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Tng hp insulin ngi t miniproinsulin thng qua vic chuyn gene E. coli
M u
Cng vi s pht trin ca x hi hin i, y hc nh ang chy ua vi v vn vn nan gii trong c nhiu bnh kh cha. in hnh phi k n l bnh tiu ng. Theo t chc i tho ng th gii, c mi 24h, trn th gii c: 3.600 trng hp mi c chn on, 580 ngi b t vong lin quan n bin chng i tho ng, 225 ngi b ct on chi, 120 ngi suy thn giai on cui v 55 ngi b m lo (Nguyn T. L., 2009). Bnh tiu ng c bit nguy him hn so vi nhiu cn bnh khc bi bnh nhn bi khi mc phi bnh tiu ng bnh nhn c th phi i mt vi hng lot bin chng khn lng khc v gy gnh nng cho x hi. Do tm ra phng php hu hiu iu tr tiu ng l v cng quan trng i vi ton th nhn loi. Trong gii hn ca ti, chng em xin gii thiu mt trong nhng phng php l Tng hp insulin ngi t mini-proinsulin thng qua vic chuyn gene E. coli.

Ni dung
Insulin l mt hormone c tit ra bi t bo trong o Langerhans ca tuyn ty (Trnh H. H. v C. H., 2007). Insulin ngi l mt polypeptide bao g m mt chui vi 30 acid amin, c mt c u ni disulfur trong chui vi 21 acid amin v mt chui v v 2 c u ni disufur ni gia

B. Chc nng chnh l lm gim hm lng glucose mu qua cc qu trnh bin dng s dng glucose trong mu (Nguyn P. N. v cng s, 2007). Insulin c hnh thnh t preproinsulin, r i proinsulin. Cc enzyme chuyn ct chui polypeptide C ca proinsulin to ra insulin (Trnh H. H. v C. H., 2007).

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Hnh 1. Cu trc insulin hon chnh vi 2 chui polypeptide A v B (Vajo Z. et al., 2001) Chuyn gene c bn thng c 6 bc: (1) c lp gene; (2) xy dng gene c n chuyn v vector; (3) chuyn gene; (4) nhn dng; (5) chn lc; (6) khng nh. (Phm T. H., 2010) Ti bc u tin, c lp v tch gene m ha proinsulin ngi (1). Sau dng phn ng PCR khuch i gene bng cch dng 5 on DNA overlapping, mi on c di 65-68 bases theo nh mu. bc xy dng gene c n chuyn v plasmid (2), chng E. coli c s dng lm t bo ch trong nghin cu ny l chng BL21 (DE3) (F- ompT rB- mB-), c nhim sc th mang gene T7 RNA polymerase (T7 RNAP) ngy cng c s dng rng ri biu hin cc protein trong chng ch l vi khun. Trong h thng ny, gen mc tiu c to dng (clone) downstream ca bacteriophage T7 promoter vi s lng bn sao plasmid trung bnh. i v cc gen mc tiu khng c s dng polymerase ca chng ch. Chu s kim sot ca promoter lacUV5 (mt dn xut ca lac promoter). T7 RN P biu hin trong chng ch khi promoter lacUV5 c cm ng bi cht cm ng isopropyl--D-thiogalactopyranoside (IPTG) (Nguyn H. T., 2009). Mini-proinsulin (M2PI) c vng trung tm peptide-C thay th bi mt chui c dng nghch o. Peptide-C c vai tr trong qu trnh cun xon v hot ng ca proinsulin. Quy trnh to plasmid c din t theo cc s di y (Chang, et al. 1998):

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Di truyn v K thut Di truyn Plasmid pT2 DNA m ha 8 -12 aa TNF- v His10 c tng hp ha hc Ct bng NdeI v BamHI Chn vo downstream T7 promoter Biu hin trong pET-3a Ct bi endonuclease Thu pT2

Plasmid pT2-hPI Gene m ha cho proinsulin ngi PCR ( 5 DNA overlapping, mi on 65-68 base, nh mu) Ct bng BamHI v HindIII Chn vo pT2 pT2-hPI

Plasmid pT2M2PI Gene m ha M2PI em PCR (gene proinsulin ngi lm mu DNA) Ct bng BamHI v HindIII Chn vo pT2 pT2M2PI

GVHD: ThS. Trn Hng Bo Quyn

Di truyn v K thut Di truyn Nu theo ng l thuyt, bc chuyn gene (3) s c tin hnh bng cc phng php nh sng bn gene, shock nhit, shock in, vi tim Tuy nhin, bi bo khng cp ti vn ny.V vy, bc chuyn gene c l ng vo bc xy dng gene. Sau khi chuyn gene, cc tc gi tin hnh bc chn lc nhng gene c th biu hin ra ngoi (4). Theo , biu hin plasmid pT2M2PI (hnh 2) c s dng bin i E. coli BL21 (DE3) t bo cha nhim sc th sao chp t bacteriophage T7 RNA polymerase. T bo E. coli ti t hp nui trong mi trng Luria-Bertani (1% (w/v) peptone; 0,5% (w/v) cao nm men; 1% (w/v) NaCl; 0.1% NaOH (v/v)) duy tr 200mg/l ampicillin (nhng t bo E. coli khng chuyn gene c s cht trn mi trng c ampicilline - cht khng sinh). Sau khi cm ng T7 promoter bng IPTG, thu t bo sau 10h. Ph v t bo bng sng siu m. Protein dung hp c phn tch SDS/PAGE. Mc biu hin ca protein dung hp, T2M2PI khong 20-25% tng s protein trong t bo.

Hnh 2. S biu hin ca mini-proinsulin trong plasmid pT2M2PI Plasmid c kch thc 4.366bp, mt v tr ori (khi u sao chp), mt gene khng ampicillin, promoter T7, M2PI l gene m ha mini-proinsulin (sau khi chuyn vo plasmid T2, plasmid mi thu c l pT2M2PI), c ba RE l Nde I, BamHI, HindIII. T2 cha 21 acid amin, mt Methionine m u, 5 peptide TNF N-terminal, 10 Histidine v tr bt buc, 5 amino acid m (spacer). CNBr tch T2 v M2PI ngay v tr nh hnh minh ha. (Chang, et al. 1998)

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Di truyn v K thut Di truyn V mc ch chnh ca bi bo l sn xut mini-proinsulin, nn cc tc gi khng cp ti vn nhn dng trong bi bo ny. Chng ta cng c th ng m hiu vic nhn dng nhng t bo E. coli c gene biu hin c tin hnh (5). Theo , dng t bo E. coli c nui cy c em i ly tm tc thp v thu cn t bo chun b cho bc tip theo l tinh sch v khng nh (6). Sau khi thu c cn, mini-proinsulin c tinh sch bng ly tm, S-Sapharose sc k trao i ion dng (Pharmacia iotechnology), tinh sch v khi lng phn t ca mini-proinsulin c xc nh bng tricine SDS/PAGE. Sau khi tinh sch, phn ng xon cun c thc hin vi mc ch protein c th biu hin cc chc nng ca n cu trc bc 3 hay bc 4. V vy, chui mini-proinsulin sau khi c tch ra t bc tinh sch trn vn dng mch thng (do s dng E. coli mt t bo prokaryote sn xut mini-proinsulin s khng c giai on hu dch m hnh thnh cu trc khng gian ca protein). Phn ng cun xon xy ra nh sau: 50mM glycine buffer Mini-proinsulin tinh sch (50M, 0.375mg/l) (bng 2 lng mini-proinsulin) (18h, 4oC) H3PO4 n pH 2.5 Dng phn ng Tinh sch (Reverse-phase HPLC) So snh peak vi mini-proinsulin xon cun vi insulin 78% S-sulphonated mini-proinsulin c chuyn hon ton thnh dng xon cun cao hn proinsulin (57%) cng n ng protein 50M, pH 11.5. Kt qu chng minh trnh t ngn ( mini-proinsulin) c hiu qu trong qu trnh xon cun hn C-peptide. 2-mercaptoethanol Phn ng cun xon

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Di truyn v K thut Di truyn Thng thng chuyn gene c 2 mc ch: Chuyn gene n thu n v chuyn gene biu hin ca gene thnh tnh trng. Nghin cu ny c nhm mc ch th 2. V vy, vic khng nh c th c xem l vn then cht trong nghin cu ny. Cc tc gi thc hin qua hai bc (1) Phn tch fingerprint (du n di truyn); (2) Kho nghim th th c hiu. Sau , vic phn tch fingerprint xc nh trnh t ca phn t insulin c tin hnh theo trnh t nh sau:

Mini-proinsulin xon cun Gradient n ng 20-30% CH3CN trong (TFA) 0.1% Np vo ct Vydac C8 HPLC Tinh sch (60mins) Thu h i ct chnh Trysin v carboxypeptidase B x l Sy kh (nhit thp) HCl pH n 2.5 Dng phn ng Np vo ct Vydac C18 HPLC Theo gradient n ng 20-50% CH3CN Tch ra (30mins) Thu h i ct chnh Sy kh (nhit thp)

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Di truyn v K thut Di truyn Glu-C endoproteinase (Dng Staphylococcus aureus V8 protease) Phn tch fingerprint Khng nh hnh thnh lin kt disulfite Thu insulin (0.2mg/ml) 0.2 M Tris/HCl (pH 7.5) T l 1:10 (w/w) enzyme: c cht (25oC, 14h) Tch ra chn lc Phn tch MS v trnh t peptide N-terminal Phn tch bng MS l phng php ph khi lng phn tch tnh cht ca phn t, khi lng, so snh trnh t protein ti t hp vi protein gc, (Mann, et al. 2001). Theo bi bo ny d on khi lng phn t insulin ngi l 5807.8, th phn tch MS tm ra 5807.0. Thnh ph n amino acid v N-terminal ca protein tng hp nh gi tr d kin. Hot tnh sinh hc in vitro ca insulin, proinsulin v mini-proinsulin c xc nhn bi kho nghim th th hot ng c hiu. Hot tnh ca M2PI ca insulin l 48%, ngc li proinsulin nh du ch l 2% (s dng ng v phng x 125I th th c hiu). T bo lympho IM-9 cng c s dng trong kho nghim th th c hiu. Phng php tip cn mini-proinsulin c gng ni trnh t Arg-Arg-Gly-Ser-Lysrg, nhng tinh sch dng mini-proinsulin rt thp, hnh thnh c u ni disulphite ring l khng xc nh c. Hot tnh ca insulin tng khi chui khng c u N. Do t nhng kt qu trn thu c c n tm ra phng php tinh sch miniproisnsulin hiu qu hn, xc nh c u ni disulphite r rng v nghin cu su hn c th sn xut insulin i tr t mini-proinsulin.

Gradient n ng 20-50% CH3CN Tch ra khi ct Vydac C18 trong 0.1% TFA UV 215nm

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Kt lun
V E. coli l mt prokaryote nn vic tng hp insulin ngi s khng c giai on hu dch m, do vy insulin sn xut t E. coli phi hnh thnh cu trc khng gian nhn to c hot tnh. V th c th nghin cu sn xut insulin t eukaryote nh nm men chng hn. Hy vng tng lai s c phng php hiu qu, kinh t hn nhm biu hin hon ton insulin ngi v bnh nhn khng phi dng thuc sut i. Mc d khoa hc c nhng bc tin ln trong vic iu tr tiu ng, nhng hin nay vn cha c thuc c tr i vi bnh ny. V trong khi ch i n khi tm ra phng thuc k diu chn ng cn bnh th k th 2 ny th mi ngi c n dng thuc theo ch nh ca bc s, dinh dng hp l v th dc, nhm gp ph n chng chi hiu qu vi bnh tiu ng.

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Ti liu tham kho

[1] Chang Seung-Gu, Kim Dae-Young, Choi Ki-Doo, Shin Jae-Min, and Shin Hang-Cheol. Human insulin production from a novel mini-proinsulin which has high receptorbinding activity. Biochemical Journal, 1998: 631-635. [2] Zoltan Vajo, Janet Fawcett, and William C. Duckworth. Recombinant DN Technology in the Treatment of Diabetes: Insulin 22(5):706-717, 2001. [3] Barry S. Selinsky. Methods in Molecular Biology, vol 209: Membrane Protein Protocols Expression, Purification and Characterization. Human Press, 2011: 4. [4] Matthias Mann, Ronald C. Hendrickson and khilesh Pandey, nalysis of proteins and proteimics by mass spectrometry. Biochem 70: 437-73, 2001. [5] Nguyn Phc Nhun, Phan Th ng, L Th Phng H ng v cng s. Sinh ho hc Phn 1: Sinh ho hc tnh. NXB Nng nghip, 2007: 151-152. [6] Nguyn Th Lm. Dinh dng trong iu tr tiu ng. VNExpress, 2009. http://vnexpress.net/gl/suc-khoe/2009/08/3ba12925/ [7] Nguyn Hu Tr. C s phn t v vai tr v c ch iu ha bi cc yu t cis trong vector biu hin E. coli. B mn Sinh hc, H Nng lm Tp. HCM, 2009. http://www2.hcmuaf.edu.vn/contents.php?ids=3963&ur=nhtri [8] Phm Thnh H. Di truyn hc. NXB Gio dc, 2010: 384-405. [9] Trnh Hu Hng v Cng Hunh. Sinh l ngi v ng vt. Tp 7. NX HQG H Ni, 2007: 28-31. nalogs. Endocrine Reviews,

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