Cn b HDKH: TS. o Xun tn Hc vin: o th ngc anh K11- Sinh hc thc nghim

1.1 Cu trc phn t insulin Insulin l mt hoocmon protein do t bo tuyn ty sinh ra. Phn t protein insulin c khi lng khong 6000da. Cu to bi hai chui polypeptit A v B. Cc chui A v B lin kt vi nhau bng hai cu ni isulfua. Hu ht cc loi chui A gm 21aa; chui B c 30aa.

Hnh 1: Cu trc phn t insulin

1.2 Vai tr sinh hc ca insulinInsulin l mt trong nhng hoocmon iu ho nng glucoz trong mu. 1.2.1. Insulin v trao i hydratcacbon -Insulin tun hon trong mu, khi gn vi th th trn mng t bo to ra phc hp th th - insulin lm pht tn hiu truyn thng tin chuyn glucoz ra khi huyt tng.

Hnh 2:Insulin gn vi th th trn mng t bo v tun hon trong mu

- Insulin thc y gan d tr glucoz dng glycogen. Glucoz b photphoryl ho nh enzym hexokinase nh glucoz b by vo trong t bo. - Insulin c ch hot ng ca glucoz - 6 photphatase. Khi khng c mt insulin, qu trnh tng hp glycogen b dng li v cc enzym phn hu glycogen hot ng.

Insulin ngn cn lng ng qu cao trong mu nn khng c c qu nhiu insulin, do enzym insulinase kim sot mc insulin. Khi lng insulin qu cao enzym insulinase s phn hu hoocmon ny vi thi gian 6 pht. Qu trnh ny m bo cho hm lng insulin c n nh trong mu v lng glucoz khng b gim xung mc nguy him.

Hnh 3: insulin iu ho glucoz trong mu

1.2.2. Insulin v trao i lipita- Insulin thc y sinh tng hp cc axit bo trong gan. Khi lng glycogen trong gan qu cao (> 5% lng th ca gan) th qu trnh tng hp b c ch. Lng glucoz hp th vo t bo gan chuyn sang tng hp axit bo v chuyn khi gan dng lipoprotein. Cc lipoprotein i vo vng tun hon, cung cp cc axit bo t do cho cc m khc nh m m tng hp triglyxerit.

b- Insulin c ch phn hu cht bo trong m m: bng cch c ch qu trnh thu phn triglyxerit thnh glixerol v axit bo t do. Enzym nhy cm vi hoocmon ny tr nn hot ng c khi photphoryl ho. Insulin lm ngn cn s kt hp vi th th trn mng t bo. Do lm tng s tch t triglyxerit trong t bo cc m m.

1.2.3. Nhng tc ng khc ca insulin

- Insulin lm tng tnh hp th ca cc axitamin. - Insulin lm tng tnh thm cc ion kali, magie v photphat v c to iu kin cho qu trnh photphoryl ho v s dng glucoz.

Hnh 4: Insulin lm tng tnh thm cc ion kali, magie v photphat v c

1.3. Tng hp insulin trong c th Insulin c tng hp trong t bo ca tuyn ty. Insulin c to ra t mt phn ca mt protein ln hn m bo s gp np ng. mARN dch m protein preproinsulin. Preproinsulin gm mt trnh t u aa tin cht ny i qua li ni cht tham gia vo qu trnh sau dch m. Ti qu trnh sau dch m preproinsulin b thu phn to thnh dng proinsulin. Sau khi hnh thnh 3 cu isulfua mt s peptitdaza ct proinsulin thnh insulin hon chnh v hot ng. Insulin c ng gi v cha trong cc ht tit tch t trong t bo cht n khi c kch hot gii phng.

Hnh 5: Tng hp insulin trong c th

Hnh

6: Cc bc ci bin sau dch m ca phn t insulin

Hin nay cng ngh ADN ti t hp c s dng sn xut.

2.1. Phng php 1 To ra cc chui ring bit, kt hp ho hc hoc to mt tincht chui n proinsulin ngi, sau phn ct to thnh insulin hon chnh.

Bc 1: Bng k thut tch gen s dng trong sinh hc phn t tch c gen m ho proinsulin ngi trn nhim sc th s 11. Tch mARN ca gen tng hp proinsulin t mu nghin tu ca ngi. Dng phn ng RT-PCR vi mi c hiu khuch i gen. Do hu ht cc mARN ca ngi u c ui polyA nn s dng chui polyT bt cp vi ui polyA . S dng sc k i lc vi polyT gi li mARN cn thit cho qu trnh dch m; cn li loi b ADN v cc ARN khc. Ct b cu ni A - T thu c mARN.

Hnh 7: Tch mARN ca gen tng hp proinsulin t mu nghin tu ca ngi

Bc 2: Tch v thit k plasmit ti t hp.- Ct gen m ho proinsulin v plasmit bng mt loi enzym gii hn. Ni bng ADN ligase ca phageT4. -Thit k cc trnh t m ho cho cc protein tn hiu gip vn chuyn insulin ra ngoi t bo cht. +Nu s dng mARN tch c t trn tin hnh nh sau: - Sao mARN tinh khit thnh cADN nh enzym phin m ngc v nh cc dNTP (trong phn ng RT - PCR) - Ci on cADN m ho insulin hon chnh vo plasmit ng sau mt promoter mnh. Bin np vector ti t hp vo vi khun E.coli.

Bc 3: Bin np plasmit ti t hp vo E.coli nh phng php trnvi dung dch ion Ca hoc to l xung in. Sau qu trnh bin np, cn chn lc c nhng dng vi khun mang gen mong mun.

Hnh 8: Tch v bin np plasmit ti t hp vo E.coli

Bc 4: Cc vi khun chuyn gen sau c a vo ln men.Nui chng trong ni ln men s dng phng php nui cy lin tc, cc cht dinh dng c b xung thng xuyn m bo s tng trng ca vi khun theo hm s m. Sau 20 pht c hng triu vi khun c nhn ln qua nguyn phn. Ch sau mt thi gian ngn, sinh khi tng ln rt nhanh v gen insulin c tng hp.

Hnh 9: a cc vi khun chuyn gen vo ni ln men.

Bc 5: Tin tinh sch Sau khi ln men cn tch t bo v kh trng nhit. Dng enzym lizozyme ph v mng t bo, sau dng hn hp cht ty ra tch lp mng lipit. Bng phng php ly tm v lc tch c proinsulin.

Hnh 10: Proinsulin c lc tch

Bc 6: Hot ho. Do h thng E.coli c kh nng biu hin gen insulin nhng khng c kh nng hot ho insulin. Hot ho proinsulin invitro bng cch x l dung dch m, gip n t cu trc bc 4, sau dng enzym c hiu trypsin phn ct proinsulin. Khi sn phm thu c mi c hot tnh cn thit.

Hnh 11:Hot ho proinsulin thnh insulin hon chnh

Bc 7: Hn hp tinh sch ch cn c insulin. Bng phng php sc k, tch v phng php min dch gn enzym. tinh sch ca insulin c nh gi qua mi giai on trung gian ca qu trnh sn xut nh phng th nghim chuyn ho. Cui cng insulin c tinh th ho.Hnh 12: Cc tinh th insulin

2.2. Phng php 2 - Tng hp ring r hai chui A v B. - Xc nh trnh t ADN sn xut chui A v B. Qua tng hp v tch hai dng gen ny. - Mi ADN c chn vo plasmit. Sau sn xut tng t nh sn xut proinsulin. - Cui cng hai chui A v B c trn vi nhau v hnh thnh cu ni isulfua qua phn ng ti oxi ho kh nh mt cht oxi ho nht nh. Ngy nay, nm men cng c s dng sn xut insulin. C nhiu u im: t bo nm men to phn t insulin ngi gn nh hon chnh vi cu trc khng gian hon ho. iu lm gim ti a tnh phc tp v gi thnh ca cc giai on tinh sch.

Hnh 13: Sn xut insulin bng cch tng hp ring r hai chui A v B.