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Kỹ Thuật Di Truyền Dna Tái Tổ Hợp
Kỹ Thuật Di Truyền Dna Tái Tổ Hợp
Hnh 6.1
6.2.2 Cc vector.
Thng thng mt phn t DNA ti t hp bao gm mt phn t DNA c bn cht l
plasmid hoc phage nguyn vn gi l vector (th ti) v mt on DNA t ngun khc
mang mt gene hoc yu t iu ha mong mun c cho xen vo; n c gi l DNA
ngoi lai ( foreign DNA).
DNA ti t hp (recombinant DNA) l phn t DNA c to ra trong ng nghim bng
cch kt hp cc DNA t cc ngun (loi) khc nhau, theo mt quy trnh k thut nht
nh, gi l k thut ti t hp DNA. Thng thng mt phn tDNA ti t hp bao gm
mt phn t DNA c bn cht l plasmidhoc phage nguyn vn gi l vector (th ti) v
mt on DNA t ngun khc mang mt gene hoc yu t iu ha mong mun c cho
xen vo; n c gi l DNA ngoi lai (foreign DNA).
Vector l phn t DNA c kch thc b hoc va phi, ng vai tr l vt trung gian
mang truyn on DNA ngoi lai nghin cu vo trong t bo th nhn (t bo kh bin)
bng con ng bin np (transformation) hoc ti np (transduction).
C ba loa vector l cc plasmid, cc phage hoc cc nhim sc th nhn to.
Trong s cc phage dng lm vector th phage lambda () c nhiu u th nht, bi l
phn gia ca b gene c cha mt s gene khng quan trng v khng lin quan vi s
ti bn ca n, nn thun li cho vic xen on DNA mong mun vo y. Cc phage
khng cha cc gene khng thuc cho nn vic theo di phage ti thp c xc nh
da vo cc vt tan dng tnh (positive plaques) trn nn vi khun.
Cc plasmid ca vi khun tn ti c lp vi nhim sc th trong t bo vi khun. Chng
c s dng rng ri hn c, nh c cc c im sau: (i) Mi plasmid l mt phn t
DNA si kp thng dng vng v chc mt khi im ti bn ring; (ii) C kh nng
xm nhp vo t bo vt ch v hot ng bnh thng; (iii) C kch thc b, thng ch
vi ngn cp base nn ddng tinh chit; (iv) S bn sao trong mi t bo vi khun thng
kh cao; (v) Mt splasmid c cha cc gene khng thuc tin li cho vic theo di v
pht hin sc mt ca plasmid ti thp trong vi khun ch.
Ni chung cc plasmid c ti bn bi cng mt b my ti bn dng cho nhim sc th
vi khun. Mt s plasmid c sao chp vi cng t l nh nhim sc th vi khun, v vy
trong mt t bo ch c mt bn sao ca plasmid. Cc plasmid c sao chp c lp vi
nhim sc th mt t l cao, v vy mt t bo c th c nhiu hn 50 bn sao mi loi.
Cc gene trn cc plasmid c mt nhiu ln thng thng c biu hin mc cao. V
bn cht, cc gene ny thng m ho cho cc protein (v d nh cc enzyme) bo vvi
khun khi btc ng ca mt hoc nhiu cht khng sinh. V nh cp chng 5,
Hnh 6.2
Hnh 6.3
Cc plasmid cc vi sinh vt eukaryote: Nh bit, cc plasmid khng ch gii
hn cc vi khn. Chng hn, mt s plasmid c nghin cu rng ri nm
men v c pht trin thnh cc vector to dng nm men. Cc plasmid ny cng
c s dng nh l mt "h thng n gin" tm hiu c ch v s iu ho
ti bn DNA cc t bo eukaryote
Hnh 6.4
(1) Tch chit v tinh sch DNA thuc cc ngun khc nhau (gm vector v DNA
mang gene mong mun)
(2) To ra phn t DNA ti t hp in vitro.
(3) a phn t DNA ti t hp vo trong t bo nhn, thng l E. coli hoc nm
men.
(4) Pht hin v phn lp cc dng vi khun DNA ti t hp c hiu.
Hnh 6.6
Trong t bo ch, phn t DNA ti t hp c th biu hin gene mong mun (cho sn
phm protein) hoc ti bn c lp nhiu ln to ra hng lot bn sao ca n, v khi t
bo ch phn chia s ko theo hin tng to dng phn t (molecular cloning). Mt
khc, do tc phn chia rt nhanh ca cc vi khun nn c th to hng triu bn sao
mong mun. V th nh khoa hc c th tch dng bt k mt gene no dng cho
nghin cu hoc cho sn xut trn quy m cng nghip mt s lng ln cc protein vn
l nhng ch phm y-sinh hc no .
6.5 ng dng cng ngh ADN ti t hp vi y dc hc:
n dng to nn mt nhnh pht trin mau l nht ca cng nghsinh hc, v cng
vi vic sn xut cc vaccine chng tr thnh nhng phng tin quan trng nht trong
chnh sch y t cng ng ca cc nc ang pht trin v xt v lu di, vic ng dng
cc khng th n dng c trin vng cha c nhiu bnh trong c cc khi u c
tnh.
*Mt s sn phm c sn xut thng qua cc vi khun cha cc gene ngi c to
dng:
Cc interferon: iu tr cc bnh ly nhim virus v mt s bnh ung th, vim
gan mn tnh.
Interleukin 2: Kch thch h thng min dch v c thdng trong iu tr ung th
v cc ri lon h thng min dch
Insulin: iu tr bnh tiu ng
Hormone sinh trng ca ngi, c s dng iu tr nhng tr em b bnh
ln hoc bnh gi trc tui.
Hot t plasminogen ca m (tPA), mt enzyme tham gia vo qu trnh lm tan cc
cc mu ng (huyt khi)
Somatotropin (GH): iu tr d tt ln thuc v tuyn yn
Cht kch hot plasminogen: Lm tan cc cc ng mu cho iu tr v ngn chn
cc tnh trng tc nghn tim mch
Nhn t hoi t khi u: Tn cng v git cc khi u ung th
Cc nhn tXI v VIII: iu tr bnh mu kh ng
Erythropoietin: Kch thch to cc t bo hng cu cho iu tr bnh thiu mu
(anemia)
Beta endorphin: Cc cht gim au t nhin do c thto ra; c th c dng lm
gim au
Cc enzyme: c dng rng ri t vic iu khin cc phn ng ho hc trong
cc qu trnh k ngh cho n vic b sung cc enzyme trong khu phn n ca
ngi
Cc vaccine tiu n v (ti t hp): Kch thch kh nng min dch ca c th i
vi mt hoc hai khng nguyn then cht ca mt tc nhn gy bnh; gim nguy
c ri ro ca cc vaccine thng thng. Cc vaccine ti t hp cho bnh vim gan,
v bnh vim mng no do Hemophilus influenza B gy ra.Octolon, mt cht c
ch min dch dng cho cc bnh nhn cy ghp ni tng
Hnh 6.8. Khoa hc M Th nghim cy ghp thnh cng gene ngi trn
nm men da vo k thut DNA ti t hp, h tr kim tra cc phng php
iu tr lin quan n t bin gen
Hnh 6.9