大肠杆菌不耐热肠毒素B亚单位基因改建及其表达产物粘膜免疫佐剂活性的研究_英文__cropped

大肠杆菌不耐热肠毒素B亚单位基因改建及其表达产物粘膜免疫佐剂活性的研究_英文__cropped () 文章编号 :1002 - 2694 200605 - 0385 - 06 Recon struct ion of the hea t2la bile enterotoxin subun it B gene of Es c he r ic hi a col i an d its immune a djuvant act ivity on mucosa 1 2 3 2SU N J u2yun, TA N Pa n2li, H U Ye,MA O Ya2f ei () ABSTRACT : To reco nst r uct t he heat2labile entero to xin subunit B L TBgene of Esc he ric hi a coi l in o r der to increa se t he ( ) o utp ut s of t he p ro ka r yo tic exp ressio n o n reco mbinant L TB rL TB, a nd to deter mine it s immune adj uvant activit y o n muco sa , t he nucleo tide sequence of t he w hole lengt h of L TB gene wa s synt he sized acco r di ng to t he p ref er red co do ns of E. col i , a nd t he p ro kar yo tic exp ressio n pla smid p E T32a2rL TB a nd it s exp re ssio n system in E. col i BL 21D E3 were reco nst r ucted. The reco mbi2 nant pla smid wa s ext racted a nd t he inserted sequence of rL TB gene wa s deter mined. Meanw hile , t he exp re ssio n quantit y of t he reco nst r ucted rL TB wa s identified by SDS2PA GE and Bio Rad agaro se image a nalyzing system , a nd co mpa red wit h t hat of t he un - reco nst r ucted rL TB . The abilitie s of t he reco nst r ucted rL TB a nd t he un2reco nst r uct ed rL TB to bind wit h bo vine GMwere 1 deter mined by mea ns of GM2EL ISA a ssay. By using t he reco mbinant urea se subunit B a s a ntigen , t he eff ect s of t he reco n2 1 st r ucted and t he un2reco nst r ucted rL TB o n t he imp ro vement of immune p ro tectio n of BALB/ c mice inf ected wit h H el i cobacte r p y l ori st rain SS1 a nd t he i nductio n of S2IgA s in inf ected mice were a ssayed. The experimental re sult s sho wed t hat t he exp re s2 sio n quantit y of t he reco nst r uct ed rL TB app roached up to 35 . 4 % of t he to tal bacterial p ro teins af ter inductio n wit h 1 mmol/ L () IP T G fo r p E T32a2rL TB2 E. col iBL 21D E3 and to be 12 . 6 time s higher t han t hat of t he un2reco nst r ucted rL TB 2 . 8 %. In addi2 tio n , bo t h t he abilitie s of t he reco mbinant reco nst r uct ed L TB a nd t he un2reco nst r ucted rL TB to bind wit h bo vine GMco uld be 1 demo nst rated by GM2EL ISA .The immune p ro tectio n rate of t he reco mbi na nt urea se subunit B in t he inf ected mice wa s 1 66 . 7 % ; a nd it co uld reach up to 91 . 7 % wit h a significa nt increa se of t he sp ecific S2IgA level , w hen it wa s immunized wit h t he reco nst r ucted o r t he un2reco nst r ucted rL TB . It i s co ncl uded t hat t he reco nst r ucted L TB gene i n t he p re sent st udy sho w s a re2 ma r ka ble increa sed o utp ut s of exp re ssio n of t hi s gene wit h a st ro ng immune adj uvant activit y o n muco sa . KEY WORDS : Esc he ri c hi a col i ; L TB gene/ reco nst r uctio n ; reco mbina nt p ro tein / exp re ssio n ; muco sal immunit y ; adj u2 vanticit y CLC Number :R378 . 2 Document code :A 〔728〕 O n acco unt of t he lo wer be neficial eff ect of t ha n t hat fo r t he i nductio n of ser um a nti bo die s. 2 i mmunizatio n wit h mo st of t he ge neticall y e ngiC TB act s a s a n a dj uva nt t h ro ugh t he Th2 p at h2 way , i n w hich t he sp ecificall y i nduci ng Ig E cyto2 neeri ng vacci ne s u se d at p re se nt ti me , i n w hic h a 〔9210〕ki ne IL24 i s p ro duced. Ho wever , L TB doe s si ngle reco mbi na nt p ro t ei n wa s u suall y u sed a s a n2 no t ca u se Ig E2mediat e d aller gic reactio n , beca u se it ti ge n , it i s de si ra ble to i ncrea se t he i mmu no ge nici2 〔9 ,11〕mai nl y activat ed t he Th1 p at hway. O ur p revi2 t y of a nti ge n s bei ng u se d by co2i mmunizatio n wit h a dj uva nt s. The heat la bile e nt e ro to xi n subu nit B () L TBof Esc he ri c hi a col i a nd c hole ra to xi n sub2 Co r re spo ndi ng a ut ho r : MAO Ya2f ei , E2mail : Med_ bp @zj u . edu . cn ( ) U nit of t he a ut ho r s : 1 . Medical School of Li shui College , Zhejia ng unit B C TBwe re rece nt l y co nfi r med to be a n ef2 Li shui 323000 , Chi na ; f ective a nd saf e muco sa a dj uva nt s , a nd t hei r a dj u2 2 . Dep a rt ment of Medical Micro biolo gy a nd va nticit y wa s p ro ved to be dep e nde nt o n t hei r bi nd2 Para sitolo gy , College of Medici ne , Zhe2 〔123〕2 Sevi ng a bilit y to GMrecep to r s o n ho st cell s.1 jia ng U ni ver sit y , Zhejiang Ha ngzho u 310031 , Chi na ; eral st udie s de mo n st rat ed t hat t he a dj uva nticit y of 3 . Medical School of t he Prof e ssio nal Tech2 L TB wa s eve n mo re po werf ul fo r t he muco sal i m2 nique College 〔426〕 mu nizatio n t ha n t hat of C TB, b ut it wa s lo we r , 3 . 0 U Taq2Pf u pol ymera se mi x , M gClo u s st udy by u si ng t he co n st r uct ed p ro ka r yo tic e x2 mol/ L 2 ( p re ssio n syst e m of L TB ge ne off ered a relativel y 100 ng DN A t e mp lat e a nd 1 ×PCR buff e r p H ) lo wer o utp ut of t he t a r get reco mbi na nt p ro t ei n o n2 8 . 3. The p a ra met e r s fo r PCR we re : 94 ? 5 mi n ;l y ma de of app ro xi mat el y 5 % of t he to t al bact e rial 94 ? 30 S ,50 ?30 S , 72 ?45 S , 30 cycle s ; 72 ?7 p ro t ei n s. mi n a nd t he p ri mer s u se d i n t hi s st udy were syn2 ( ) 2 t he o utp ut of t he reTo i ncrea se efficie nt l y t he size d by Bio A sia Sha nghai , Chi na. The Hi gh ( ) fi delit y PCR Kit wa s p urcha se d f ro m Ta Ka Ra co mbi na nt L TB rL TB, t he L TB ge ne wa s reco n2 () Dalia n , Chi na. The re sult of PCR wa s o b ser ved st r uct ed acco r di ng to t he p ref e r red co do n s of E. co2 under U V li ght af t er elect rop ho re si s o n 1 . 5 % a ga2 2 wa s e st a bl i , a nd t he n t he a ssay of GM1 2EL ISA ro se gel p re2st ai ne d wit h et hi di um bro mi de . The li she d to det er mi ne t he a bilit y to bi nd wit h bo vi ne GMof t he reco mbi na nt L TB e xp re sse d by reco n2 e xp ect e d size of L TB ge ne a mp lificatio n f ra gme nt 1 st r uct ed L TB ge ne a nd t he o ri gi nal L TB ge ne . B y wa s 375 bp . u si ng t he reco mbi na nt urea se B subu nit a s a nti2 2 The a mp lificatio n f ra gClon ing and sequenc ing 〔12〕 ge n, t he eff ect s of t he reco n st r uct e d a nd t he o2 me nt of L TB ge ne wa s clo ned i nto p la smi d vecto r ri gi nal L TB ge ne s o n t he i mp ro ve me nt of t he i m2 p U Cm2T by u si ng a T2A clo ni ng kit off e red by mu ne p ro t ectio n o n H el i cobact e r p y l o ri2i nf ect ed ( ) Bio Colo r Sha nghai , Chi naacco r di ng to t he ma n2 mice a nd t he i nductio n of sp ecific S2IgA i n t he i n2 uf act ure r’ s i n st r uctio n . The reco mbi na nt p la smi d αf ect ed mice we re i nve sti gat ed i n t he p re se nt st udy. p U Cm2T2oL TB wa s a mp lified i n E. col i D H 5a nd 〔13〕t he n e xt ract ed by Sa mbroo k’s met ho d . Bio A sia Material s and Methods co mp a ny wa s re spo n si ble fo r nucleo ti de seque nce E. col i L T2p ro duci ng Bacterial stra ins and culture a nal y si s of t he i n se rt e d f ra gme nt . 2 Acco r di ng to t he p rest rai n 44815 L T a nd H . p y l o ri st rai n SS1 were of2 L TB gene reconstruct ion f ere d by t he In stit ut e of Dr ug a nd Biolo gical Pro d2 f er red co do n s of E. col i , t he nucleo ti de seque nce of ( ) a reco n st r uct e d L TB rL TB ge ne wa s det er2 uct s of Chi na , wit h LB a ga r u sed a s t he medi um fo r t he cultivatio n of E. col i st rai n . Col umbia bloo d mi ned . The rL TB ge ne i n ser t e d i n p la smi d vecto r a ga r co nt ai ni ng 10 mg/ L va nco myci n , 5 mg/ L p U Cm2T wa s synt he sized by Bio A sia . TM P , 2500 IU pol ymi xi n B a nd 5 mg/ L a mp ho t e r2 Expression and identif ication of target recombinant ( ) Two reco mbi na nt p la smi ds p U Cm2T2 ici nB bio Me rie ux wa s fo r t he cultivatio n of H . proteins p y l o ri st rai n . oL TB a nd p U Cm2T2rL TB a s well a s t he e xp re ssio n ( ) vecto r p E T32a No va ge nwe re a mp lified i n E. col i L TB gene a mpl if icat ion Ge no mic DN A of E. col i α st rai n 44815 wa s e xt ract e d by co nve ntio nal p he nol2 D H5re sp ectivel y. The se p la smi ds we re e xt ract ed chlo rofo r m met ho d a nd DNa se2f ree RNa se t reat2 a nd t he n di ge st e d wit h e ndo nuclea se s Eco R I a nd me nt a nd t he co nce nt ratio n a nd p urit y of t he DN A X ho I. The f ra gme nt s of L TB , reco mbi na nt L TB p rep a ratio n wa s det er mi ned by ult raviolet sp ect ro2 a nd li nea r p E T32a were reco ve re d a nd t he n li ga2 〔13〕t ed . All t he rea ge nt s u se d i n ligatio n were off e red p ho to met r y. The p ri me r s were de si gned to a m2 by Ta Ka Ra . The reco mbi na nt e xp re ssio n vecto r p lif y e nti re L TB ge ne f ro m E. col i st rai n 44815 p E T32a2oL TB a nd p E T32a2rL TB we re t he n t ra n s2 ba se d o n t he p ubli she d dat a o n Ge nBa nk a nd re2 ( ) st rictio n e ndo n uclea se a nal ysi s. fo r med into E. coli BL21D E3 Novagen, and t hese The seque nce of two exp ressio n systems were designated as p E T32a2 se n se p ri me r wit h a n e ndo nuclea se Eco R I wa s 5’2 oL TB2 E. coli BL212D E3 and p E T32a2rL TB2 E. coli CC G GA A T TC A T G A A T A A A GTA A A A TA2 3’ , a nd t hat of t he a nti se n se p ri mer wit h a n e ndo2 BL 212D E3 re sp ectivel y. The se t wo e xp re ssio n sys2 t e m s were relativel y c ultivat e d i n LB medi um at nuclea se X ho I wa s 5’2CC G C TC GA G GA G C TA GT T T TC CA T A C T GA23’. The to t al vol u me p e r 37 ? wit h i nductio n of 1 . 0 mmol/ L IP T G. SD S2 μPA GE p l u s Bio Ra d a ga ro se i ma ge a nal yzi ng syst e m PCR wa s 100 l , co nt ai ni ng 2 . 5 mol/ L each of t he wa s u sed to mea sure t he o utp ut s of t he reco mbi2 dN T P , 250 nmol/ L each of t he t wo p ri mer s , 20 na nt p ro t ei n s e xp re sse d by t he o ri gi nal L TB ge ne A nd t he i solat e s were i nde ntified by Gra m2st ai2 ( )ge ne ni ng , urea se a nd o xi da se t e st s. Af t e r t reat me nt roL TB a nd t he reco n st r uct ed L TB μ( ) wit h a nti2p ro t ei na se a nd a dditio n of 20 l PM SF r rL TB. () 2 NiBio A sia 100 mmol/ L , ga st ric j uice sp eci me n s 1 2EL ISAAff in ity chromatogra phy and GM were ce nt rif uge d at 1000 r/ mi n fo r 10 mi n , a nd t he N TA affi nit y c hro mato grap hy wa s u se d to e xt ract sup e r nat a nt s we re collect ed to det ect t he p re se nce r rL TB a nd roL TB . Ba se d o n t he met ho d a dvocat ed 〔14〕 μ2EL ISA wa s GMof S2IgA by EL ISA . B y u si ng 20 g/ ml of r U reB by De Haa n et al , a a ssay fo r1 e st a bli shed a nd u sed to det er mi ne t he a bilitie s of a s coat ed a nti ge n , 1 ?200 dil utio n of ra bbit a nti2 〔12〕( roL TB a nd r rL TB to bri ng wit h bo vi ne GMSi g21 rL TB ser um a s t he fi r st a nti bo dy a nd t he H R P2 ( ) ) la beli ng ra b bit a nti2mo u se IgA Zymed L a bser um ma. B rief l y , 200 ng of GMp er well wa s coat ed 1 o n 962well s p lat e at 4 ? o ve r ni ght , a nd 0 . 01 mol/ a s t he seco nd a nti bo dy a s well a s O P T a s sub2 ( ) L PB S co nt ai ni ng 5 % v/ v bo vi ne se r u m a nd st rat e , t he ODval ue of each well s we re det ect ed 490 ( ) ( ) 0 . 05 % v/ v Twee n220 p H7 . 4 wa s u se d to by u si ng Bio Ra d mo del 680 microp lat e rea der . It block well s. roL TB a nd r rL TB were t he n a dde d i n2 wa s co n si dere d to be po sitive w he n t hi s val ue i s μgreat e r t ha n t hat of t he mea n val ue of 5 ne gative to well s wit h fi nal co nce nt ratio n s of 1 a nd 5g re2 sp ectivel y a nd i ncu bat e d at 37 ? fo r o ne ho ur . B ywell s wit h 3 st a nda r d deviatio n . u si ng t he ra b bit a nti2rL TB ser um at dil utio n of 1 ?Results 〔15〕200 a s t he fi r st a nti bo dy ; t he H R P2la beli ng () sheep a nti2ra bbit Ig G J ack so n Immu no2Re sea rc hPCR : The t a r get f ra gme nt wit h t he e xp ect ed size a s t he seco nd a nti bo dy a nd O P T a s sub st rat e , t he wa s p re se nt i n t he PCR p ro duct a mp lified f ro m t he ODval ue of each well wa s det er mi ned by u si ng490 DN A t e mp lat e of E. col i st rai n 44815 . Bio Ra d mo del 680 microp lat e rea de r . In t he GM21 EL ISA a ssay , 5 rep eat e d well s we re set up fo r each co nce nt ratio n of roL TB o r r rL TB , a nd a no t h2 er 5 well s wit ho ut t he se p ro t ei n were to be t a ke n u sed well wa s great er t ha n t hat of t he mea n CD490 〔16〕 val ue wit h 3 st a nda r d deviatio n s . Indent if icat ion of a djuvantic ity of the reconstructed The reco mbi na nt reco n2 an d original L TB genes st r uct ed a nd o ri gi nal L TB ge ne s collect e d by Ni2 N TA affi nit y. Chro mato grap hy were di ssol ved i n ( ) PB S 0 . 01 mol/ L , p H7 . 4 . Healt hy B ALB/ c mice wit h bo dy wei ght of 18 ?1 g we re divi ded i nto 4 gro up s wit h 12 mice i n eac h gro up s. The t hree Fig. 1 The ampl if ication f ragmentsof L TB gene f rom E. coli t e st ed gro up s of mice were o rall y i mmu nized t wice stra in 44815 μμμwit h 100g r U reB , 100 g p l u s 10 g roL TB a nd ( ) 1 ?250 bp DN A ma r ker Bio Colo r ; 2 a nd 3 : t he μμ100 g r U reB p l u s 10 g r rL TB , re sp ectivel y. O n ta r get a mplificatio n f ragment s of t he L TB gene ; 5 : blank co nt rol . t he 28 t h , 30 t h a nd 32 t h da ys af t er t he fi nal i m2 mu nizatio n , mice i n t he se t h ree gro up s a nd a no t he r Sequence analysis and compar ison In co mp a ri so n gro up of mice wit ho ut a ny p re2i mmunizatio n were ( wit h t he repo r t ed L TB ge ne seque nce s N CB I : μ challe nged wit h 200 l of f re shl y c ult ure H . p y l o riABO11677 , S60731 , A F242418 , X83966 , J 01646 10 () su sp e n sio n s 5 ×10C FU / ml , a nd 28 days af t e r) a nd M17874, t he si mila rit y of nucleo ti de a nd p u2 t he fi nal i nf ectio n , all t he mice we re killed to col2 t ative a mi no aci d seque nce s of t he clo ne d L TB ge ne lect ga st ric j uice s a nd to t a ke t he biop sy sp eci me n s were 99 . 12 %, 99 . 71 % a nd 97 . 58 %, 99 . 19 % , of a nt r u m i n w hic h H . p y l o ri wa s to be i solat e d. re sp ectivel y . The nucleo ti de a nd p ut ative a mi no ( ) aci d seque nce s of t he o ri gi nal L TB ge ne oL TB ( ) a nd t he reco n st r uct e d L TB ge ne rL TB were sho w n i n fi gure 2 . Expression of the recombinant protein s roL TB a nd r rL TB were 2 . 8 % a nd 35 . 4 % of t he to t al bac2 () t erial p ro t ei n s , re sp ectivel y fi gure 3. The fo r me r o utp ut wa s app ro xi mat e 12 . 62fol d s of t he lat t e r . The results of GM2EL ISA Acco r di ng to t he re2 1 sult s of GM2EL ISA , ODmea n ?SD of t he neg2 1 490 ative co nt rol s wa s 0 . 10 ?0 . 03 , re sulti ng i n t he po sitive st a nda r d a s 0 . 19 . The ODmea n s SD of490 roL TB a nd r rL TB were 1 . 03 ?0 . 19 a nd 1 . 10 ?Fig. 3 Expression of roL TB and rrL TB under inducement 0 . 25 , re sp ectivel y , i ndicati ng t he t wo e xp re ssed of IPTG . GML TB s co ul d co mbi ne wit h t he bo vi ne 1 ( ) M : p ro tein mar ker Sha nghai Shi sheng; 1 : blank co nt rol ; 2 a nd 3 : t he exp re ssed roL TB a nd r rL TB , resp ectively. The mucosal a djuvant ic ity of rrL TB and roL TB roL TB o r r rL TB sho we d re ma r ka ble eff ect s to i n2 crea se t he i mmune p ro t ectio n rat e s of r U reB f ro m 66 . 7 % to 91 . 7 % i n t he H . p y l o ri st rai n SS1 i nf ec2 t ed mice a nd co ul d elevat e t he level s of t he sp ecific ( ) ( ) S2IgA a gai n st r U reB P < 0 . 01t a ble 1. Discussion Vacci ne s develop ed by ge netic e ngi neeri ng t ec h nique s have several a dva nt a ge s o ver t he t ra di2 tio nal vacci ne s o n acco unt of t hei r clea r a ntige nic co mpo sitio n a nd lo wer si de eff ect s. Ho wever , i n mo st of t he se vacci ne s o nl y a si ngle p ro t ei n wa s u sed a s a nti ge n w hic h f reque nt l y i nduced lo wer i m2 mu ne p ro t ectio n a gai n st i nf ectio n s. Co2a dmi ni st ra2 tio n wit h a dj uva nt s may be a n eff ective wa y to i m2 p ro ve t he i mmune p ro t ectio n of t he se vacci ne s. It wa s rep eat edl y co nfi r me d by ma ny of t he rece nt st udie s t hat L TB wa s p ro ve d to be a muco sal a dj u2 va nt , a nd t he a dj uva nticit y of it s reco mbi na nt Fig. 2 The sequences of the L TB gene f rom E. coli stra in 〔122 ,4〕44815 and the reconstructed L TB gene p ro duct s i s al so well defi ned. () () 1a nd 2: t he nucleo tide sequences of t he o riginal In co mp a ri so n wit h t he 6 repo r t e d L TB ge ne () a nd reco nst r ucted L TB gene s , resp ectivel y ; 3t he ( seque nce s N CB I : ABO11677 , S60731 , A F242418 , p utative amino acid sequence . The underlined indi2 ) X38966 , J O1646 a nd M17874, t he si mila ritie s of cates t he p ri mer po sitio ns a nd t he sign " 3 " mea ns nucleo ti de a nd p ut ative a mi no aci d seque nce s of t he t he stop co do n. clo ne d L TB ge ne s we re 99 . 12 % , 99 . 71 % a nd 97 . 58 %,99 . 19 % ; a nd a mo ng t he 6 repo r t ed L TB Ta ble. 1 Eff ects of rrL TB and roL TB on increasing the immunoprotection of H. p yl o r i r Ure B ( )Ca se s r U reB r rL TB roL TB S2IgA p ro tective rates H. p y l ori i solatio n n Gro up s ( ) ( ) ( ) ( ) ( ) ( ) ng/ mo useg/ mo useg/ mo u se+ / %+ / % Te sted : A 12 100 / / 4/ 33 . 3 3/ 25 . 0 B 9/ 75 . 0 3 12 100 10 / 1/ 0 . 9 C 12 100 / 10 1/ 0 . 9 8/ 66 . 7 3 Co nt rol 12 / / / 11/ 91 . 7 1/ 8 . 3 + : H. p y l o ri co uld be i solated a nd t he sp ecific S2IgA co uld be detecta ble. 3 : P < 0 . 01 vs gro up A co mp a ri so n wit h t ho se of mice i mmu nized wit h r U2 , 100 % a ndge ne seque nce s , t ho se were 98 . 53 % 97 . 58 %,100 % re sp ectivel y. Th u s , t he mut atio n reB alo ne . level of o ur clo ned L TB ge ne i s wit hi n t he ra nge of Reff erences t ho se of p revio u sl y repo r t e d. 〔1〕Verweij WR , De Haan L , Holt rop M , et al . M uso sal Fo r o bt ai ni ng a p ro ka r yo tic e xp re ssio n vecto r immunoadj uvant activit y of reco mbina nt Esc he ric hi a col i wit h hi ghe r o utp ut s , t he L TB ge ne wa s reco n2 heat2la bile entero to xin a nd it s B subunit : Inductio n of st r uct ed acco r di ng to t he p ref er red co do n s E. col i. systemic Ig G a nd secreto r y IgA re spo nse s in mice by in2 The SD S2PA GE a nd Bio Ra d a ga ro se i ma ge a nal y2 t ra na sal immunizatio n wit h inf l uenza vir us surf ace a ntigen zi ng syst e m de mo n st rat ed t hat t he o utp ut s of 〔J 〕. Vaccine , 1998 , 16 :206922076 .r rL TB e xp re sse d by t he reco n st r uct e d L TB ge ne 〔2〕Do uce G , Giulia ni MM , Gia nnelli V , et al . Muco sal im2wa s 35 . 4 % of t he to t al bact erial p ro t ei n s , i ndica2 muno genicit y of geneticall y deto xified derivative s of heat ti ng t hat t hi s o utp ut wa s a bo ut 12 . 62fol d s hi ghe r la bile to xin f ro m Esc he ric hi a col i〔J 〕. Vaccine , 1998 , t ha n t hat e xp re ssed by t he reco mbi na nt o ri gi nal 16 :106521073 . 〔3 〕Pizza M , Giulia ni MM , Fo ntana M R , et al . M uco sal () L TB ge ne 2 . 8 %. It wa s repo rt e d t hat t he eff ec2 vaccines : no n2to xi n derivative s of L T and C T a s muco sal tive do se s of L TB a s muco sal a dj uva nt wa s a mo ng 〔8 ,17218〕 adj uvant s〔J 〕. Vaccine , 2001 , 19 :253422541 .μ Therefo re , t hi s a mo unt of o utp ut s 1 - 25g. 〔4〕Weltzi n R , GuyB , Tho ma s WD , et al . Parenteral adj u2fo r r rL TB i s quit e sati sf acto r y to t he reque st fo r vant activitie s of Esc he ric hi a col i heat2la bile to xin a nd it s f ur t her i ndu st rial p ro ductio n . B subunit fo r immunizatio n of mice against ga st ric H el i2 The a dj uva nticit y of t he reco mbi na nt L TB wa s cobacte r p y l o ri inf ectio n. Inf ect Immun , 2000 , 68 : co nfi r me d to be dep e nde nt o n it s a bilit y to bi nd 277522782 . 〔123〕wit h t he GMo n t he surf ace of ho st cell s. Fro m 1 〔5〕Do uce G , Fo nta na M , Pizza M , et al . Int ra na sal immu2 t he a ssa y wit h GM2EL ISA , it wa s de mo n st rat ed 1 no genicit y a nd adj uva nticit y of site2directed mutant deriv2 t hat bo t h t he o ri gi nal a nd t he reco n st r uct e d L TB atives of cholera to xin〔J 〕. Inf ect Immun , 1997 , 65 : 282122828 . co ul d st ro ngl y bi nd wit h si mila r a bilit y of bi ndi ng 〔6〕Ya mamo to S , Ta keda Y , Ya mamo to M , et al . M uta nt s to bo vi ne GM, sugge sti ng t he p re se nce of muco sal 1 in t he AD P2ribo sylt ransf era se clef t of cholera to xin lack a dj uva nticit y i n t he se t wo reco mbi na nt L TB s. 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New( )Receivde :2006203 - 03 Yo r k : Cold Sp ring Ha r bo r L a bo rato ry Press , 1989 :212 大肠杆菌不耐热肠毒素B 亚单位基因改建及 其表达产物粘膜免疫佐剂活性的研究 1 2 3 2孙菊云,谈潘莉,胡 野,毛亚飞 (1 . 丽水学院医学院 ,丽水 ,323000 ;2 . 浙江大学医学院病原生物学教研室 ,杭州 ,310031 ; ) 3 . 金华职业技术学院医学院 ,金华 ,321000 () ( ) 摘 要 :目的 改建大肠杆菌不耐热肠毒素 B 亚单位 L TB基因序列以提高重组 L TB rL TB原核表达量 ,确定重组改建 ( ) L TB r rL TB粘膜免疫佐剂活性 。方法 按大肠杆菌偏爱密码子合成全长 L TB 基因的核苷酸序列 。构建 p E T32a2rL TB 原核 重组表达质粒及 p E T32a2rL TB2 E. col iBL 21D E3 表达系统 ,提取重组质粒并测定其插入的 rL TB 基因序列 。采用 SDS2PA GE ( ) 和 Bio Rad 凝胶成像分析系统鉴定 r rL TB 表达产量 ,并与未改建的重组 L TB roL TB比较 。采用 GM2EL ISA 确定 r rL TB 和1 ( ) roL TB 结合牛 GM的能力 。分别以幽门螺杆菌重组尿素酶 B 亚单位 r U reB,检测 r rL TB 和 roL TB 对 r U reB 对幽门螺杆菌1 SS1 株感 染 BALB/ c 小 鼠 保 护 作 用 及 产 生 特 异 性 S2IgA 的 增 强 效 应 。结 果 p E T32a2rL TB2 E. col iBL 21D E3 经 1 mmol/ L () IP T G 诱导后 ,r rL TB 表达量约占细菌总蛋白的 35 . 4 % ,为 roL TB 2 . 8 %的 12 . 6 倍 。GM2EL ISA 结果证实 r rL TB 和 roL TB1 均能与牛 GM结合 。单一 r U reB 对感染小鼠的免疫保护率为 66 . 7 % ,但与 r rL TB 或 roL TB 合用时 ,保护率可至 91 . 6 % ,并 1 ( ) 可提高感染小鼠特异性 S2IgA 产生量 P < 0 . 01。结论 改建的 L TB 基因能明显提高表达量 ,所表达的 r rL TB 仍保持较强 的粘膜免疫佐剂活性 。 关键词 :大肠杆菌 ;L TB 基因/ 改建 ;重组蛋白/ 表达 ;粘膜免疫 ;佐剂活性 中图分类号 : R378 . 2 文献标识码 :A