CJhxh>CJ"h$h&hh奾抙榟Zi\i頸jmmmmmmmmmm$mBmDmFmLmhmjmlmxm榤歮甿瞞膍蕀躮鋗黰0n@nBnHn蜷噠咚噠咚唄吒吒攉洬帿帿帿洬帿帿帿巶帿帿閥hxh+?CJhB*CJo(phh躉\h~ SB*CJph j答h躉\h+?B*CJphh躉\h+?B*CJphhxh>5丆Jhl5丆Jo(hxhJHs5丆Jhxh鯨`5丆Jhxh!5丆Jhxh!CJhxhx5丆J\乷(*m\m╩鰉@nBnhn妌畁衝襫鑞鴑oo`o妎 d,G$H$gd! & Fd,G$H$gd! d,G$H$gd躉\刪d,G$H$WD`刪gdl剼刪d,G$H$VDWDV^剼`刪dx d,G$H$gd; & Fd,G$H$gd;HnJnRnXnxnzn磏纍衝躰鎛鑞靚頽鰊鴑ooooDoFojolo抩攐瞣磑6p8pNp琾畃纏鈖qqXqZqnqzq|q~q榪綦愴愴愴匐譯綦屹腖淪毀毀①樫媫嬞胭牖牖q渾h躉\h躉\CJH*o(hxh!CJH*mHsHhxh!CJmHsHhxh!CJH*hxh!CJOJQJ j答hxh!CJ h躉\CJo(hxh躉\CJ h+?CJo( hlCJo(hxh!CJhxh~ SCJhxh+?CJ j答hxh+?CJ+妎榦秓竜Hp刾纏豴趐$q琿*rjrlrzr|r $d,G$H$a$gd! d,G$H$gd; & Fd,G$H$gd;剼d,G$H$WD`剼gdx & Fd,G$H$gd! d,G$H$gd!刪d,G$H$WD`刪gdx榪歲╭鋛鎞rr"r$r0r4rCJhxh!CJ h躉\CJo( jmhxh+?CJhxh+?CJhxh+?CJOJ%|r妑磖 s^s苨*tbtdthtvt u爑鎢v聉膙dhG$H$gd>dhG$H$gdS5$刪剺dh7$8$G$H$WDj^刪`剺$gd両$dh7$8$G$H$a$gd> $dhG$H$a$gd> $dhG$H$a$gdS5dslstsvszs|s剆畇秙 ttttt&t(tTt\t`tbtdtfthtvtzt蹣芪課茜艹攬澻躹obVLChxh>CJhxhB5丆Jh躉\hS5CJKHo(jhxhS5CJU hS5CJo(h+?CJKHo(h躉\CJKHo(h両h>5丆JKHh両CJKHo(hxh>CJKHh躉\h+?CJKHh躉\h躉\CJKHo(h?hS55>*CJKHo(h?h+?5>*CJKHhxh+?CJKHhUTh+?5>*CJKHhxhyCJKHzt巘瑃皌磘纓苩蕋蘴謙鎡鷗uuuuu(u,uuJutu倁巙杣榰皍瞮簎紆膗萿衭襲趗躸鎢靧v vvvvv(v2v4vh>CJo(hxh1>CJh1>h1>CJh1>h1>CJo(h1>h>CJ h1>CJo(hxh>CJOJh躉\h>CJhxh躉\CJhxh>CJ h躉\CJo(:聉膙衯講豽剋坵巜榳渨瑆畐皐竪鄔鑧陊JxLxPxRxZx\x^xrxvx坸瀤爔瑇膞苮y yyy0y4y:y蹼廑庹肆朔水廉廉ギ洃泩洰ギxnhxh舙CJH* hxCJo(hxh舙CJhxhCJhxhM!6丆Jhxh6丆JhxhM!CJhxhCJhxhI~5丆Jhxh5丆Jhxh舙5丆J h/sVCJ h1>CJo(hxh>CJhxhB5丆Jhxh躉\CJo('膙衯Nw唚瑆畐紈辸靪苮0zdd$勦d,88G$H$WDXDdYDd`勦a$gdx$d,88G$H$XDdYDda$gdM!勦d,G$H$WD`勦gd$d,88G$H$XDdYDda$gd $da$gd舙 $dhG$H$a$gd/sV dhG$H$gd>dhG$H$gd>dhG$H$gdS5 :y.z0zTz恴榸歾瞶聑詚鄗陑騴魖鰖{{({*{剓唟攞杮▄磠鑬饆騵鰗鴞鷞|蜷拚獸練練賃論姕妢pg`摐揧 h蕋CJo( h1CJo(hxh1CJhz)h舙CJmHsHhz)hCJmHsHhz)h舙CJhxh1CJ hz)CJo(hxh?>CJhxh?>CJPJhxh6丆JhxhCJhxhCJPJhxh舙CJhxh舙CJPJhxhM!CJhxh齅B*CJph0zJzTz恴歾陑鰖({唟▄鑬*|,|.|0|2|@|d橚gd< & F d橚gd< & F d橚gd1 & F dgd1$勦d,88G$H$WDXDdYDd`勦a$gd 勦dWD`勦gd舙$d88XDdYDda$gd舙| ||(|*|,|0|2|@|F|X|h|z|||剕唡妡寍抾殀竱簗纜聕膢詜謡鈢鎩鑭饇} }}}"}.}F}H}h}l}x}~}瞹竲衹黟麋噘夏徊槦粦粦徊姛矡矈不懟矐矁z儂儾徊徊徊hcf?h嶤NCJ h嶤NCJo( h/CJo( hqCJo( h;<CJo(hxhCJhxh6丆Jhxh舙CJhxh2CJhxh舙CJPJhz)hz)CJo( h'CJo( h<CJo(hxh1CJ h蕋CJo(hxh蕋CJ-@|x}瞹鴠鷠鼄 ~~~魚N:<>妧詟RT騺悅 刪剺dWDj^刪`剺d菳 dgd舙$ da$gd舙dgd^D*d,gd/刪剺d,ゐWDj^刪`剺d/衹襺鑮饈魙鰙鷠鼄~~ ~"~&~~~~剘魚鰚鷡NPT:<FHRVZ^`dfpv妧拃爛黝甾疹思摷集⒓集⒓妬zs鉉掮sz頹顏hxh_CJ hVCJo( h_CJo(hxhI~CJh"hI~CJh_h舙B* CJo(phh_h舙B* CJphh_hI~B* CJphh_h"B* CJo(phhxh5丆Jhxh^D*CJhxhqCJ hqCJo(hxh舙CJhxhrvCJ)爛詟軃鈥鋩HJR\`bnp纴蝸騺鷣 ^l悅拏杺槀瀭爞畟皞 0VXZ\^r|~們唭垉寖巸拑攦槂騭嗩仫揚尕曙曙仫尕曙曙仫扛尕曙曙仫乇ф劓铩棌嫃嫃嫃hrjhrUhxh舙CJo( h/sVCJhVhVCJo( hI~CJo( hACJo(hACJOJQJo( h CJo( h(ACJo( hVCJo( h1(CJo(hxhI~CJhxh舙CJ h CJo(5悅0XZ~剝唭妰寖悆拑杻槂爟 dgdI~ dgd舙 刪剺dWDj^刪`剺d菳槂爟蜩hxh舙CJo(hrh= h= o(:&P 182P:p*&皞. 捌A!皀"皀#恘$恘%癝班 惄5zDd =P0  # Ab鴇yL S鵥絜i忞韣衠=峺Dn饏yL S鵥絜i忞韣衠=塒NG  IHDR招攔sRGBy.IDATx^頋y%EuH6妧臘*u個鴄1@擡Y\0, 寑,*0殙`FeQYE!盛c$奃d (寛0 _x癱桀鄯o過烚銅[]u戛>}SЕOz巰#8=B酲/Gp佺pf鬟#8}C罊給#挲qG罊巰# g鼉崹髑pgv 8巰#7溬6⑥Gp溬78嶡pf鏜坺G載祚焾#0L2e鑒鬏g鐔ü蛶#!嗵羈Gp鷨3{逨喳8巰3G戊}Q飶#8酉呃奎絲斜鹴瞼涍舺U 弽)[79撤<7?35嬤3妃轄碤X嫗p8硍馱崽夼Qh硺侄撎鉻 ;棍陊Pig溬+叧∈溬侯fX 饑X ,&諶<艡篏猻;硍n0貦綃a滁n榋2栓匪/w蛓!嗵蕖2S=6磞LZ椃n曯飪#08硍~a衈鴱覞佡節Jり敭莖tXh螠91蒯6擣鄷瘄邐;锛趈Yn )fw羮0鹴1]M蹤8j冰燻曯*糍訨癆A%戊]Dg.広焟_^響吉+r鑣彛:訚佾貌箽\go霘Z獪鉰{M沐: >{ 滓D:Lh*/_K骷襤p=殹髻G+灑80>氚J詹瞟幨絅Gg齪ヰg覡曌8U 嗵^娡幀╯撌靄4Qkb姝9搮3{髕憚G揨櫡&潷{p慌嶡pf鏞燳ζ顧;嶡#87su岪隞P.{墠褙頃單kr欳罊9+iI﹝洃J歩XGpf镓铩謵槑a徭:幚溬霍親l擰烽h>u7狨;嶡8匙乯]u娰獼c&i憓|抖魖:pf軯脛菊嶚褮狄Nxe巰#戊M<~;76柗熭 ?v^#<戊蚦^睧l 豎鮁湈掓鷐巰#炿=霨i橽入寰疾僢盞S=B麥獨鰖:剙鐉煨`櫓戊51絆H>{黦fm9犯迧逬誚:砏峢97侐榤裙娉凡#R豛-2兲 ?柳E,匙~{$8矴2#欋>\>礉v _a挀O沋t>tK X*窔炸尡#絃磧7Z g鯮暗}訜";l饢O 慺p=dU吻.$陳膋Ah勵瓄 Q!C9鷺樁跅R'W 8漵 5:汃澝鎑=缽犣3#擠罊$p嵼ΕIZ1仮虉韞n|冖阭皆%∧Z鞌島\迱#腱 ,6癬K蓪諫瘢蚼絀瀼j墬o蹤[趕^存n乤-I亥hLrQ?藋軖kHQ.H#r/竟煄4趕貘;6鄀[[K慇遆w汨峴=!0衹靂G蜆]rvc渾祲\M賧沢己隨~霐7L戊駧礷#穎11X,]迸"29崾P櫧Q煔e崱Sn#\崏w(%変!5Y?*<曼駛啓槪勞倃y&p5傳j藑霆惉Q^勎)0鄭ǜ贇 謨dS1腲#衉溬c[ E匥矆腄)' 睯畐q崊jQ鯿p乹p5f鬸筗!0{Z崙'>抈RZ櫋艉栣靱ギ苩q 輌弎閱k癜%0 蕦/.鎸dv&g齦Q亂h峅 鰰幑4efh虒篒yJj}S w亷"鄇L\gs塟?掹QaO騝cJ4T/&F嫡誡-tq|輌弅月罀x詷1*.)6%j{ 嶡87忶m駧-Pj8觽婁窀 ) 6=j&5f嬐C-: 嗵蟈艱-oy 職]煭,Pl仍竐蹣軨玝 'J ?G戊%@栃aok4鐀R4i駂鱖蚌J伨#嗵kgQ躑钑篋肬踤鏤x鈸n12n#溬cI;mEd椃:孚粑r 枸嘯納tc,?罰_8矴1"V疊閏h鱨A :盲!N 埓擩H贂櫋8硹[攆T蔞獺磻某D柸&'C}H'嵮頃P挨欘?餹#P戊礎[竍D5黔瑣镚葼GS觓鷸曯*9El畻偶洃厑魝巰#餲溬)H鶳T孨甓蓆旸C娤蒷]1嬉aB;+ t虪Z&叐1鼴&pfo韺賭s襽梣-}&寭<蠩3碿9N齘G犛8撤<|h >婣g5-SG蠓(I烮 ↗橯X渦 朑螞w"F罊藉翍鎌硵酴1洙=<].7激脛簩q翥樶:軧罊酵 蘭犫 >R,cz{岲犓$黤|3G燙8撤5W綊y忍4p鎋噊繕>t焱`hI/'_驐1靻nXo錦8y琛囂+掚醧W旌氘梍~y渳夁*g鮒迫;蕚 冑U記喥хK3伱甩d" R蠀z喨謣琧4|袳靈w遾7ul魁鰷\}跽\佂-^x虡1?鮐cw遾1Z炶[澷n藹仨Z#劣螗i4儦囫0鴊-p蕨U莵鄙{!磽餇]澁糕l癆z(?齣滾~擟罊n堀愋 茠緔璱蘆楢鏚3蓾9跠孧卻侑傫*闍張dZ9'憤~箋of+偖獁遼笸踟陣Z澷`X弦 `<蕎榼麶"凍沼順!蚑f器贛蠶P辰@@搿0w頫d甛|衽÷螘鬟骹鳡)G諐仂 鳱蕟杗T4Z訐^鋄+u(Q,縉0├檿桭蟧藾`蕯) 冞-=<冑v踡譠k[,裾Fmt諽g*4犄合蘼o 3t}|)F=﹋\姶牛+]坍屬kU瓳"{橵x粞G蚈=鮐鯧.箘膝朲 K掖迠y絣詸絽aU:丏b艤<#橷AX[妸Q茢])橝 岕 iヰ菜.K或孽+~詣3{M缼P眸罕蕳魢禕TR氚13+# )W曔O>鵧 腸嵉揎啘賉嘰3T.ae匥鼑.殗罃橮!緽%ZJ3詒/P+3g蜠d:u*嬰;锛3a鞗m熀 v,奮rL玁6m檈柀摘墾<秛稥;w絃"慇厃α? h鄄-牖Y歓蝜U轑'&)4O<馜-齙-髻泘~薦E擧鋋s.1_-巔J桒K/ C蹅 M妁增xC/R纑!!s1浪擣`楗楒i找鑵7z詂%0嶱"翱Du& 蕶傳 圱na懟瀡壭稀Q忶茍儕杮cF=:灜 >{ '|蒢'\Z"愰闀5嬊猣S -q浗#溬JqY3幊=3l姴%I"2@v漻尒ㄘ:1雐鶺忭9!嗵>~_]珖瀒-嗼3彼蓁(z嚨5 搈|J 馡}褑價鰧qr/仦咭Q溬[4艑熱v澬:JH(淥,Cu晙3磿Z鶚亞pfop$dA%訡%冊綣,寞jP覯'n1軫*庨n{鷣鷨@l釡2燨;拈v\ 5敭9}壁謘:掃S{聟5d^ +k4鸋昄r嶝(馬)個-?鬎3瓧2╢i賶浜(C7澗宀.硧銍-z2取冣z墍3{用 $詷Jt槾緫出G驁6鰃彊刟墻胦披?駨y惕函x沠聬汘y{嶡.戊嶠@DU l嶢+纏 5圩6齈弎卻 攭錇汀誠 J'9杲^戊blDc徂&g哨X d墻+3dvw齋呍k戊,+ J鴨胄瑣曍6㎎4!啰袣J茴8紸荔4:旈)A]岢u3懪e蒜蔞c喚瞍'K=礍!)9!XV俇<暩限鬤d苉種腧柱4o]L:4kc%鷓tf袷焵1]:閃嫢鸮|糳<87=蚅煐T╩硤aW俽祶tWiXlb 睄肏瓄aG爂877 劃T覚D8紓0~P9M3T 戊崕TW葦$iu+3碆[i椞拤泧7跗:厐3{獳cヌ擑C$2%[瑪)砐z葌4橙稜1^g齪顅 !葴錕#閔嘪O -p眥`鉻嶡a溬 C5 r礉1檳穮嫏)X~";軨溬{8ㄕvI{b剞 罆fbf戮S0怓 屒蘐鹻疒:亐3{'啯}#磼!RUQ祙嶡u8砏噀遦2撐櫣霆B0裻AEe則氷U9 嗵葽t脤 懭2鮁粵\伭槀寤伝[寛3麍峎>襲楊N允: UgAWR5v狥罊絡D囌W湩喺棗鲀2DVヌ槩R惁曞備b&v薅#P?戊鮟苡L橧$紻5WTp淼蠁+0=y稦F罊}d賽丏喨1剚驟pVyP/L灍焦Q66l2骲s6U識"蕫? )f灤, c{]u働KMS━^N躦軮獅 懶鬑<鯿襜拁&拮pfo鐸hfP喨k驌2i決 &a紡巰pf鱛B3DZZ偺f爄M~叓,_e嘸.G;8硍gci槆 潠 幀歿沓$躌G溬哿竭璮f4吘K).瑳bS1衞劷w嶡>榆/$3GU<摐r簠鳫]"姚筿韠K優v熃嫞諹涘w照O=悔g粉hg鰷Pwz k)03`$薌#饻?(8q"`133 r玝F罊=嫜榧mo~鬀K眛`濓縲h g鰱煂fK港&d 綏幚h8硰啑棶%yW鼂}瓁蛶$ 嗵> \>B隠*r婧墡@8城=>揳)0撗]銃#P;戊礐< 劔2f!摭:pfU/$舵”W`T媭g霞邡襟芇k尯掀喧紷Mxv仛Z髻k呑+侒n9 ダ寯:啤t佲8鞘K帇鍊魟鉈軺;8雛X織X獵〞迾#衠溬;>n#8)溬鼼8嶡pf鏜坺Gpf鬟#8}C罊給#挲qG罊巰# g鼉崹髑pgv 8!磊{HU旨W戊p狝@檖嗘冬1菶罊焦;K4讟吩 柎扡8 磠 嘛Me 嗵捃下椩7噓-壞饍伉r桂~8 ?綱`鱂{個匁t~y礞隇{O喒冚pf鏇牴 &'辮掮T#獻4翙}G蓰<&滹e鹼侍榾"嗵>*b^~P鬕櫃鬫L齱fouo硾H伭鯭w円c{=>獩#=珴倩7fnq+萓'睽阜諫]茺柗譏g侍>鬂飢G=錮螠9檠徠g湣隱m嫡-奮Rz憇麠奎鍕.篐瀨蟶D嬸曇朌~3{銷\P爕屁湯灝g婏擉~戛М逼"雼/絕諿oM鼴戢寨~跣C+N啋;鬽Vx%<韼;罾灈n ~?蛉#?饋$J騪同獨癄cE镺娛湄颮M%<>眭\ 6痡r誈昣嗏佟q2eJfw鵡(飜塹譤麏W尖鈍n>韃椊歆?)燂鹮湛鶻鍾璲鄷-p譟g甽魁鰴> 哢{lw磛o均f=3鈯6諶'z琛踤豁z氕敲c藜y{淼譩-0>]陵b髻8jnss訟隭希㈦y鄀攸o鰋梈jo紂v嘞3<~囆&鋨6囀唣7郪锝鬢\質笸3G衱H冎衲g虡鼻{皓冸7躳僓r諽ga蓕鬏wC雓:灮+ㄤ[l羃<*汒 笛3{m悍掾 h^怣1k舶yg=髂U⒊>楖圻6酏櫹|B"7縹跽W7e啹~過y鍟ily0刕yX`|暙l~譣w伶0蛩/辻鐗9y鈮'璓^X影FR3{$醘膮簛xG謂鬢薟箸?衲y挜;7徆)楝峂詮 懓9摙8閭帰贅饒樾wH 徺o魁(瀨r@s }a蚿&父鬇行> 飋錦謨陝袟^zi鈐戟揙>ct;n;l.仦JP犺8N鷴K.9H)m鶚7z糟巔{f詂媾*酡!D旼7巎]効嘇Q徯7l雧熎嚡?Orra 6 妖昖Z _x(&疰圭瀷 錨0J斒#$紈羵;V 夲癲qlC虶}酤o5-|懲6兪am! 翑M纓O棾徫C<猙ZG個淽v賲{瘍=C籠y髻+嚧諑vO 幋(<唂ml菜)QE旯h漵Dパ箿t\眘&]u%}@) Z椑b  鎎庳u譣韖B3g翁WK鴧@8 ~嚮%'O/1I 蜽*LX7輙&3檓瓜捃t譯g+z」拾 阹M+9萭WUx,J蕅並碭.裗/絕釶&lO;4&Wy藜顄i粢K)脣紖痄嘖N@CL 蠟Gd莔:Q@\A)m7:砏鎼褥貙獽錈宕堋鸔N tD侁囍&{T<茟捈鎰y9淬僔獎戈盔/.混28傓d揗^W噉~8聥鄬簋仾拫根E1}蕸 殳瘧0;2 L憾.;7黧廠W'捩+跼︾k綀D[巷\難騷霾#S讅O=1*轅梓躶ヽ恾犻E]碻撔:淒1B鑘雙:憤嶤纼A 鬗` K^%裁膦{愹褏 6WS1蹤k扼)婝1@0鑁d q#O()r#[k躶拝}簦E{a捤qG|Y硃珛:7鐅闍絹!w."7妔3 (瑜揘漏G厙謾h宓竳剎tv-饊搡DgM柌v<1v%9Γf@嫛賀柛B輰鶋湛 慤 zA:;呱滍/鵎r1G鷷瘄?嚜.僱離琴'袇宀腉>肞958飏啤uN勵H40>j Z 佛奝UK足jLi鐵頸jLB燞袾9嗐62#謌隥*3暴1禊熁磯壙搛6r旀駪;r凔é蘈o贉pf摪3頀J趽 r眩*歿N扝h]K惼t+1,蜫溬椫琂-欜K鼈 鯇R耹籩)癁J檳硟娗鍤摰抇0銖鍓a$$@=?鋹C6辺c-+i8櫥rX藈粍邭筀)過=wba氋欩TF澱x熟華⑶ew 珅 Q蘍?簟釣3j鏋栩777姿 澷{9;曢&婦nc諶4'j7}疲倘咇$ .?覒輎99!.XGIw螷彃3{i鑪{#喣圝.pz檦T圚W M2.R&-糷U鶯朧宮*眻.t獗Z鼦@儲躶22鉃傀醑'婞[啁顈2硯MLhI嶊(2鴉虌#惗彺瀛蹤o@羄せ(Cg[=昺猞秎X毾J"B孔_}磉銳蔥欚 輌熜'zf稢HHL鼩o慯EZx裇翑橵z*0賷 酁硴9媭豑跑蒹 酠T逄^禲猋J.)/pg "-nに鬧醾綈賢?M潶(跮c-吜#KZW嘑EU?釩鯗鍫B=g1!.ど踵朡,^迿8V-!翊X>^韋狫冑i- S3gM鶘JW撋,詩,7b3n'B鋼_9:陏壍;!:砄菮gw桺B凚筗Xa%縡4鵎觧{:訋|[h饊j:th{:詽+n!筙夙A魸闣$詛紱. bm65Х秵鳒臘哥噸斤}緹F撮q塵灐璱趰pU瞠瀙N;(>嫊彪閅怢7a啣< 危螞:8╛*嵜窒cP迤 濷w辻'涐t蠥$!`T瀻\墠U&髻泘擣貫躹(脪瓢?n;嬏@晝脦剷"?彑2鍈E蛪懦薽7現0О p捨]c鮓課閦闔臮1崁gk頦壽謏媾苐采R 籖*t偛 脋磲V岫f$JH脎q`秾,巑嶴8rJ8+恲搷衷/'?QT{b:A h忨7顮M枽kS[R趥胿摒T\嶟cV蠵l)憧誇[兘nfJ4島!|-1礎緗鑼C蟯H齂嚷L)GG捗-S氧肮f禸pc6T6粎`sB <'n娫祫⒊橽l堅$p5?獝蝋+?WD Iμ⊿! 沘i蓸允2.,虶5椝C 磚{μⅩrc 's,rRqA駠zhX?[輡笸-矙湺嘚琞猗懼E%℡(亐3{ 惺邟鎺騯誴а"/趘C;垎MI7H4UrDz!"胒寫恿?0壆A〥嗚3/黇je訦se>P攱絕╮檴痛婡:'<箵)wP*o糛e焥9燩3\1s鍸v昪9!n鈑u畚襇庀趭s>楫宏*f騥O@暠腳岓z㎡n迦娘d^HC 墻臹5k瓋銴4阰B搼楷*QfTONm兲tK?濧4虨{顈G(F杻s濬麵朷濷簈豁#鯭 ,f9]鋅_Q 帆B齣蹌芓戂><紾銚0N脺V *銃盺&魮處p硯d瑳"鰽位翏颯桭敥鐳i6`U-o5w哀37綁sv輌焳栒Nr痔b-6塖/哭z氕赺敐鐞9亮緄f3x氍艡絅t嬚--b>]9倀"蝕a鯣寳噶?s譿辻gd,P錇L楽?H>p襓C&A隭栬Qz:薓醗4垬滲%秳 <鵠:溛W"'{Fs騌)b{Ж踅草1$趖(b楯cBM侶〉兆!M婻C斯b鶵BY倅M傁Qf2籧 撿黑MC蛅s罕>鑶<"<驦TI+/RZt^溲R`j)3湴%帊︹非}v阽! 6T&搫慁獞!_SfL61;m螬娰;蝱2擃J加 L8{≯-M L剡w軕躮渒N羈~綞T危J~ $p秒'#o豁6窩夔螡藍來i"t _┰軔?|葔6gG蠷瀒 J5譢Cv'L蹷T珫 v聶[DG+]w/| 聛艝g焳6蘣嫎t呬ǎ帓C潂葯0e B揯魟%T⑿+&Yd懼贏貸ツ內"XN>鵧4栱憤|垊A:嚄W]uU詖曠+鮒 蕝韁扠.p#9脈豤铻x榦|飤骰yT 覵暝2=5p綍巒7踜判w妙琚搻)腷 &Q*甒&灓紆i2μ@緳R夻s僰$&澄VD 屷y21 0惠蜞ⅷ 2 醄﨑肣1F匱W芪塲6惟!6庩=t埭0孕0l糏蹔伐^P^}I希娛[祋妍>N4畉貣e圠蠚覑蛓&j衲テ柘5譢缶8}鷗胙襅/娃I$r&寪酧渨#}5中* 萈鏭{恁j(>竈/嚙黫+iρ鞝.葷 ? W^軆 鶓Z磥0od] 錦9邘曇緼s出嘟aUf|娎箹襲CN G踻v+#/烵 嵎獦5>6髻1g .n敘)A懜&W韛&2兙Zf镠8ォ^,盓興譚入墍H汛淑;-}睼$ O=]4鶃>v肛辱mTu譅=聭7'16*aE鯩鈆媍炸4i勵m9硰廰j(1t壖 0軩鼬zD%燮\蘥墒亾mF巸攴2陥z!2 3e&QLKU閡&尅 P%:je 埛具~鸌虘2搸x勺Or"d諿o僥殾!E*dFw鯪賒;9~<`t_お8撤~\Mk?<%屚9磱卪(鱖糛(騄b貁氕挹Mev+#BW/*u枆溼<3 ~t槍聈爬刟6Z匘m娖佊90軍哢(Q>Zr忞w\5齴揁翮椞翣<鍰褭戊駨QC'炦裍浮Tx葈媜Vfd)EE:"芓_Lv嘪噿(Rぬ凞,((瘻狟k蚈_x釁鵍蠟孕膝淄S/o漊堤 Cb扜立9#罊=j曇矑&戤某 詬 闛s蚉雯>#e%穄泩猧3姡Cc磤^_塅底╩@:-;婸淇濼TSG$矰%Cerz伒zY檙悳庫J腞纈bz殑u&K+÷28欏 d彇蔄+擼犩頋+<暽婡%3j蕍Z筘忭kZmI礲栃橍&M5c%]學'E( 抄寠;臙[謫. иn氋謻,旕f錉^ 輳uM4恀$蟏襹罜UP謼jZ[lQ=d5 NJm'&HTR揩1漵)j1X惟韗牴DM刏{ -Ee8襶冕 -嶹zw估減a焀辮OHS敗 +\疂爸 {墓;R/蘥W E鎀 F鳯 雙 哵戁紝#P暨覒7b垌16U(T<寴Q滕u 8敡f昗笆覱?]疝"臫ZNtQ屘+鯜R饘凊<ElT菬7宼|(|絨刕q;潛絑渋S帣薳巡徊L sf弇D詒Gd(^K蟆H'&EI朲牋軻Cq伝`蚮挷#':挌爿淿l">筊|?pI_猱$滵_L +;24萺]袳跛4p拑g鎇儈 >糶C.礙T~笸7+鄤oI撍苮C駚鰭Cpf 諏勌"eF,c^$腥lB5徾Iá戡)3Vq}蓏14=峪A 孶+溍旿鰷1tyQr恎-l_FYK,Ir鄎齀,嘆w,賕_蜔?_;砼悕潔錸s艤;7d,橞熲n揧賯` t;$_H0撞牔QG7B比l6馟k痙6v悑鈠S淔K妸苔Z桰`k8 Lㄏ[緯U=G濊@6,疥爻魡n 5?5嘍面$W漞>瀪8镵.筪撾#殿咉pf鱛葻 嶳*p㈦R6t匫1惠譎氞じ9舨修<澳z!YF駧殘海^c犏H綘Nm擰冎 璉棗#擒菊;勀"劈i208 碞6D韘}=鰼遷遾鱢沵咾v 嫣$鈕_I^梑撠9' g鏨爰灇J篈虙)\韜)*燌矷|錿gZk423j/寧髸駏鍾Μ覌T揱m媤 fW* 6貈O铔E柱箚繴Vr4?鵍匶晾鮉7輙h+^戊錺脎]&艅!1D(傣Ge8Q饐r藞-%讒氞S隭"旗怨$ 跛 諥6#魾弴A蕦)0 ZW胴,=F{鷦]塲Q傘鏁VZI(3:" n;K鸕寭銶D嶀呫鼻 j尀滀飿Z$顆pf胹楡 &epE叴晳b困6攆R<8憇:h飵欯棎bR唞!W?碝潚聱蕑』瑑m蜹fp鋝`mɡh#斬fM[H炁(:3╘A栣梊%耔賣匋-".R挏丐kAl絏q溬媍甄掟慭Z睂&蚈T劯43 藚紡奿V鍜踕a55焆]憁歒諎*趮則Cyy逺f襓諓龜睤濯<3釁捁湱煓,臶囍y&ICLN諼c 責bj嬥nDp8借褰戊謶;彉嚠xp罐" >uE蘚.歿2o菰d<匽弆謭7X艚LU皔:b蘚9殛爉汀7兟躽#饊^垼Cc箲:Qf8蒊幛(鮤:砼張D舚澗M浂媉{淼 妻磰啦觳藃N 瞴敉\+ ";B /y犤牫貏貴l塲';-9耚WsT9鄙=1N釰x嬢e%UI\鈑$浂D8:!&顬詠P呴緞醥01鑄崏鶙粼SO 3o藜?湀u6~嚒rR疸汲k(艭霚喙螟0>13((镘2};豬蹨;=|o2)6*瓷ISZ竛鷨蕋蔚^麝O>%n勄 /\Y'G蜖*\?aeFɡH啋齛/詑UbO坱/$繄苡紵鄑[$Nナ訬(0葰竨槡葰 UH滍8蛉螒駱a y "賐+N2s!pQ紜D)3{溈$硤4"Til繼M蠞J/+ 3栽=)71姪鶹fHq2-gYu嬫6諟稛嬫z1W= t睏 k7楚骿!p1 +壃塳睮偘<庖}鬏g9豊/rwf鬟鰏剴J^7慪四bUq8.邠璲 d 雲*譝閪呏:h彺V鐮e 劥鰲膂H碽Ua怩寠88w鵆~蝭嚎镸o"拠9閨瞔 勇Dy瞫>縑!S>扡gU 強牀艕卦(!<贓;Q副弰疘蛺葼骦R>{竍BP=帶T縟篿8f鈔X迏P挗zg 蜹'獡F%跣J #凜鎮'J4熗<鮐#攄3/蒲:12魩uW鑟*bR=!j瓴l 烠F獠M嗹Н鶜2I蟭/TsB^媴f$Z$/峹E軾駨滙 濷捽)%僉禘eA諷鈍Vc冰5鐖蛉25媁胩^珵棓j∝N+橏"6(銩I 坯(嚨報 "]鋹 s絜\珫 钜晞y(鴑*黇 授HJ[嫭褥m]湬鳥彧拲78}_v賓沷9鬗 粉]k&🤏🏼。蹱,翟R>W葇I ?鍉掊蘜! 鶳& 5>*仭竍擢 槺e瘛Gl" *︰d=妑M踩枔-奮採,fZxouB,M"nc摜>&=T!辡A2'氌4S^鲄踟9'3r霻筰a婩膊9袮甔O 詐踫N[0緶A ﹔NtQa幒獺n,2S&Qa竛朄u坁Wp凵潬|8I琢;g襏W=溬叟蠎'-!尾堿  ' 6I裙論u@:膯*Rwe烎3^塥8追陸融碶\詼昂`幬鶘痩Z:匔望;奜五b藑3Q媱伏i帨sE廔N姄;院睗鐵蓆苩(唲D蠠鴰XvN瘃鞳t0塀[9撤厊韸8l狿r茀'P釘d9J1疺2坹鉛J"y膠鯇瀕穸澰2)篳霠*WOM~r濟伵2.郻}粞篞擀閄徝4t獣轚+勘z7鄓f/25杋齙4岨嘜枎rQ1竽シ0檖殕pup4洫踂劯_‰渟E穭_qoz 'o(i+!+切+ 戊黍I( "w+,F釀paaI]4飿痳K蕸菌凩m譽"#eLx媫e獘7_>QhsZ蓱z#,i聑Z[縅"*棸兿F%爕%}E罊蔣#;Z 婸'1UZ-6嗠8孻鑎70 G's2歲匥%毢內0蕝+燦%cm覺t摟i/j#6 1矇中5止f)詐勱"瀓隠恗N鎻璽B_;⒄る.Dl裐S薣BG6P|k/\Qdぞ ̄V驠{3{遍.hvT-賭&@|J+<*mA湠葲h茮zc敔d翹t)'z癷K i鍦皈槡"0怮 $慚彣紛;溬{7wH筍5!i 9讙zK昜RM泌Z鷐,詡趼?4閖(fqg鯞醮H潿銸9殫暅]8aZ斬,/ @罊藉烣3"矤U"睵澃hS-L 憯1% 敆'n$M侁+a挒澧V杛"吩郻橔1L*0'> 7Q9戊旵歐a:"f瑭H龜聽88梌!鵅 p,bB >3魾拺聏,l勗Y褄溎P%&0kx袖圤B[戊崕睧X7陘鑽咣i籕X/2溏*f\ F>%鍐 矪ff圠b鈗+d>s穽D諪m~鬫嵾戊Fk>.X趵\R*b0={dべ3 *2垝w襋4壃峼 j/A罊J晻 *暤]涪r釅 _輸>脡4崢}v肍蕦 栧#N縵4>9[jX,cf製rP哨 >糶C磓w%^踟鷜P巃腌贏墸g鮒屏vm蛡y.礂EW淪雲2貇闝暢淵}39 糍砱)"5E<鮐9A黯+{鈮'}鬛齀螅&溬k睹語~莚}恉a $$歳6|梱觬枕匔rM.ZD丏諪櫘<鋯b菌戢+蜱鈍n冣9v辻鐂=棑/綳馮寒躾氕:昗^9磣/P),f-}s7苀O厎醋y阣諵|◆瑯蟾K 燭-魬M!訅V璔?.犙F(贕o溣#>睚肍恖_-鄳矒o蜤E硼(乬環虣3'箂繅鍄篋漵9Gy潸氙焨諽瘂驀謀sM輚笸7C鑋l蹇駦ox鄟Rl敢n縵込Ui LqVG0潉烸/轑-mXX^y羇搵疹糛卩A7啠&鹐v8俛f楧姌0薇r蹥﹑鋅鰝 8>粉n;潾$ Ok瀙v!聼<鎁唎|鵒秒蒁勒樦炣6G讱7,崅v澠[栿鶾磏☆ $68g*W\q臵k鋼Or幵#獬磯壢}鬏?㏒y擇硪K//颮{?皺蘜渡欳鵘+錈貅唾Q傖}E**Mwt榧B擵!<$ q(L縮H7wd k>QZ$ 鉀蜳疙譤{韞鐫藄耱/w┙瓤O2戊錺+sW鋒*dv12Q?鏌,蠝>M1a製楖舤復燽峽沶簤┭C=詌勥礱7l_Ps:z荽趩,潞-s缊>W酔Y x)d聞$杊[綁oT/娵iY睘2L揭J+許O"喣餾{梋vA勧%&'娼1潉V溬鍦4嚈n`Cf nF9u0F揧鮏 ~} _滓 %&tXl6農-t觝掄髻g85tP劐釤37?0縠蓖,73葸|+6 驥 賗 岑+o急* oO_)Wycw 媷Q籿13ZihE館耊加肼逫磳錅x$*崾n誕8u厄踡燪q態勌 J( 侰=凾U=|6y礛.休^斃d鋅矱.B邇輸|Ep8D狩#嗵^猝^鏂0:領!藯﹝靮寁:r毲〆sG+堵閃,縲.":{sz^/関鬉b(d踭d/325猵峲J-齟嬊$紡H#嗵捃疊殕6v泲.m{婃Ly%氈陏睍I.鶭9呿 )d|9~o笍逌#0x迾喥=#GX敧婀b3r$^N/執ys蟯⒙2U]h劓f愸VL21畛0d%眏iR婊~:瑵c庰痹賃#寗3鸋p釃0詼f9癢(瀕踥儦髂戊Tl猼 嘲嶅;镘f沵^W矘呾" §8幚87鬋(RR箎08 仗煔族揷蘄輌焳鰹揙>鏄c瀥鏅:埬X'減kR瀦戛墅:嶡&戊 0l■俧,'|揦訌觫+Ti2%?Y剅訯G}骰啐靏?稽蛩巢ξn浮D潂#寠3垗U>3囕D蜌&fn捧ョr纓>),G>J熙豺#徖鳿Gf8"嗵>.#輔!!'-+閘幟囍*'_(錻T闌孏侚/睞輠锝,(沸}+'椚??}鬅gs礘憪髀幚D!嗵摁p+禗尾憔歀袾4$岴;'櫳 嬝<幰p(塐|bv89闣縅萂Z虘 晨%/!鷞憜紝#擟罊n戕%OY來闃U鄜厽酈W懟],PAF[灸頷K,蹦躴清wG筤賃噄誶瞼]巰#0g觥酩-EBJz?愨綍:3豙2誳V-M/揀0鸅 -剒蟮瘆瓩g[駘yIG8櫥`G鑲h枑RnW kr-k搘 t.$j>畂采&旖@d$蜋3gxQG(3{:^$慔褼榬,t2t-艶'毎蛠蚻囓賐基嶡\8城5昜? }m]詎p癭 穭踋j誒65U豅~_溲渺l騸豠噛偸G+淴溬?鬦侱?Cr譋4M恊%|揾nNX欭鮪o啕辸作<紘嶡8匙弎-饋殤幎槰笉a%獸*旙蝩綏灟蘌x鯶m嫡$ 捳L悍窾^pB溬'梓P:g@垜傎u穄蝵B輟宖P嘻 彬+7I ;I*眝侳諄87鷖哎I嵍ZCc橸  誘j殾0蔝W]晞bg渜F粱j铚Wtg鰷礪庢Z% 瓉%褎蕦吳敹$ ]O; s尓天菜.疙爨襇C57g鮒喴K2#m,e慂Q氒*才j 旾譯鯰 坴'壺n豁迄E]鋣鸘xC]G罊降Ds@線鮖7P庺@鹵勌獿V6舭 憠暠塿U^u罱鬢K/4弰c=秷y^p溬埸 2應蟧!5F燍+T迫荽︷1`蔐(絢>鳹螊_K.逛儉蝛+o戊"兣(h窰厒 Yz跼#%+ 圍挃毽Kl惍D咍3蟥s袳Em_癭令夥拋 媁tg鯢GP1謉庀FM-8斃嫤maΤKG耎E聞軹>Hab$鼥殟覥筠齠pd<鍞S<絳每o甤)睠決L耡聇c漊媣槓蚈6.柗:颩& 'J+Un_匨*矴%(強o岮7 EnN!躦o鞩琗斨堊詟橨欋$s%寗憼TN櫋!Sf寓\u6c/f儀e驁款弘匚閣葵]kC ;"P,4Q蜵鑽6旭聰疅e2'韝v蒯┃関/g鮫 迦)払 ]i▕坤镴し埊ヌ "w 8S"嬅M 2澉S~G肪#骺龖q 踇効讎@B罤伲秌nu(絢$4@wG5p%翦蹣PI <筇Q繪錍b閥懫1}濾擿4崇胄&そ佤f帋o柗ロ屑94斨U這0C'瀤bhw粿&盅罨觾鱒騗P\J"預xub[%萒f,}偔NRy但@裻e,]u瓾 C暀㏒骺蒏^2{鯪+瞲準#衜b{"蒶埻)醏]s z羶q3%q}縣u!Q /&輰趒貮4$侍9鐪3覯^>繘4髻積`觶灈w\6馴渨價洄黴k聹qe+%pW惢鯋HcjL"猁P涻蠷z鱗o錦S燏擥 t仭苓p髻 ^聴1/襜鑒踶Z? 漽呈$鳯?!鑷1蘬羹I"v鐫w妓麬z報p熃笙妯:*1醫'?e抽靸篳☆V@.u>齧>氾e=敊Y砯E厸T媭3{祒zmR$倧M殎D:*=-藙Z蜖揀儐A H飄3>Z嶡_pf鐙榷/(沵饘攓禐H欇騚m>)畵2:<貰A輴甹mG近>玓k盡糾G爁溬k孬6P檮5嫨E+瀗戲3T櫛j V鉭t仦0;o謜躴G蜨軀笸呔/鴻'焲RUq'\LWV-釐濜M(VMgd~j&朶w隲緑夂茳PJ誋ぬ(嚁烲yИ=嶡$魟資2w鈷L/揀B-T脙 &{癌*備N?齮N輔gC孺[m禃 > e驘. 箼買3A(6j~轂荁6纘1汧]懧c蔐X ;\こ艧cZ宸;"f惱W_婚(9c 矽o_~7鋸瘈&哆~鹹篋Q 皥慮嬴 S2黶5,bCW蹨=9ごX滬澇亞駛:"衋f噟<蛉/?\q:鐎婱Q^9咿}鬏擾v賓*翬^嶙[o=]/仙 7軃踾趇ы菜.;锛3 汧鶣N卣!>Mp嬚@+襭"?BB瘻m檬渺\3QUZ5訍j2*0fy?駢O渦諽K-礣滏90?t{麋>醼雦笸7g?膛[L,奮r|>呄抵Zl 梶踡返?q辯澌摀w葵輝<0鴵Glp襂'A*l>盜鉑,眲n b呼x4y]>{貗t垕y謡殺5~擤橯暝*±p馦oz兯綬屵醋D匑嚈 ($ &9[la 4鼨3g^}跽纘怯蒓~r遾麽裺穾D礫扠.〧o撿F9閿鋓兵鈰險籺=W 賜柄8n3#|═血Q ML柺;蕖綬嶡6硾1_|qb缾V謔G}T* >5R沶z)К傀蟀硁詭弘[fV9\鏥-Q<$濢U靛E吰+L 摦稼籲'h]Ow浼轚豐乶3)0 5&挽鞝垛+釿k&E;た>麡嬐[鼏n垏^0ij屧鵓gu鬺枘懗 齔叅y詒5Tx椞怚<筇甖a錨#'銔 亞kPI滛~螅:j嫡Vc:魞 '淍a轟6@衃n攻螜)E=谉< 鶩9OG佯◥禢鋇譤沇窩y>鴃i旗Z椕烇蹲軦洩祎`箸蟔f檈阯纂wZG犤蘮j q/v庼 蒢9/郌纏鍴錄m:t/餡v*懫鋁),<(<滙嗪 o<堼h壴v 鐺 +榘`D軺SZ鸋4}b鯒奎逅^霾舓v"A犆處籌鈞屢(-s<顂=W уn#P!fvT驕旳虠J i0s"d窴駤輯#沝啛/緊牁v紆l漹陂菜.:?u懨S?m+B厬|鰻(o澇&爑嫕鹹"皙W姜u#G:天!:/R m農- /奎rv屍奧 2!\pe賂_C<鋻Sx羵Rcx6楞'u"咣r'埨a井C3齗褂郋Yぢ>zU嶡魟佟Z8Z+Pt鄋鋦銕喕?!h靄嚞--89毤Ny踤冸%萖 H1DI哤x'x蠒D:"籗>赒7zXy I蕪 刜Q巽85!f8訐.楶寇js +\]侺x$" d奃潡買訌J璣 -畉忺FG燙魢;z$χu窀砑c1 鴑#P+戊德{艴(墕繹a鉚梓(捜Z8z"0咠秌m`允e蠑)SF絨沂8o(D鍇份ō研匫蹆y墝籞薺燍茋bC"B壒2棝5H3gNDi8GE1日懜樃潻,{轂CパN闃戊礅_覺 2瓅![昘疶+崇w劍g蜌錸抗7媗z (潽鏂BfO#磥T慃g淎魨譾迶w57CY#朚蜂5 5崉菔罽菆T['擆,說弑tz塔焗Z鏓霢F1O鈫;睓A蹈5P3{ 且剛aX+k!+/p豷嵟韝镵_ P荈0U妸#釡e}づ&-[Xi珨!6袂<%~辵譣Y糾y;pbksf煚0 g格\鄢"濸柺苅絩H懚X葤lV怭鵍+%梊翬謘橉N拾).紀>賄噯C襻J懯頊 澷'p衉|' /柉d匭T戩賧SO=5墐]g焿rA]A娧迵k哯=j'?6垱2w輚桼0呟焣軻謎靌枕>Y悱迒:< ?j6u滛<3b藴?蛉#8豼鰴?2b ;豀!u覅^6No犚貇砰Mr4v碴vf煬{k記槔"za|ef糑強 L彰/蒃{T戨l瑍8#謾)% 抜U篳I蜛%訥覐jpf蠋咋橛勓w辻鏦掘曌決礬G隂\9唾巘c槁蟧~_Z厶錳H8槎儘躹綧;3(钅J舛驣畖.簿㈱銂_3{飮x瀩鮭8_鋨6軿B橝嶀橯>|9潯艤℅^'o礋擄倚1 S g摬Pr狨垍H芬e邸汷鼄鋄廽鋈;硰 Y弌 #馩笙?煬6弞鏅g義慭嵇!8躤=~cBY謰聨~h=t&pfo韝埴蒿~w菗鷵;kQ[H22<憚=\{淼lZt2m睻揌@Ц,鞁爉佸壦戠uJ蕦E攙'4H1qtb瑴;1L?宋,箭鮪礍_yZ蠷/ |A巛v鎘R惿伾S 棡r~圀稐朧槼鹽鏢 若k淟鴰饡弾3{鍚v珺vkXx躴菂禂+敊偺 瘇醌c3 痝頛愞;!睍B;瞞睃9砄趫タ+?釀>喜{3`|憟搼.*WZ[醃眧鏅g&wそ鐡噣3嶚=撅鈞x&3燍 : JK:澳\xRf詞墦0EA 闣繆逪沸 g霆愳L=>,lK泀C俑釞炙=譝r珥O) 4*71\r!槱髛o(溬c呮lx?駨 鬈s;囀Ny邇&9鍨C柎閔wb!鳹9渝s儇跅)5#髺"@罊=侫h椌=镔x銔拋Ev鄹粢Kg蠟崄 oO+6JQ0埦7診紺'O-譫P 8城0 帝篚齵啕迚'濰1屢PZg嶸W窺U/湙嘆v3D*﹢揖鄒 痍硚8個3{3ビGn6m苂巷6Yu操f淉&沴鳒[岟ヌ鑑礫8領<猂r瞺_戹:噣3{ 燌'擷U~t>G婰Q`!m蘺'j0G4盡%譞滑免荀+=~cpf鐔鏟骺齣vG喟_s5C叢@璜^酹<恌唺6 SO=0掄K2錌 V''%{鯝!髷o═鐶銋鷤3{_F騾鼲!9*!t瀉氘;T亼Sl3珄锝峰 薈 fU愥emT燃jZI巒眉1筂F韰梬:崁3{/踴h齝悶 K驃SA/a櫋⑼P淵瓬刡8>k脂 Y丄}$3s蓒駳餞#9溬;7dC &a]L椣玘Zd*,榿@拫狟xA繟蟖d準5三_譌匓玴媹幚 嗵薹&Z ; 昐銼忥B悮 顔*F鮬揅縞9嫫o3gN筿"葒踶筡%~#9溬;7dC &<咈蒺"*Mc袠d踼労鵤篃z戛Z*蹙鶻飴o|悒7zyG犽8硍}擌襯e2筏戶62尡5MX龐Hj寣擠仇 撹鎵'瀈s潲錕?初鬰=棛l圊e沵訂衊戊ρ孴0- o)栔14赿&藮講滑R, '葎U'"s拮Zk瓂篋‘0?L,c頱8涍前W2^^I'鳶d9*}絆QxQjq2冾喝"K琗篅{J%濱勱?Of/Mbi嬜1成}鯪>弸-瑿瀚J2j腴v\嵉楜ayq=.,"墸霃~#_q 餞#蠯溬64&$eC訏|RS酚齏n驄鍔)0E忤U3滆a2s襂'}顂焎陣_S9{壈`M}鬸 P痰o當膠怴愋Ck誰"LB)nFBP矄罱鬢珞#俳瓬&蹊伲 {萌G鍧攼5㈡U;1!嗵觝Dc8T吺(婺鎀跼f0僜2鱌 Q|鮭g傀醑矨沁-揀卵靻8"嗵^/據祒v澯饑|Z崫7諱嬠聈騝`H槁覰;M>澰氕穅cFzC嶡肝搡4>=aP◤g娸F唙c|澼崄'?T6Gj華'膠p熃嫞吱蚃梙 m+ 艂R旒V長綐4刄9窪z氕Gy銿[m艦y礪鍟;q">{溿2塥苵v3%t撚胥<3若"^鰻{語 僇⒗\p~7抉 匁誠>{v帵寑]黸趕夼Qk莊 cQ4!簲iT籲籂,哿燡詈>歍合棊 禇畛wu4涽賲擡戨O龜F9巠 熭"雵` W\1w鈷pD輌黜>6_凔焱c揄3夭"S鹢釙Eh7o轪V皉/g鰚弌-匠iL龜Ri,趠ó恲晗y`\u誙_豖#S乊hj伬+u"G楘?帹L娪晄|飡遚[K!0刺麞狣玲硼" dV蝽o{鯪9諡{罟E29涅鹷p='顯筅屹%⑤-蒐:丶犖産#8鈱3穩,e涳m頓Λc囕1崽>^駭熟L'襃睯就 wP%P9k齧"-鶺a.哶7$酔(&g/'焲挏-敦晦醍盔9tf胩>>喭奏:{髽7遭RK-蹌O暫瑪v扳R駔+1NW蕤咥7>沒觳薣啁主訣靳碞 <驦紝#3溬{6燪tGq怰Tl7薒謾L~b`菿尪掘+G裊7g(嚗鸉)-e磦I 懘軌Z荗:槨珞"2c茖#=pjD罊紽p惘候%棔!以r旯堭2T亂粞Gw踡販魁(0.篽簠嶡8撤蜆8詏蠌P檻C鍁2!Rx鄟9~鸇!嗵>Q冪Ng磲!誆 a3岫|櫀±渦諽s獺ZN乮`L経G gv4亐I橯8 '意5尺~倒宏笪;溯鑧9滷罊接米1鉓~蒓@餅v趇燓臾7趆u彝u"@罊=侫$攼 '\0絕/獍蠚5k瑰枦$l集嶡eD熟b(朂"珗腲,/蝽2"7D熟C淹剼.鴻廹邔8`禚灚 X_蠺 抆Ipf镘愼余: "辿锝墳b"薞嫚瘥迿P^8櫥oL蛔#楨Y橆崪[+戊睅蘢豼w躴S輜遾'G轐G 溬汙氽菵<,A>鴝方!r pfL痡 D1~靋圾鈰 鹒~85魅#徏%/┅rg鰳G踠?蔭韅刖漺迿㏎觥営奎/C鑜嫳藦_鶢垶\r%氆遦v罊=凇i0 ;攸h眾l懲4儥逸w咻楯鮆丩溬龂慏@桓ZwH囖pjB罊&`籞-倝播玟覡j噦nG`bpf煒諵鞟屆蕍沱活簨蚡籥玔T妧3{v2mo1z妾粵#鎂;Cpf鐲O,>蓄хgKZp亁pf廹,*禿迸{*朁絈`*勛s簤3{7莔l獷雔Y'a=伿SO=敵B 昗tg鯡嵸=肫0鮚椌籟q6QV輖欭}琛噉均鎩("XQ蠐O>橸掛嗅uU如狉E殝繉3{點T眳#槉戸 0w頫v*鏚(-p≮#搃昆梌JJ <>鷋8夥辴覯瘢A懵 /勑 鋥鵒薈黔啿9x饡蝍aSf(虩|舃軤諿弢s澮>}:鐪渪鈮|證倆n瑰W\q笸7鏞}R橗觵呼r\溬{9N3_'酼謰監戟篃z戛('踤豁7搌鵜f5T{轂渠;o洪3f(a'瑡^|衽E魘R'升9侇WZi%餿&圄燑'\'7=*魕遾鱭弼踥 ,毬NTS]辟S]蠚畨啁5譢刜.摓,+?宻h濯j鍁┏慕羒鱚w輚瘂驀JT宸虣34 x謭x觸瀞92Ix*\癭焅菞_e昒 n習嗁<餈<w軕[鴸媹鯏淝啻覰潰b揗6軌菜#嶂[oE-UHa╩5駐(鐫w^:Kw0a舀︴~咪甕疛傷楪XI%巌忀~鏅gVU$3?癡滵8躔 蒼羔啘 S糲~3k?!hnQy 隱蓀=鬈Sb!-Tu襂'q*銸 l份v| 煖玓]:情f匃顲wL塔戩﨡螏嶡[,緊襵謭軾盆$ 梍~k&T9碳K.箘9U孽f禨[地抬c?t${逛|虼纆骰邖@O ︷ 鵍來憤塮詿i|扟歫-@Wゐ麩pf鏃橚笹d@澻f沵羆笪;櫀JZ7 h錀c9'0Gdq膓5<*J:隋競xM齜垮a罒氍碃氷鋭 e檈栄硤燫樭?'常p: 鎌酔艩-奮tj郲&Q脙5卓脵c鷹襝p蠥聯>9枸 閕摧G7橋F4嚵qA錎瑣染趈+PpKxP兟'僣午菜.磝陝琛r融鉬醓.2e獕Y唣7D"籛詐渮x鎦鞕-"N鋁舂 @駵娓錁麨r)_( 橸-h*q_aIh潷q惔肧d愄Y殲黼狁靰鳧~1汮2[NW+E憍&爿q 櫜Cd'-0}財粖轓2e蘪垔佨!_f鮛讍摉2 攔阦S並畱錎諉e楿 浝厳Y︷+忮烴瓜>鎻禼礬簡駛磯+Gv豠?f綎篚鄝)蕊堜河N;9璆20+鼪圗騷u霴:md哀&l曤壇鶭霠崽蓮縌3鴚"愚骶笙?粲O呷療Y諪崹1薜壉5冑kㄞ玹:儉3{g喪 uG 戊侁[1&T塃5=SI^j━攫/tT杫鮭g橜c}#y鐷逨他寛3麍酲8#AL@-V]擉歱c卵崎e笏M兕昳冏兼5腅付>嗚紸罊7CYAG滼;過X馫鋲鯽 N:W瓽~#蠩溬8j5贚鶥7巋M矇≌ぷ冊仞鑅@|_'熷w倔澀囤j G牱8櫥vhKwl釁&T} n8C&q崉oI滼彔哞8雛o 钂 卍塦^d#!邇喢m_釥膃;10)BMl74譞UkP貼揢榭醑ILf5&逓瓜镓Db﹢橲厷Y禑iR摮i碂齦$喔嶡8城9.qYE&R,0N螜,#櫎垇賩锝 h o戊峔n瑰f蜌u=3蛹5l猵蟶'摚晐R頭-:滳罊絪C枝領善4梌zi揊 唥?黳w諞勢坳4戊孏s蚾~C靨骺齻統!}翏塨歃啢疁続R9 蒁罊}2墻|瘔!I$☆LI婍超溷.秇饻錑孫淭溬'u淝7滬訜|鞖2*P>辻篋_u鮷潕@pf秣芻蟶3aZ德f3峳\仼U痡pf煷快稧偼7嘸狫哨h9戊韽乕8嶡8砏嫥祖8嶡8撤?n#8"娞怴E>IEND瓸`z?懾蔏⑶細(*z?懾蔏⑶細(*z?懾蔏⑶細(*b 2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~PJ_HmH nHsH tHJ`J ck噀 $1$a$ CJKH_HaJmH nHsH tH<< +?h槝 1$@&XD 5丆J\$A`$ 貫祂=刉[SOBiB nf恏@0 +?h槝$a$ 5丆J\(B( +?ck噀噀,gCJ.﨩!. +? tpc_content(' 1( lyb鑜_(uCJaJ*B* lyb鑜噀W[$a$*jAB* lyb鑜;N槝5乗.b. lyb鑜Fh噀,gCJaJe@r +f HTML 剺緥贗3縗`?/[G歿\!-跼k.搒曰..椃碼嬋?PK!ブх6 _rels/.rels剰蟡0 囷吔冄}Q頤%v/C/(h"脎O 劋秣=畫旂 毆免C?薶醰=偵叅%[xp啠{鄣_糚眩<10堎O糝瓸d焉襃E4b$q_槥6L籲R7`畯ㄉ趁0虨O,錏n7擫i鋌〃/鉙綈╡械根鑄PK!kytheme/theme/themeManager.xml 蘉 @醹愘7c(Eb菜CA菭覠圩邈7蕪諞K Y, 奺.埛饇,ㄚH,l崆駢x紗逫萻Q}#諓叚遞 值+!,較$j=婫W櫨)釫+& 8PK!mQtheme/theme/theme1.xml靁MoE#F{oc'vGuh覨盵糟x=逎zvg53Nj廐H垈z飴昛 $石I)*E阓酀欇鮊&IA!裎>秣羌3緓镹男>掤擱U蟇L0斈屵嚦z迃rx鰾脅稔h9T8奣/魁煜?鍃Y雒'蟸3S琦棌~蜩鶅O_|w)馉鎘圚t=乤+d NG1-Rl苼1諶J鴚T锠疢1Kc柩"o he浪撣幝絇L-憒%絎臙甴Y7'qP.\L姼=岟薲穛炷3I爋fi墸.帽墘BRb-J款P_p蒅 茛▍i㎏鷗郿訉h汧梚櫷o7;7Q嫵2染媱珼>a/銐翾>嶺厭W 藬霱卂膗H剄)薶 胺+:Vi豾4r態裶汐橌"r嫃!帓2l徠a麃C奲此U|嚮!8^顩8>軤仯,A魶壭眲V韙鄨讕厏ls囔14犁_=,澀返o聻TV 跥陲"苧鎎H唼灮'.4熯x薜躻-戲蹺鮸褾;氕衯躑`嘼3"G 'e\昮H柊O 話ㄩ恬愪'$劘i_wp伬 >*靺8魂i&丩Y%\鋁,楎謝視=矚伭壵阱綔 r6f 提3儲淭厥厰)橗:陋ZK誏玸ゅ&C 鏜兣軟0 [浪玴@註醏j扣7 媺耏咹唜Hi葷cT5A蕆跑@顢腍驇馴AZC硙i' RQ\m伕,zo,僩Q襲{Y\,Nψ/=溷ら嵿L _.跆嘫7C6韽-fS宄h62密"5咍麥罭H凾[X65太4X%Y龡豚殖2纅鷎h輩紳痠~tCKF#猥b +趙1m鑵4`眹!:U翞!昿5a:倊{4韒笫m蝘o 萎c杽8m泛D矹秔S槍姍豓1鋣?#S奿?3E'pS2疳W`る甸q]( ┻08樲w別 n撏A鯴[s枃)k8皓= Aa?R d趻刪c楿詠瞬d)#換ueb恾滿捷C!ず&i0福>4魫S7鋥z蟣 F箎 4sKvUKo瘸椒h垀1砵YU奧V蠬塔5U8錠k;譁潘鮈9堚寂皹D 荀 ?*|FL 迪鳡"A3兇伂>g類dm2iV值殍そ杕謌<殞r8[kv抶熞蠃p鎶sj,潩z傴礭[鑚堨叆Qv11縤u鈨-高0%M2羙J描3u舘%覎PK! 褠煻'theme/theme/_rels/themeManager.xml.rels剰M 0匃倃oo雍&輬協勪5 6?$Q祉 ,.嘺緳i粭澤c21h:闀q毩m胳嶡RN壻;d癭値o7g慘(M&$R(.1榬'J摐袏T鶂8V"&A然蠬鱱}狇|$絙{朠除8塯/]As賲(⑵銻#洩L蔥漢倪PK-!倞[Content_Types].xmlPK-!ブх6 +_rels/.relsPK-!kytheme/theme/themeManager.xmlPK-!mQtheme/theme/theme1.xmlPK-! 褠煻' theme/theme/_rels/themeManager.xml.relsPK]  L  T&t@!j#&'(篹g"hHn榪dszt聉:y|衹爛槂 !"#%&')*+,.01BDEGIKLMOQSTV~ :|j#'鴈m妎|r膙0z@|悅$(-/CFHJNPRU餈  @餒 0(  饞0(  養 S  ?E[x嵠\x攳]x詫^x攲_x敋`x敊ax詷bx樒cx詶dx敆ex敄fx敃gx敇hxT樒ix櫰jxT毱kx詸lxT櫰mx毱nxT椘ox詵pxT嵠qxd庿rxd岠sxd嶘txuxvxd忴wxd孁xx$岠yx$孁zx{x鋲|x鋴}x~xx鋳xd婙亁$婙倄d夬儀錁剎d楕厁鋽唜$戻噚d擑坸鋻墄$擑妜$桒媥寈峹d橒巟$橒弜恱鋼憍d旞抶搙$旞攛d戻晉$掵杧d掵梮錀榵鋺檟d桒歺鋶泋d書渪鋾漻瀤焫鋹sy!8ORs`lBb: V 5 , O ~ ~ V ,4F=NuSB&5Qmu       !"#$%&'()*+,-./0123456789:;<=>?@ABCDw{$:QVzepGh< X : $ / S Y 0:L CUw WD(9Sqy   !"#$%&'()*+,-./0123456789:;<=>?@ABCD;;*urn:schemas-microsoft-com:office:smarttagschsdate<E*urn:schemas-microsoft-com:office:smarttagschmetcnv 葴1.01.1.1425.175.5.75.8011.211.5101211522.420201029.230.2530.33337443.3555060669!cmDayFalsegHasSpace IsLunarDate IsROCDatelmmmMonthNegative NumberType SourceValueTCSCTrueUnitNameYearE/,#!*.+-E/',#!*!+-E/",#!*!+-E/,#!*.+-E-+*!#!,/"E-+*!#!, /"E/',#!*!+-E/&,#!*!+-E/, #!*!+-E/,#!*!+-;0) $!%!E-+*!#!,/"E-+*!#!,/"E-+*!#!,/E-+*!#., /'E-+*!#.,/'E-+*!#., /"E-+*!#!,/E-+*!#!, /"E/",#!*!+-E/,#!*!+-E/",#!*!+-E/',#.*!+-E/",#!*!+-E/,#!*.+-E/,#!*!+-E-+*.#!,/E-+*!#!,/(E-+*!#!, /'E-+*!#., /'E-+*!#!,/E-+*!#!,/'E-+*!#!,/(E-+*!#!,/"E-+*!#!,/"E/",#!*!+-E-+*!#!,/E-+*!#!,/'E-+*!#!,/(E-+*!#!,/'E-+*!#!,/"E-+*!#!,/"E/,#!*!+-E/&,#!*!+-E/,#!*!+-E/&,#!*!+-E/,#!*!+-E/,#!*!+-E-+*.#!,/E-+*!#!,/'E-+*!#!,/'E-+*.#!,/E/,#!*.+-E/',#!*!+-E-+*!#!,/E/,#.*!+-E/',#!*!+-E/(, #!*!+-E/',#!*!+-E/(, #.*!+-E/',#!*!+-E/',#!*!+-E/,#!*!+-E-+*!#!,/'E-+*!#!, /(E/(, #!*!+-E/',#!*!+-E/(,#!*!+-E/',#!*!+- -.45MNTUadu|#',589:ADEIRW[]clnswx%1237;=?@COQRVZ\_t~ %)*-05:<?LSTY]aghvz{NTajs $*05RU<?SV  %(,028;@ADMNORSVX^_`fiqtu}$&(+4;>@ABGHPQVW]abmpt~   $ . < A G V X _ a d k p t y       " ' @ C K L e g   $ , / 6 9 < > D H L M O S V [ ^ g n x        " ? B C F L n q t u x ~   . 0 1 7 9 : ? @ A V Y ] _ a c %,OS\b "#%*.8;>@ILxz   !&).257@chiuz{#aeghjm*9:ABFMNTV_`ilo/3U\_`cdKbi#24>BCPQTUV_d()/0\cg)*ktyz|*0suwz89GHwx#$%(/03  =>BCEFIKOfjkpqy  "489;@FJR]`nx|~   !#.8BLTVWY\]i!(59:?@JQS[\_   $ & , 3 4 = > ? C F K N R V i q u v { +0  14X`  " & ) , ~ f j ,[%)14NQNP\_%&?@NOefv{|8=_c17=GNU@Cmt =?KM8; ss3s333333333333333s3333sssss33s33ss333333333s333333333s33.5NUJ`g,9;K e  > E  F  (6aCx]_ijqrVbc+,24;*1X^>q y   K\                  *聨c                 劮榃                          鷻        縝鸗焞&1< UT47镮l=  - 繳 靐 莔 ; M HN 蕋辬搟-(S蒳恴抜鯿V7腒測r繳 M! "#馝$觭$薼%z&*&'37'j (1( d)^D*笹*///錵/*01 2羉2 3籔4縍5耯5Is6莄7;>1>AhBC(C::CL~C獸D8KH;II"cIz6K/ L嶤N鋙N豑O8kP*Q~ STU齗UV/sV騄WF1Za&[ w[躉\<}]^$^*{^_ A_鯨`剗`跙c穖d+f8f;gl !lRem%Xoq訫qJHs薦stt沀wx,tx鑕yy{齲{{{ O(Cvd`菳F/;t膍E)a1汮繷楿]h欽?>xu 55\=癮靌盢纕VGD3x7!箉;鉝渧泘/}rv%0S5I~B1鉛訬籚kn v4蒨僡<h燞鷞?>&z/l{d*,颒+uP8>.se?)蝊 s)\榢+秨@QU(lu"*Pr2 1錮(A攁!pp 訟鈋舙;c dv;<!7-C} 贜篖k 訤T餤+?z)1齅9両ry"餚訿]1T6鎛$$; @{{薩{{L  xx xx"x$xdUnknownUSERG*郃x Times New Roman5Symbol3. *郈x Arial;(媅SOSimSun;= 鷟8褳SOSimHei;WingdingsA$BCambria Math 1hBS)gBS)g"&g ; ;!-!),.:;?]}    & 6"0000 0 0 00000 =@\]^([{  0 0 00000;[nn個  2僸HP ?訿2!xx瀃寶烻8h駤黐h埦徥S纇KmR恎ju儽傢V恄'Y哵y 5zEzUz 這駃 嶐Z'櫟`激禝嶏Z'櫟<         鄥燆鵒h珣+'遲0   ( 4 @LT\dl 實驗🤽‍♂️:原核誘導表達及檢測分析 番茄花園 Normal.dotm徐大慶2Microsoft Office Word@寙G@C@ThZ刺@ThZ刺 Gㄕ Rt6 胘&" WMFC huhl&6 Rt EMFhW&6)3(   &6% % Rpj媅SO\薈肋@鎣薕C]肋杠(搛みOC]肋杠 獒絖杠肋 @宂d飩_$  ;(媅SOSimun燔8礯%(鎣(鎣0z砡%P鎣@宂dv% % % Rpj@Times New Roman\薈 肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 !{d飩_ G*郃x Times ew Roman蠨=燔8礯%(鎣(鎣0z砡%P鎣!{dv% % % Rp @Times New Romanよ瞋vP!綺ˊ={綺 獒絖麗!腮! {d飩_ 2d pG{{Ax Times ew RomanPC]赧!8礯%0!0!0z砡%X!{dv% % % % % % % % % % % % % %  % % %  T`aAq]@眡@aNL&6T KKK% % % TTBq]@眡@BNL&6P !&6 '% Ld)# !??% " Rp8媅SO斳x鎣薕C]癤`搛苓OC]癤 獒絖癤 @宂d飩_$  ;(媅SOSimun諢C]b5#{谼=$鎣8礯%`鎣`鎣0z砡%堓@宂dv% % % Rp8@Times New Roman\薈 肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 {d飩_ G*郃x Times ew Roman銪=燔8礯%(鎣(鎣0z砡%P鎣{dv% % % % % % % % %  % % % Tx;]@眡@;zL&6\RP[uirf[瀃寶% % % TX]@眡@zL&6PIINM% % % TT]@眡@zL&6P % % %  % % % Tx `]@眡@ L&6\藛}v(崉vh埦廩tT`a]@眡@aL&6T0瘇S[tTd]@眡@L&6T蔛纇Km % % % TTs]@眡@L&6P  % % % TTa=]@眡@aL&6P  % % % TX v~ =]@眡@ "L&6P瀃寶TT vG=]@眡@ "L&6PN6TTHv=]@眡@H"L&6PTlv=]@眡@"L&6XY恘鶺郪(WTdv=]@眡@"L&6T'Y爛Fg虄TX v=]@眡@ "L&6P-N剉Tdv=]@眡@"L&6T駤黐h埦徤% % % TTkiL]@眡@"L&6P Rp8媅SO斳x鎣薕C]癤`搛苓OC]癤 獒絖癤 @宂d飩_$  ;(媅SOSimun諢C]b5,*{郉=$鎣8礯%`鎣`鎣0z砡%堓@宂dv% % % Rp8@Times New Roman\薈 肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 l,{d飩_ G*郃x Times ew Roman鳧=燔8礯%(鎣(鎣0z砡%P鎣l,{dv% % %  % % % TT x ]@眡@j L&6P0% % % Tdy  ]@眡@yj L&6T瀃寶顅剉% % % TT c ]@眡@j L&6P0% % % TTd  ]@眡@dj L&6P [t% % %  % % % TA  ]@眡@A L&6p惽,g瀃寶哊銐Y恘鶺郪(W烻8h苸迉-N[tTX  ]@眡@ L&6P駤黐TX  ]@眡@ L&6Ph埦徣T`  ]@眡@ L&6T臽礠0[t% % % TT  ]@眡@ L&6P % % %  % % % TTOx]@眡@L&6P0[t% % % TdyO]@眡@yL&6T瀃寶烻t% % % TTOc]@眡@L&6P0% % % TTdG']@眡@dL&6P Rp 8媅SO\薈肋@鎣薕C]肋杠(搛みOC]肋杠 獒絖杠肋 @宂d飩_$  ;(媅SOSimun諢C]5 ({xC=燔8礯%(鎣(鎣0z砡%P鎣@宂dv% % % Rp 8@Times New Roman\薈 肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 &{d飩_ G*郃x Times ew RomanlC=燔8礯%(鎣(鎣0z砡%P鎣 &{dv% % % % % % % % %  &" WMFC hUh% % % T|A ^]@眡@ACL&6\\Y恘鶺郪KQ問(WTX  ^]@眡@ CL&6PO(uTX ^]@眡@ CL&6P+T g% % % T`n]@眡@CL&6Tlac8d% % % Td ^]@眡@CL&6T蚫\OP[剉T!~^]@眡@!C L&6h/T≧P[剉h埦弣廠O-N vQ(W% % % TXz*n]@眡@CL&6PE.z2% % % TT+n]@眡@+CL&6P [td% % % Tdn]@眡@CL&6TcoliYd8m% % % Td#^]@眡@CL&6T-Nh埦0% % % % % % % % %  % % % T ]@眡@ L&6d枡HQ(W N燫駤黐BR剉ag鯪 NTX ]@眡@ L&6P鵚{QTd]@眡@L&6T縖;N虄 % % % Td5]@眡@L&6TlacI8dY% % % Tl6]@眡@6L&6X鶺郪h埦弰v[tTx]@眡@L&6\u剉;朞愃唥v[t% % % Td]@眡@L&6TLacIzYY% % % TT]@眡@L&6PN[t% % % T` ]@眡@L&6Tlac8d% % % T` #]@眡@ L&6T蚫\OP[[t % % % TX@h]@眡@ML&6P觺TTAh]@眡@AM L&6d 蜰 N齹蹚LY恘鶺郪剉TX0h]@眡@ML&6Ph埦徣TT1h]@眡@1ML&6P6Th]@眡@M L&6d6qTT鵚{Q鶺-N燫eQ駤黐ir% % % Td]y]@眡@ML&6TIPTGBoz% % % TT^%h]@眡@^ML&6P TX&h]@眡@&ML&6Pdk鰁% % % Td y]@眡@ML&6TLacIzYY% % % T` #h]@眡@ ML&6T;朞愃啍 % % % TT&x]@眡@L&6P}vTly&`]@眡@yL&6X豐刧1Y;m [tT`a& ]@眡@aL&6T N齹N[t% % % T`  ]@眡@ L&6Tlac8d% % % T` &i]@眡@ L&6T蚫\OP[6Tj&9]@眡@j L&6`觺T Y恘鶺郪h埦0% % % TT:]@眡@:L&6P [t % % % TTxv]@眡@[L&6P0% % % TyP v]@眡@y[ L&6`瀃寶闚hV0Pg檈N諎BR% % % TTQ  v]@眡@Q [L&6P0% % % TT  ]@眡@ [L&6P  % % % TTx]@眡@L&6P瀃T`y]@眡@yL&6T寶闚hVTT]@眡@L&6PT]@眡@ L&6`厤繯錧\O餝0諎0T|]@眡@L&6\Gd奮0粂胈:gI{0% % % TTv]@眡@L&6P [t % % % Tl?]@眡@L&6X虄*h孴(崚|TT?`]@眡@L&6PTxa? ]@眡@aL&6\虄*h'Y爛Fg虄% % % Td 7]@眡@ L&6TBL21zed% % % Tl?]@眡@L&6X蛻膥(崚|[t% % % T`7@]@眡@L&6TpETdzh% % % TT37u]@眡@3L&6P-C% % % T`v7]@眡@vL&6T30addY% % % TT7]@眡@L&6P-[tB% % % TT7h]@眡@L&6PX[t% % % T`i?]@眡@iL&6T鶺郪% % % TT7]@眡@L&6PX% % % TT?]@眡@L&6PN% % % T`7]@眡@L&6TpET[tdzh% % % TT78]@眡@L&6P-[tC% % % T`97]@眡@9L&6T30add% % % T`? ]@眡@L&6T蛻膥0% % % TT &" WMFC h5h7x!]@眡@ L&6P  % % % TdO]@眡@4L&6T;N亯諎BR% % % TTf`]@眡@4L&6P Rp 8@Wingdingsx薈苓\鎣薕C]苓攢D搛肋OC]苓攢 獒絖攢苓 .{d飩_ ;Wingdigs諢C]F5l.{霥=鎣8礯%D鎣D鎣0z砡%l鎣.{dv% % % % % % Rp 8@"Arial敗 圉斴薕C]圉 鎣 |搛OC]圉 鎣 獒絖 鎣圉 2{d飩_3.*郈x ArialC]`咡諢C]52{E=@鎣8礯%|鎣|鎣0z砡%ぽ2{dv% % % % % % % % %  % % % TTy !]@眡@y|!L&6Pl% % % TT !]@眡@|!L&6P % % % T| !]@眡@|!L&6\50mg/ml ded98d% % % Td , !]@眡@ |!L&6TaS }TT- & !]@眡@- |!L&6PM% % % Td' !]@眡@' |!L&6T10mlde8% % % TT !]@眡@|!L&6P TT !]@眡@|!L&6P(u% % % |!Rp 8@Times New Roman肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 l,{d飩_ G*郃x Times ew oman鳧=燔8礯%(鎣T鎣+Q蓋T鎣bQ蓋霸dv% %  Tp !]@眡@|!L&6X0.22md2ddl % ( % % % Tp !]@眡@|!L&6X鋘渷菑鋘d柼內T` T!]@眡@|!L&6T Rb% % % TXU N!]@眡@U|!L&6P10d% % % TXO !]@眡@O|!L&6P齆 T` 6!]@眡@|!L&6T.峏[嶯% % % TT7 y!]@眡@7|!L&6P-C% % % TXz A !]@眡@z|!L&6P20dd% % % TTB !!]@眡@B |!L&6P!TT ! !!]@眡@ !|!L&6P0% % % TT! g"!]@眡@!|!L&6P !&6" Rp {媅SO|倀噗`裒薕C]噗刎H邛嫩OC]噗刎 獒絖刎噗 @宂d飩_$  ;(媅SOSimun諢C]J 50{鬌= 裒8礯%H裒H裒0z砡%p裒@宂dv% % % Rp{@Times New Roman|倀 噗`裒薕C]噗刎 H邛嫩OC]噗刎 獒絖刎噗 4{d飩_ G*郃x Times ew Roman E= 裒8礯%H裒H裒0z砡%p裒4{dv% % % % % % % % % % % %  % % % TTy " "]@眡@y"L&6Pl% % % TT ""]@眡@"L&6P % % % T` "4"]@眡@"L&6T1M dH% % % Td5 ""]@眡@5"L&6TIPTGCoz% % % TT" "]@眡@"L&6PTT " "]@眡@ "L&6P\% % % T` " "]@眡@ "L&6T2.4d2d% % % Tp "F"]@眡@ "L&6Xg IPTGdHCoz% % % TXG""]@眡@G"L&6P秐嶯% % % T|  ""]@眡@ "L&6\10mL ddHdesHdd% % % TTb""]@眡@"L&6P2C% % % TX ""]@眡@"L&6PO d% % % TX"F"]@眡@"L&6P-N TTG"@"]@眡@G"L&6P(u% % % A"Rp8@Times New Roman肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 l,{d飩_ G*郃x Times ew oman鳧=燔8礯%(鎣|薈+Q蓋|薈bQ蓋|(霸dv% %  TpA ""]@眡@A"L&6X0.22md2ddl % ( % % % T"!"]@眡@" L&6`鋘渷菑鋘d柼 蟢齆皸% % % T`! "H""]@眡@!"L&6T1mle8% % % TTI""#"]@眡@I""L&6P 6!&6"  % % % T`X#.$]@眡@$L&6T.峏[嶯% % % TT/P#q0$]@眡@/$L&6P-C% % % TXrP#9 0$]@眡@r$L&6P20dd% %&" WMFC hh % TT: X# $]@眡@: $L&6P!TT X# $]@眡@ $L&6P0c(% % % TT P#_ 0$]@眡@ $L&6P !&6"  % % % TTy$ r%]@眡@yI%L&6Pl% % % TT$t%]@眡@I%L&6P % % % TX$s%]@眡@I%L&6PLB% % % Tl$ d%]@眡@I%L&6X瞞SO鵚{Q鶺% % % TT $ s%]@眡@ I%L&6P !&6"  % % % Tl % &]@眡@ &L&6X饊藛}v鑰% % % T| % &]@眡@ &L&6\tryptone8Cdd8dcY% % % TT %S &]@眡@ &L&6P % % % TT %:&]@眡@T & L&6` cddddddddd% % % TT;%&]@眡@;&L&6P d% % % TT%&]@眡@&L&6P d% % % TX%&]@眡@&L&6P10dd% % % TX%&]@眡@&L&6Pg dd% % % TT%(&]@眡@&L&6P  % % % Tp %' ']@眡@ 'L&6Xu懲ku懲k弫% % % T 'n']@眡@ ' L&6dyeast extracdYYN82Yc8BYY% % % TTo'']@眡@o'L&6Pt8% % % TT%'n']@眡@'L&6P % % % T`o'']@眡@o'L&6T ddd% % % T`'']@眡@'L&6T dcd% % % TT')']@眡@'L&6P d% % % TT*'']@眡@*'L&6P5d% % % TT'']@眡@'L&6Pgd% % % TT'']@眡@'L&6P  % % % TdQ(@1)]@眡@)L&6T dddd% % % TXAQ(1)]@眡@A)L&6P dd% % % TQ(1)]@眡@ )L&6Nacl YY8ddddddddddddddddddddddddddd% % % TXQ(1)]@眡@)L&6P10dd% % % TTQ(:1)]@眡@)L&6Pgd% % % TT;Q(1)]@眡@;)L&6P  % % % T)P*]@眡@5* L&6dGd≧筟hV魐髞秐(崒[hQ秐銐% % % TT)Oa*]@眡@5*L&6P,2% % % TTP)>P*]@眡@P5*L&6P(u% % % T|<)a*]@眡@?5*L&6\NaOH(1M)YCdC% % % TX)P*]@眡@5*L&6P寕側% % % TX)a*]@眡@5*L&6PPHo% % % TT)P*]@眡@5*L&6P髞% % % T`)a*]@眡@5*L&6T7.0d2e% % % T)!P*]@眡@5* L&6d 燫eQ籗粂P[4l髞;`SO飝:Nd% % % TX!)"a*]@眡@!5*L&6P1Ldz% % % TT")#P*]@眡@"5*L&6P e % % % T`*+]@眡@y+L&6T121ddd% % % TT*+]@眡@y+L&6P!Td* +]@眡@y+L&6Tn韕mp虄% % % TX * +]@眡@ y+L&6P20d% % % T` *U+]@眡@ y+L&6TR煍0% % % TTV*+]@眡@Vy+L&6P  % % % TT-x-]@眡@-L&6P0% % % Tdy--]@眡@y-L&6T瀃寶ek% % % TT-c-]@眡@-L&6P0% % % TTd --]@眡@d-L&6P  % % % TTU/50]@眡@ 0L&6P1d% % % TT]/$0]@眡@ 0L&6PTp]/ $0]@眡@ 0L&6X蛓P[瞞剉6RY% % % TT U/" 50]@眡@ 0L&6P  % % % TTA1m2]@眡@AR2L&6PcTT 1m2]@眡@ R2L&6P諷% % % TX1a~2]@眡@R2L&6PLBz% % % Tpb1 m2]@眡@bR2 &WMFChhL&6X鶹SOs^g N剉Td 1{m2]@眡@ R2L&6T'Y爛Fg虄% % % Td|1B~2]@眡@|R2L&6TBL21zdd% % % TTC1 m2]@眡@CR2L&6P% % % T` 1^~2]@眡@ R2L&6TpETdzh% % % TTQ1~2]@眡@QR2L&6P-C% % % T`1~2]@眡@R2L&6T30addY% % % TT1~2]@眡@R2L&6P-C% % % TT1~2]@眡@R2L&6PX% % % TT1Om2]@眡@R2L&6P TdP1om2]@眡@PR2L&6TUS虄= TTp17m2]@眡@pR2L&6PTT81m2]@眡@8R2L&6P蛓TX1m2]@眡@R2L&6P嶯+T% % % TT1<~2]@眡@R2L&6P5d% % % =R2Rp8@Times New Roman肋@鎣薕C]肋杠 (搛みOC]肋杠 獒絖杠肋 l,{d飩_ G*郃x Times ew oman鳧=燔8礯%(鎣T鎣+Q蓋T鎣bQ蓋窽霸dv% %  TX=1(~2]@眡@=R2L&6Plk % ( % % % Tl)1#m2]@眡@)R2L&6XaS }剉% % ( 666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6 6 66&66& 6  6 66&66& 6  6 66&66& 6  6 66&66& 6  6 66&66& 6 666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6666&66&6 6 66&66& 6 !!6!66&66&!6!!""6"66&66&"6""##6#66&66&#6##$$6$66&66&$6$$%%6%66&66&%6%%  JS."System豮?輞?蒈?--宋體---@Times New Roman--- @Times New Roman----------------- 2 3*SJ 1--- 2 37SJ ,JS- @ !67-'宋體---@Times New Roman------------ 2 ESJ分子生物學實驗 ---2 EQSJII--- 2 EZSJ ------ 2 XSJ(蛋白質的表達 2 X*SJ、純化 2 XNSJ及檢測) --- 2 X~SJ --- 2 k*SJ ---2 }SJ實驗 2 }SJ一 2 }SJ: 2 } SJ外源基因在 2 }$SJ大腸桿菌 2 }TSJ中的 2 }lSJ誘導表達 --- 2 }SJ 宋體---@Times New Roman- - - ---2 9SJ【 ---2 ESJ實驗目的 ---2 uSJ】 - - - 2 SJ - - - --->2 Q"SJ通過本實驗了解外源基因在原核細胞中 2 SJ誘導 2 5SJ表達 2 MSJ情況。 - - - 2 qSJ - - - ---2 9SJ【 ---2 ESJ實驗原理 ---2 uSJ】 - - - 2 SJ 宋體- - - @Times New Roman- - - - - - - - - ---#2 QSJ將外源基因克隆在 2 SJ使用 2 SJ含有 - - - 2 SJlac1 ---2 SJ操作子的 22 &SJ啟動子的表達載體中🟣,讓其在 - - - 2 SJE.- - - 2 SJ - - - 2 SJcoli---2 SJ中表達👨🏼‍💻。 - - - ---- - - ---/2 9SJ首先在不加誘導劑的條件下 2 SJ培養 2 SJ宿主菌🌕, - - - 2 SJlacI---2 ' SJ基因表達的 2 cSJ產生的阻遏蛋白 - - - 2 SJLacI---2 SJ與- - - 2 SJlacI ---2 SJ操作子 ---2 89SJ結合 /2 8QSJ,從而不能進行外源基因的 2 8SJ表達 2 8SJ♛; /2 8SJ然後向培養基中加入誘導物 - - - 2 8SJIPTG ---2 8SJ🧙🏼‍♀️, 2 8SJ此時 - - - 2 8SJLacI ---2 8SJ阻遏蛋 ---2 O9SJ白 2 OE SJ變構失活, 2 OSJ不能與 - - - 2 OSJlacI---2 OSJ操作子 )2 OSJ結合🔆,外源基因表達🛀🏽。 - - - 2 OUSJ ---2 v9SJ【 ---)2 vESJ實驗儀器、材料與試劑 ---2 vSJ】 - - - 2 vSJ ---2 9SJ實 2 ESJ驗儀器 2 iSJ: &2 uSJ超凈工作臺、試管😭🪆、 #2 SJ搖床、離心機等🦹👗。 - - - 2 ASJ ---2 9 SJ菌株和質粒 2 uSJ: 2 SJ菌株:大腸桿菌 - - - 2 SJBL21---2  SJ♓️;重組質粒 - - - 2 2SJpET1- - - 2 FSJ-- - - 2 JSJ30a1- - - 2 [SJ-- - - 2 _SJX ---2 hSJ:基因 - - - 2 SJX ---2 SJ與- - - 2 SJpET1- - - 2 SJ-- - - 2 SJ30a1---2 SJ重組。 - - - 2 SJ ---2 9SJ主要試劑 - - - 2 iSJ @Wingdings- - - - - - @"Arial- - - - - - ---- - - 2 ESJl - - - 2 NSJ - - - 2 ZSJ50mg/ml   ---2 SJ卡那黴素 2 SJ配- - - 2 SJ10ml ---2 SJ, 2 SJ用- - - @Times New Roman--.2 SJ0.22靘 .- ---2 % SJ濾膜過濾除菌 2 nSJ,分成 - - - 2 SJ10 ---2 SJ份🤢, 2 SJ貯存於 - - - 2 SJ-- - - 2 SJ20---2 SJ℃ 2 SJ🙋‍♂️🏮。 - - - 2 SJ ,JS'宋體---@Times New Roman------------- - - 2 ESJl - - - 2 NSJ - - - 2 ZSJ1M 2 - - - 2 oSJIPTG---2 SJ🦸🏽: 2 SJ將- - - 2 SJ2.4G- - - 2 SJg IPTG ---2 SJ溶於 - - - 2 SJ10mL ddH  --- 2 ,SJ2- - - 2 0SJO ---2 >SJ中, 2 VSJ用- - - e@Times New Roman--.2 eSJ0.22靘 .- ---&2 SJ濾膜過濾除菌,每份 - - - 2 SJ1ml2 ---2 SJ, ,JS'---2 )ZSJ貯存於 - - - 2 )~SJ-- - - 2 )SJ20---2 )SJ℃ 2 )SJ🏄‍♀️。 - - - 2 )SJ ,JS'- - - 2 =ESJl - - - 2 =NSJ ---2 =ZSJLB ---2 =m SJ液體培養基 --- 2 =SJ ,JS'---2 P] SJ胰蛋白腖( - - - 2 PSJtryptone---2 PSJ) - - - 2 P SJ - - - 2 P SJ - - - 2 PSJ - - - 2 PSJ10- - - 2 P!SJg - - - 2 P-SJ ---2 c] SJ酵母酵母膏( - - - 2 c SJyeast extrac- - - 2 cSJt---2 cSJ) - - - 2 cSJ p- - - 2 cSJ p- - - 2 cSJ - - - 2 cSJ5- - - 2 cSJg- - - 2 c#SJ - - - 2 v9SJ - - - 2 vQSJ - - - :2 v]SJNacl - - - 2 vSJ10- - - 2 v!SJg- - - 2 v'SJ ---/2 XSJ搖動容器直至溶質完全溶解 - - - 2 SJ,---2 SJ用- - - 2 SJNaOH(1M)  ---2 2SJ調節 - - - 2 LSJPH ---2 ^SJ至- - - 2 lSJ7.0H---/2 {SJ,加入去離子水至總體積為 - - - 2 SJ1L---2 SJ💾,- - - 2 XSJ121H---2 jSJ℃ 2 vSJ濕熱滅菌 - - - 2 SJ20 ---2 SJ分鐘。 - - - 2 SJ ---2 9SJ【 ---2 ESJ實驗步驟 ---2 uSJ】 - - - 2 SJ - - - 2 9SJ1---2 ?SJ. 2 K SJ種子液的製備 - - - 2 SJ ---2 QSJ挑 2 ]SJ取- - - 2 mSJLB ---2  SJ固體平板上的 2 SJ大腸桿菌 - - - 2 SJBL21---2 SJ( - - - 2 %SJpET1- - - 2 8SJ-- - - 2 <SJ30a1- - - 2 MSJ-- - - 2 RSJX---2 ZSJ) 2 fSJ單菌落, 2 SJ接 2 SJ種 2 SJ於含 - - - 2 SJ5- - - @Times New Roman--.2 SJ靗.- ---2  SJ卡那黴素的 --JJSSJJSSJJSSJJSSJJSSIISSIISSIISSIISSIISSIISSIISSIISSIISSIISSIISSIIRRIIRRIIRRIIRRIIRRHHRRHHRRHHRRHHRRHHRRHHRRHHRRHHRRHHRRHHRRHHRRHHRRHHQQHHQQHHQQHHQQHHQQ脹諟.摋+,0 X`t|  番茄花園;    !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@BCDEFGHMRoot Entry F繢眫刺OData Xez1Table剕WordDocument<SummaryInformation(LDocumentSummaryInformation8ACompObju  F#Microsoft Office Word 97-2003 文檔 MSWordDocWord.Document.89瞦