ICA69基因工程融合抗原的制备_英文__cropped

ICA69基因工程融合抗原的制备_英文__cropped Tra nsa ctio ns of Tia njin Unive r sity Vol . 8 No . 4 De c . 2002 Prep a ratio n of Re co mbina nt Isl et Cell Auto a nti ge n 69 kD Fusio n Pro t ein 1 1 2 2 HUAN G He, GAN Yi2ru, HUAN G Nai2ping, ZHAN G J ing2yu (1 . School of Chemical Engineering , Tianjin U niversity , Tianjin 300072 , China ; )2 . Instit ute of Endocrinology , Tianjin Medical U niversity , Tianjin 300070 ,China ( ) Abstract :To get reco mbinant antigen Is/ et Cell Autoantigen 69ICA69 which was exp ressed in Escherichia coli ( ) st rains E. col i by means of t he gene engineering technique so t hat it can be used for early diagnosis of and screening in t ype ? diabetes mellit us , t he cDNA f ragment of human ICA69 was amplified by PCR , and t hen clo ned into p SPOR T 1 vector . Af ter DNA sequencing , it was inserted into p GEX22 T bet ween t he sites of Eco R ? and Sma ?, t hen reco mbinant plasmid p2 T2ICA69 was co nst ructed and int roduced into E. col i . The GS T2 ICA69 f usio n p rotein was exp ressed by t he inductio n of IP T G. The reco mbinant ICA69 p roteins were used to de2 tect t he antibodies against h ICA69 in 100 healt hy subject s and type ? diabetic serum by t he use of indirect EL ISA. The sequence analysis showed t hat t he amplified f ragment s co ntained 1449 bp , encoded 483 amino acids , and had been correctly inserted into p GEX22 T vector . The reco mbinant p roteins exp ressed in t he p ro kary2 otic cells had immunogenicit y and could be used to detect antibodies against ICA69 in type ? diabetic serum. Fi2 nally it can be co ncluded in t his paper t hat t he exp ressio n p roduct s obtained by t he met hod of gene engineering are reco mbinant ICA69 antigen and may be used to imp rove t he forecast rate and t he diagnostic rate of type ? dia2 betes in co mbinatio n wit h ot her test s. Key words :islet cell autoantigen ; 69 kD p rotein ; GS T f usio n p rotein ; IDDM ; EL ISA Ξ A novel 69 kD p rotein located in t he cytoplasmic 1 Material s and methods co mpart ment of human islet cell have been identified as o ne of t he autoantigens of beta cells of t he pant reatic islet . 1 . 1 Material s No minated as islet cell autoantigen 69 kD p rotein Bacteria ho st ,plasmids and DNA were co nserved by ( ) ICA69, it is a 483 amino acid p rotein and nearly associ2 o ur gro up ; all biochemical reagent s were analytic grade ; ated wit h t he cellular and humo ral immunity of insulin2de2 2 sera of co nt rol subject s were rando m o btained f ro m unre1 ( ) pendent diabetes mellit us IDDM . By now , t he islet lated healt hy members ; all IDDM patient s regarded as cell autoantibo dy against ICA69 is regarded as o ne of t he st udied subject s f ulfilled t he following clinical and labo ra2 mo st impo rtant immunological and early diagno stic target s to ry data : first o nset of keto sis2acid to xico sis symp to m , in t his autoimmune diso rder . The p reparatio n of reco mbi2 O GT T and insulin2releasing t rial clue o n bad islet f unc2 nant ICA69 antigen holds p ro mise fo r imp roving o ur un2 tio n ,age below < 40 and age at o nset below 2 years. derstanding of t he pat hogenesis and t he early diagno sis of 1 . 2 Methods IDDM , w hich is still blank inside China . 1 . 2 . 1 Amplificatio n and clo ning of ICA69 f ull lengt h The p resent st udy was undertaken to apply t he gene sequence reco mbinatio n and exp ressio n technique to clo ne and re2 ( ) U sing human islet cell t umo r H I T cDNA as t he co mbine t he ICA69 gene , and exp ress t he autoantigen in template , all t he operatio n sho uld be do ne acco rding to t he kit′s manual . The amplificatio n reactio n was perfo r med 30 vit ro successf ully. Then using indirect enzyme2linked im2 ( ) cycles under co nditio ns as follow s : started by t he befo remuno so rbent assay EL ISA, we have o ur handle o n de2 2 tecting t he antibo dies against ICA69 in healt hy subject s hand denat uralizatio n fo r 7 in ; 92 1 min , 54 and early t ype ? diabetic serum , w hich is t he p rimary ex2 1 in , 72 3 in ; and ended by t he extenuatio n plo re of t he feasibilit y of it s clinical applicatio n in early di2 fo r 5 min at 72 . Af ter identified by agaro se gel elec2 agno stics fo r IDDM . Ξ Accepted date :20022 042 17 . HUAN G He ,bo rn in 1971 ,female ,docto rate st udent ,lect urer . 2PA GE. Af ter elect rop ho resis , stain analyzed by using SDSt rop ho resis , t he PCR p ro duct s were filled in recessed 3′2 wit h Coo massie Blue and decolo r , t he p rotein exp ressio n ends by Klenow f ragment of DNA polymerase ?. U sing TM Advantage PCR2Pure Bind Kit , t he differential amplifi2 yield was measured by screening light2densit y and co mpar2 catio n f ragment was reclaimed. Acco rding to t he met ho ds ing wit h molecular weight standard. Ot herwise , measure described in Ref . 2 and mo dified b y t he aut ho r app recia2 t he t ransfer distance of exp ressio n p roteins and calculate bly , t his f ragment was clo ned into Plasmid p SPO R T 1 t he related t ransfer f requence and molecular weight w hich was first subjected to digestio n wit h rest rictio n en2 ( ) M W. αzyme Sma ?. Then 2co mplementatio n was adop ted to i21 . 2 . 4 Serological detectio n of t he reco mbinant ICA69 dentif y t he po sitive bacterial colo ny w hich had been no mi2 p rotein )1 nated as p S2ICA69 subsequently. Af ter f urt her sub2 Identificatio n of antigencit y of t he reco mbinant clo ning , nucleotide sequence of clo ning ICA69 cDNA was ICA69 p rotein : The wells were coated wit h lysed cell pel2 identified. let′s super natant in carbo nate buffer , p H 9 . 6 over night at 2 . Af ter washing and blocking , add app rop riately di4 1 . 2 . 2 Co nst ructio n of reco mbinant GS T2ICA69 f usio n luted sera of diabetic patient s and healt hy subject s , and in2 p rotein and it s induced exp ressio n in E . col i μL . Wash t he plates , add 100 cubate fo r 1 h at 37 Af ter t he ro utine operatio n we have mentio ned above , ( t he ICA69 gene f ragment o btained f ro m p S2ICA69 was in2 seco nd antibo dy —goat anti2human Ig G2HR P ho rseradish ) () , add pero xidaseantibo dies 1200?0, incubate at 37 serted into p GEX22 T plasmid exp ressio n vecto rs wit h cer2 an enzyme2specific subst rate to develop colo r and read t he tain o rientatio n . The co nst ructed reco mbinant exp ressio n plate spect rop hoto met rically at 450 nm. vecto r co ntaining t he inserted gene in p roper o rientatio n ) 2 Detectio n of anti2ICA69 antibo dies p resented in was t ransferred to Esche richi a Col i BL 21 . Pick up single DM patient s′sera : Set ting up a blank co nt rol , detect t he colo nies of t he co nt rol st rain and t he po sitive t ransfo r mant antibo dies against ICA69 in 100 healt hy subject s and 30 ( medium Ap f ro m t he plates , inoculate into LB/ Ap 50 t ype ? diabetic sera by t he use of indirect EL ISA . μ)g/ mL respectively. Grow over night at 37 wit h shaking. By t he dilutio n rate of 1 ?10 , inoculate t he over night cult ure into f resh LB/ Ap medium. Transfer t he 2 Results and discussion cult ure to 37 and incubate wit h shaking to an OD 600 of 0 . 4 —0 . 6 . Divide t he po sitive t ransfo r mant s into t wo 2 . 1 Construct ion and analysis of recombinant gro up s : t he induced gro up was added IP T G wit h final expression vector GST2ICA69 2induced gro up was mol/ L and unco ncent ratio n of 1 Af ter f ull2lengt h sequence analysis , p S2ICA69 was re2 not added , t hen co ntinue to grow . At time t = 1 h , take a st rictio n digested by Sal ? and filled in by Klenow f rag2 small aliquot and place into t he microcent rif uge t ube la2 ment of DNA polymerase ?. Then DNA f ragment ICA69 beled ”t = 1 ”. Cent rif uge t he cells , decant t he super2 was o btained by cleaving t he above linearized p S2ICA69 wit h Eco R ?, w hich was linked wit h linearized vecto r t natant , and f reeze t he cell pellet at - 20 . Repeat at p GEX22 T/ Sma I + Eco R I in successio n . The co nst ructed = 2 , 3 , 4 , 5 and 6 h . Then t he cell pellet s can be ana2 reco mbinant exp ressio n vecto r was no minated as p 2 T2 ( lyzed by SDS2PA GE so dium do decyl sulf ate polyacry2 ICA69 . Screening of po sitive t ransfo r mant s by rest rictio n ) lamide gel elect rop ho resisto o bserve t he difference be2 digestio n wit h Eco R V , Pst ?, Bgl ?, Eco R ?and N he t ween t ho se t ubes and figure o ut t he op timum co nditio n . ? can app rove t he co nst ructed reco mbinant exp ressio n 1 . 2 . 3 SDS2PA GE analysis of t he exp ressio n p ro duct s vecto r p 2 T2ICA69 was 6 . 4 kb , co nsistent wit h anticipa2 Wash t he above cell pellet s wit h cold PB S solutio ns tio n , and co ntaining t he inserted gene in p roper o rienta2 t wice . Af ter t he cent rif ugatio n , t he depo sitio n experienced tio n. N ucleotide sequence of t he po sitive interf ace bet ween 5 rapid f reeze2t haw cycles at - 20 . Resuspend each t he vecto r and insert was identified to co nfir m t hat t he in2 sample in cold PB S buffer and keep o n ice . Af ter t he so ni2 ( ) sert was not o ut of f rame not show n. ( ) catio n and cent rif ugalizatio n 10 000 g , 5 min, decant Vecto r p GEX22 T offer a p ro moter fo r chemically in2 t he super natant s and mix wit h equal volume of 2 ×SDS2 ducible , high2level exp ressio n and t he dow nst ream gene is PA GE loading buffer , it was called t he induced super2 ( ) Glutat hio ne S2t ransferase GS T . There is a t hro mbin natant p rotein sample ; mix t he po st2so nicate pellet s w hich p rotease recognitio n sites fo r cleaving t he desired p rotein was not cent rif uged wit h equal volume of 2 ×SDS2PA GE f ro m t he f usio n p ro duct bet ween GS T and t he multiple loading buffer , it was called t he induced mix p rotein sam2 ( ) clo ning sites MCS . When ICA69 gene f ragment is ple . Thus , t he solubilit y of t he exp ressio n p ro duct s was TRANSACTIONS O F TIANJ IN UNIVERSITY Vol . 8 No . 4 2002 clo ned into MCS wit h t he p roper f rame , t he f usio n p rotein of GS T and ICA69 can be exp ressed by t he inducement of IP T G and cleaved into GS T and ICA69 by t he use of t hro mbin , it is very co nvenient fo r p urificatio n . 2 . 2 Recombinant GST2ICA69 f usion protein′s induced expression in E. col i Transfo r m p GEX22 T and p 2 T2ICA69 into t he st rain BL 21 respectively. The result of SDS2PA GE displayed t hat IP T G2induced p 2 T2ICA69 had an expectant distinct p rotein band wit h 98 kD co mparing wit h negative co nt rol p GEX22 T , but t he lat ter had a mo re abundant p rotein L ane 1 —st rain E. coli D H5a/ p2 T ICA69 ; L ane 2 —st rain E. coli band at t he po sitio n of 29 kD . There was no specific band D H10/ p2 T ICA69 ; L ane 3 —t he mixt ure solutio n of st rain E. coli BL 21/ p2 T ICA69 induced by 1 mmol/ L IP T G ; L ane 4 —t he supernate at t he M W of 98 kD in un2induced p 2 T2ICA69 and nega2 of st rain E. coli BL 21/ p2 T ICA69 induced by 1 mmol/ L IP T G ; L ane tive co nt rol p GEX22 T , no r mo re change at t he po sitio n of 5 —t he mixt ure solutio n of st rain E. coli BL 21/ p2 T ICA69 induced by ( ) 29 kD not show n. 0 . 1 mmol/ L IP T G ; L ane 6 —t he supernate of st rain E. coli BL 21/ p GEX system can exp ress fo reign pep tides in E . col i p2 T ICA69 induced by 0 . 1 mmol/ L IP T G ; L ane 7 —st rain E. coli BL 21/ p GEX22 T induced by 1 mmol/ L IP T G ; L ane 8 —molecular mass as f usio n p roteins by inductio n of IP T G , w hile t he mar kers , f ro m top to bot to m : 94 000Da , 67 000Da , 43 000Da , 30 parental p GEX vecto r is enco ding GS T gene and GS T oc2 000Da , 20 100Da . curs as a 29 kD pep tide , but here t he f usio n p rotein of re2 0 /0 2ICA69 GSTFig. 1 8SDS2PAGE analysis of f usion protein co mbinant p 2 T2ICA69 had an distinct p rotein band wit h )( The arrows indicate t he p utative f usio n p roteins 98 kD . Measure and calculate t he related t ransfer f re2 quence of t ho se p rotein bands acco rding to Weber′s fo r mu2 There are t wo impo rtant p ro blems in t he exp ressio n la. Finally co mparing wit h molecular weight standard , of f usio n p roteins. One is t he solubilit y of t he exp ressed wo r k o ut t hat t he total p rotein M W is 100 . 2 kD and t he p rotein . It is well2accep ted t hat t he fo r matio ns of t he co r2 M W of ICA69 is 72 kD . Because t he allowable range of rect tertiary st ruct ure of many p roteins can not be finished 0 /0 detective erro r in M W measurement by SDS2PA GE is 8, spo ntaneo usly in o ne step , t hey are gradually fo r med by t he result is co nsistent wit h t he references. 3 using a set of p roteins called chapero nes . Because t he 2 . 3 SDS2PA GE ident if icat ion and induc ing dyna mics yield of f usio n p rotein may be adversely affected by t he analysis of the expression product ICA69 p rocess of denat uralizatio n/ refold , and so metimes t he re2 The yields of f usio n p rotein at different time point s fold can not carry t hro ugh p roperly , Jo hnso n′s met ho d of were analyzed fo r op timizing exp ressio n co nditio ns. The 4 f reeze/ t haw instead of denat urant was adop ted in t his result indicated t hat af ter reco mbinant plasmid t ransfo r2 st udy to imp rove reco mbinant p rotein amo unt in t he po st2 mant s were induced by IP T G , t he yield gradually in2 so nicated super nate . The result displayed t hat mo st part of creased f ro m 0 to 4 h and t ur ned to a platfo r m at 5 —6 h . f usio n p rotein GS T2ICA69 can be exp ressed as soluble p ro2 In t his platfo r m , t he SDS2PA GE of IP T G2induced p 2 T2 tein in E. col i . Anot her impo rtant aspect of f ushio n ex2 ICA69 had an expectant distinct p rotein band wit h 98 kD , p ressio n is t he stabilit y of t he exp ressed p rotein . GS T can so 5 h was regarded as t he op timum inducement ti me . Af2 avoid fo reign gene p ro duct being degraded by p rotease in ter elect rop ho resis gel of t he po st2so nicate pellet′s super2 any bacteria ; exp ressio n ho st lacking of certain int racellu2 nate induced by IP T G fo r just 5 h was stained wit h lar p rotease may also stabilize t he fo reign p rotein f ro m p ro2 Coo massie Blue , decolo red and screened light2densit y , t he 0 teolytic degradatio n . It had been co nfir med t hat t he differ2 /0 reco mbinant p rotein was show n to acco unt fo r 11 . 7of t he ence of E. col i st rains affect not o nly t he yield but also bacterial total p roteins. Ot herwise , cent rif uge t he bacteri2 t he secretio n amo unt of p roteins. p 2 T2ICA69 i n E. col i al so nicated solutio n wit h 10 000 g and investigate t he BL′21 p ro duced mo re f usio n p roteins t han t hat in E. col i super natant and mixt ure solutio n by SDS2PA GE respec2 D H5аand D H10 . tively. The result showed t he induced f usio n p rotein 2 . 4 Ident if icat ion of ant igenc ity of the recombinant ICA69 was mainly in t he super nate and implied it was a ICA69 protein ( ) soluble p rotein Fig. 1. Coating wit h t he exp ressed p ro duct s of t he parental p GEX22 T vecto r and p roteins of E. col i BL 21 , t he sera of — 233 — ) healt hy subject s and patient s had no distinctly colo red re2 22 T vecto r 2 GS T exp ressed by t he parental p GEXactio n ; t he result was similar to t he reactio ns bet ween ex2 have no cro ss2reactio n wit h human serum ; ) p ressed p ro duct s of p 2 T2ICA69 and t he co nt rol sera . But 3 Proteins f ro m blank E. col i BL 21 also have no special adso rp tio n wit h human serum ; w hen 5 samples of t he IDDM sera mixed wit h it , t he lat2 ) ter coating p rotein was o bserved remar kably tincto rial re2 4 The IDDM sera existed anti2ICA69 antibo dies w hich can specially react wit h t he reco mbinant exp ressio n actio n w hich had OD values higher t han general value of co nt rol gro up plus duple Sd. That included 3 patient s wit h p ro duct s ICA69 . age at o nset < 3 mo nt h and 2 patient s wit h age at o nset be2 At p resent , anti2ICA69 antibo dy is o ne of t he mo st ( ) t ween 3 mo nt h and 1 year Fig. 2. It can be recko ned impo rtant autoimmunological target s wit h high diagno stic 0/ ( ) significance . The aim of t his investigatio n is to exp ress re2 f ro m t hese data t hat abo ut 170 5/ 30of t he IDDM sera co mbinant ICA69 antigen in bacteria by using met ho ds of existed in anti2ICA69 antibo dies and mo re of t he Abs ap2 gene engineering and apply t he exp ressed ICA69 as au2 peared in t he p rimary p rocess of insulindependent diabetes mellit us. The result was co nsistent wit h t ho se f ro m Mar2 toantigen to establish an EL ISA test t hat can detect t he 5 tin. anti2ICA69 antibo dies in t ype ? diabetes mellit us. The reco mbinant ICA69 will help understanding t he molecular pat hogenesis of IDDM and p roviding credible gro und fo r early joint diagno ses and screen of IDDM . Meanw hile , t he p resent undertaking make it po ssible to get o ur handle o n p reventio n and cure of IDDM wit h new immunological t herapeutics. Ref erences 1 Piet ropaolo M , Castano L , Babu S et al . Islet cell autoantigen 69 kD ( ) ICA69: Molecular clo ning and characterizatio n of a novel diabetes2as2 sociated autoantigen J . J Clin Invest , 1993 , 92 :359 . —t he antibo dies against ICA69 in 100 healt hy subject s ; Gro up 1 2 Sambroo k J , Frit sh EF , Maniatis J et al . Molecular Clo ning : A L abo2 Gro up 2 —t he antibo dies against ICA69 in 30 t ype ? diabetic sera . rato ry Manual M . 2nd Ed. New Yo r k : Cold S p ring Harbo r L abo rato2 The beeline indicates general value of co nt rol gro up plus duple Sd. ry Press , 1989 . Fig. 2 Indirect EL ISA detection of the anti2ICA69 3 Hockney R C. Recent develop ment s in heterologo us p rotein p ro ductio n ( ) in Escherichi a coli J . Trends in Biotech , 1994 , 12 11: 456 —463 . antibodies 4 Jo hnso n B H. Reco mbinant p roteins can be isolated f ro m E. Coli . Concl usions 3 cells by repeated cycles of f reezing and t hawing J . Bio/ Technolo gy , ( ) 1994 ,12 13:1357 . 5 Matin S , Kardo rf J , Schulte B et al . Autoantibo dies to t he islet anti2 ) 1Only t he IDDM sera mixed wit h t he f usio n p rotein gen ICA69 occur in IDDM and in r heumatoid art hritis J . Diabetolo 2 was o bserved remar kably tincto rial reactio n , t hus t he anti2 gia , 1995 , 38 :35 . gencit y can be identified ; ICA69 基因工程融合抗原的制备 1 1 2 2黄鹤,甘一如,黄乃萍,张镜宇 ()1 . 天津大学化工学院 ,天津 300072 ; 2 . 天津医科大学内分泌研究所 ,天津 300072 摘 要 :基于基因工程技术 ,用 PCR 法扩增出编码 ICA69 的 cDNA 片段 ,直接克隆到 p SPOR T 1 质粒上 ,经 DNA 序列测 定 ,插入到 GS T 融合蛋白表达载体 p GEX22 T ,构成重组质粒 p2 T2ICA69 ,得到的表达产物 GS T2ICA69 融合蛋白用间接 EL ISA 法检测其免疫原性 . 测序结果表明 ,所获 PCR 产物已正确重组到 P GEX22 T 表达型质粒中 . 重组质粒在原核细胞 中表达的融合蛋白具有免疫原性 ,并能应用于 ?型糖尿病病人血清中抗 ICA69 抗体的检测 . 所获得的表达产物为重组 ICA69 融合抗原 ,有助于提高 ?型糖尿病的预报率和确诊率 . 关键词 :胰岛细胞自身抗原 ;69 kD 蛋白 ; GS T 融合蛋白 ; ?型糖尿病 ;酶联免疫吸附试验 () 982 2002042 2312 4 中图分类号 : Q 786 文献标识码 :A 文章编号 :10062