ng dng cng ngh vi sinh trong k thut di truyn.6.1 Gii thiu v k
thut di truyn (Cng ngh DNA ti t hp).Cc nghin cu ca di truyn vi sinh
vt v cc enzyme gii hn v plasmid cng nh vic s dng chng to ra cc phn
t DNA ti t hp u tin trong ng nghim (in vitro) trong nhng nm u ca
thp nin 1970 l c s cho s ra i ca cng ngh sinh hc hin i: k thut di
truyn (genetic engine ering) - cng ngh DNA ti t hp (recombinant DNA
technology). S ra i v pht trin nhanh chng ca lnh vc ny khng nhng a
li s hiu bit su sc v cu trc v cc c ch hot ng ca cc gene v b gene m
cn tr thnh lc lng sn xut trc tip ca x hi, gp phn gii quyt nhng vn
thc tin t ra trong y dc hc, nng nghip v mi trng. K thut di truyn
thc s m u cch mng Cng ngh sinh hc.Khi nim: DNA ti t hp (recombinant
DNA) l phn t DNA lai 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 cng ngh
ti t hp DNA. DNA ti t hp (DNA recombinant) l phn t DNA lai, to ra t
in vitro, bng cch t hp hai DNA t hai hay nhiu loi khc nhau.Cng ngh
DNA ti t hp (cn gi l k thut di truyn hay k thut gen) l mt b phn
quan trng v l cng ngh cha kha (key technology) ca lnh vc cng ngh
sinh hc. Cng ngh DNA ti t hp c hnh thnh nh s pht trin ca cc phng
php v k thut dng trong nghin cu cc qu trnh sinh hc mc phn t. T ,
cho php phn lp, phn tch v thao tc trn cc nucleic acid theo nhiu
phng thc khc nhau, gip hiu bit su sc cc lnh vc mi ca sinh hc nh cng
ngh sinh hc phn t. Nm 1972, nhm ca Paul Berg to nn phn t ADN ti t
hp in vitro u tin t ba ngun khc nhau: nguyn b gen virut SV 40 gy
ung th kh, mt phn b gen ca phage v cc gen ca operon lactoz ca E.
coli. Nm 1973, nhm Cohen, Helenski, Boyer ln u tin thu nhn c cc sn
phm c hot tnh tADN ti t hp E. coli (Gii Nobel).Trong y hc, cc k
thut ti t hp DNA c dng thm d bn cht ca ung th, chn on cc bnh di
truyn v nhim trng, sn xut thuc v iu tr cc ri lon di truyn.Khng c mt
lnh vc no ca cng ngh sinh hc thc nghim li pht trin nhanh chng nh
cng ngh di truyn (genetic engineering) hay cn gi l cng ngh DNA ti t
hp (DNA recombinant technology), cng khng c mt lnh vc no khc c th a
ra nhiu loi sn phm mi v hu ch n nh vy. Nguyn l c bn ca cng ngh ny l
thao tc c nh hng v c ch (a vo hoc loi b) DNA v cc loi nguyn liu di
truyn khc nhm lm thay i c tnh di truyn ca c th sinh vt. Hu ht cc k
thut u bt u bng s la chn mt gen mong mun, tip theo l phn lp n v ct
n bng cc enzyme hn ch. Gen ny c gn vo mt vector to dng (plasmid) v
sau a vo mt vt ch, n s c dch m thnh mt protein c bit.6.2 Cc cng c
thit yu ca k di truyn.6.2.1 Enzyme ct gii hn v mt s enzyme khc. a)
Enzym gii hn (restriction endonucleaz): T 1953 ngi ta pht hin thy
rng, khi a DNA ca mt ni vi khun E. coli ny vo t bo thuc mt ni khc
thng th DNA c a vo, gi l DNA ngoi lai hay DNA l, mt hn hot tnh di
truyn v hu nh bao gi cng b phn ct thnh cc on ngn. Ch trong mt s t
trng hp DNA l mi khng b phn ct v do n c th ti bn trong t bo ch. iu
chng t DNA l c sa i bng cch no di s kim sot ca t bo ch. Cc hin tng
ni trn xy ra ch yu khi cc phage ly nhim cc t bo vi khun.Nm 1962,
W.Arber ln u tin chng minh rng c nhng enzym c bit hn ch
(restriction) sinh sn ca phage trong t bo E. coli. Nm 1968: Enzym
ct gii hn u tin c khm ph: EcoR I. n nm 1970, Hamilton Smith v cng s
tch c mt loi enzym ct gii hn mi t vi khun Haemophilus influenzae,
gi l Hind II. Enzym ny l cng c u tin trong k thut di truyn (Gii
Nobel). y l mt trong nhng khm ph u tin cho php pht trin cng ngh DNA
ti t hp. Cc enzyme ny u c i tng nhn bit l cc on trnh t ca DNA vt ch
v DNA ngoi lai, nhng c vai tr khc nhau. Cc enzyme sa i (methylase)
ng vai tr bo v DNA vt ch bng cch gn thm nhm methyl mt s base nht nh
trong on nhn bithay on ch(recognition/ target sequence). Trong khi
cc enzyme gii hn li ng vai tr v hiu ho hot tnh ca cc DNA l bng cch
phn ct cc v tr c th chng no n cha c sa i cho ging vi DNA vt ch.Hnh
6.1
Nh vy, enzyme ct gii hn l cc enzyme c th ca vi khun c kh nng nhn
bit v ct DNA si kp ti cc v tr c th, v c coi l hng ro bo v t nhin ca
cc vi khun chng li s xm nhp ca cc phage l hoc DNA ngoi lai.Cc
enzyme gii hn khc nhau c hai kiu ct: ct lch v ct thng: Vi kiu ct
lch, to ra cc on DNA c cc u si n gm mt s base b sung gi l cc u dnh
(cohesive/sticky ends); v dBamHI, HindIII v EcoRI. Vi kiu ct thng,
tc ct cng v tr trn c hai si ca DNA si kp, to ra cc on DNA c cc u
bng (blunt ends); v dAluI, Hind II v SmaI. Cc enzyme gii hn ny, c
bit l loi u, c vai tr to ln trong vic xy dng cc phn t DNA ti t hp
in vitro. Tt c cc c tnh trn c thminh ho s sau:
b) Mt s enzyme khc thng dng trong k thut di truynBn cnh cc
enzyme gii hn l cc enzyme ch cht trong lnh vc cng ngh DNA ti t hp
ni trn, cn c nhiu enzyme khc. Tu trung c ba nhm chnh: Cc DNA
polymerase v RNA polymerase, k c cc enzyme phin m ngc (reverse
transcriptase): c s dng khi cn to ra mt s lng ln cc bn sao ca cc
nucleic acid. Chng hn, to cc vt d (probe), xc nh trnh t nucleotide,
tng hp mi, oligonucleotide v DNA bng PCR hoc cc cDNA v cc gene tcc
RNA, ... Cc ligase xc tc cc phn ng ni cc u 3' v 5' ca cc on DNA si
n (DNA ligase) hoc cc on RNA (RNA ligase), c s dng trong kin to DNA
ti t hp v to dng ni chung. l cc DNA ligase ca E. coli; cc DNA
ligase, RNA ligase v polynucleotide kinaseca phage T4; ... Cc
nuclease thuc nhm cc enzyme phn ct DNA (DNase) hoc RNA (RNase) mt
cch khng c th nh cc enzyme gii hn ni trn. Nhm ny bao gm cc enzyme
DNase I to cc "vt nt" (nick) trn DNA trong k thut nh du mu d, pht
hin cc gene trong cht nhim sc; cc nuclease S1 c kh nng ct DNA si n
nn c dng loi b cc vng trong tng hp cDNA hoc tch b cc u mt si n so
le trong cc DNA; cc exonuclease III ca E. colic hot tnh exonuclease
3' 5' c dng to cc cu trc si n mt s vng trn phn tDNA hoc gy cc t bin
mt on ti cc vng c bit khi phi hp vi nuclease S1... 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, cc plasmid i vo cc t bo vi khun tng
i d dng. iu ny xy ra trong t nhin v c th l gii cho phm vi v mc khng
cc cht khng sinh cao cc bnh vin v mi ni. Cc plasmid v vy c s dng mt
cch hiu qu trong cc th nghim bin np v to dng cc t bo vi khun.Di y l
mt s c im trc ca cc plasmid vi khun v mt s eukaryote n bo thng c s
dng trong cng ngh DNA ti t hp: Plasmid pUC19 c xy dng t pBR322v khi
im (ori) t pBR322 bt ngun t plasmid pMB9. Vng k hiu ORI trn hai
plasmid pBR322 v pUC19 l ging nhau, nhng pUC19 c nhiu hn 100 bn sao
trong mi tbo E. colitrong khi pBR322 c khong 20 bn sao trong mi t
bo.Hnh 6.2
Plasmid pAMPc kch thc 4539 bp, c mt khi m ti bn ring, mt gene
ampr khng ampicillin, mt trnh t5'GGATCC3' c nhn bit v ct bi
enzymegii hn BamHI v mt trnh t5'AAGCTT3' cho enzyme HindIII (Hnh
6.3). Vic xl pAMP bng hn hp ca BamHI v HindIII ssinh ra c hai on c
cc u dnh vi c im sau: mt on 3755 bp mang c gene ampr v khi im ti bn
v mt on 784 bp Plasmid pKANc 4207 bp, mt Oriring, mt gene kanr c
khnng khng kanamycin, mt vtr ct c nht bi BamHI, mt vtr ct c nht bi
HindIII (Hnh 6.3). Vic xl pKAN bng hn hp ca BamHI v HindIII ssinh
ra chai on c cc u dnh vi c im sau: mt on 2332 bp v mt on 1875 bp vi
gene kanr (nhng khng c Ori).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
eukaryoteHnh 6.4
6.3 Phng php xy dng ADN ti t hp in viro. 6.3.1 Phng php s dng cc
u dnh.Bt k on DNA no nu c ct bi cng mt loi enzyme gii hn (v d,
EcoRI) cho cc u dnh th c th dnh lp li vi nhau v c ni bi DNA ligase.
Phng php thnh lp phn t DNA ti t hp kiu ny ln u tin c a ra bi J.Mert
v R.Davis nm 1972 bng thc nghim trn cc virus. V sau , ln u tin nm
1973, H.Boyer v nhm nghin cu ca S.Cohen to ra c phn t DNA ti t hp
gm vector l plasmid nh pSC101 ca E. coliv DNA ''ngoi lai'' l mt
plasmid khc. Chnh s kin ny t nn mng v m ra trin vng to ln cho kthut
DNA ti t hp sau ny.
6.3.2 Phng php ni trc tip hoc tng hp cc u b sung: i vi cc on DNA
c to ra bng cch x l enzyme gii hn ct thng nh HindII chng hn, th vic
ni cc on DNA c u bng c to ra c th thc hin theo hai cch sau: Ni trc
tip bng DNA ligase ca phage T4 hoc tng hp thm cc u dnh vo cc u 3'
mt s nucleotide b sung bng cch s dng cc enzyme end-transferase, ri
sau cc on DNA nh th s c ni vi nhau bi DNA ligase ca vi khun. C s ca
phng php kt hp DNA ny c thc hin ln u tin gia DNA ca virus SV40 vi
DNA ca phage bi L.Lobban v D.Kaiser (1972), v D.Jackson v P.Berg
(1972)6.4 K thut ADN ti t hp. V nguyn tc, k thut DNA ti t hp hay to
dng (cloning) gm cc bc chung nht nh sau:(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:Mt iu lm cng ngh di truyn tr nn c bit hp dn l cc k
thut ca n c th kt hp vi cc k thut ca cng ngh ln men sn xut ra nhng
s lng ln cc cht ging khng sinh hoc steroid, hormone, vitamin,. Hu
ht cc protein ti t hp ang bn trn th trng hin nay u rt hu ch trong y
hc.Hnh 6.7
6.5.1 Sn xut cc ch phm y sinh hc bng cng ngh ADN ti t hpNu nh vo
nm 1972, gio s Roger Guillemin (France) phi dng ti 500.000 no cu mi
tinh chit c vi miligram somatostatin, mt hormone sinh trng ca vng
di i th (vi cng trnh ny ng c trao gii Nobel nm 1980), th vo 10/1977
Hebert Boyer (USA) ch to thnh cng hormone ny t E. coli bng phng php
DNA ti t hp. n 8/1978 chnh Boyer li l ngi u tin cho sn xut thnh cng
insulin ngi t E. coli. Cc thnh tu u tin ny m ra mt k nguyn mi pht
trin rc r nht trong lch s cng ngh sinh hc, cng ngh DNA ti t hp. Sau
l hng lot cc ch phm khc nh cc hormone tng trng ca ngi (human growth
hormone = HGH), b (BGH), ln (PGH), cc vaccine v cc interferon phng
chng cc cn bnh nan ngi y nh ung th, vim gan ... v mt s bnh quan
trng gia sc nh hin tng 'l mm-long mng' do virus gy ra... tt c u c
sn xut t E. coli.
c bit l, s ra i ca cc hng, cc cng ty ln u t mnh m cho s pht trin
ca k ngh ny. Nh vy gp phn gii quyt mt cch c hiu qu nhiu vn thc tin
t ra trong y hc, trong chn nui-th y, v nng nghip ni chung v.v... nc
ta cng c mt s cng trnh nghin cu v cc vn ny, chng hn nh nghin cu s
sai khc di truyn gene hormon sinh trng ca mt s ging g Vit Nam. Qua
cho thy intron I ca gene hormon sinh trng cc ging g Ri, g Ma, g c,
g H di hn kch thc d on ca gene hormon sinh trng g c Tanaka cng b nm
1992 (Trn Xun Hon, 2004). Bn cnh vic sn xut cc hormone, vaccine...
ni trn, ngi ta cn sn xut c cc khng th n dng (monocloning
antibodies) dng : (i) xc nh vi khun gy bnh (nh thng hn chng hn);
(ii) xc nh mc hormone t nh gi chc nng tuyn ni tit hoc s thay i
trong qu trnh tng hp hormone do khi u gy ra; (iii) pht hin mt s
protein c ngha trong chn on khi u hoc mt s tnh trng trc khi sinh;
(iv) pht hin cc thuc b cm c trong mu, hoc kim tra nng thuc trong mu
v t chc nhm m bo liu thuc s dng sao cho khng vt qu ngng gy c v.v.
Nh c tnh c hiu v chnh xc cao v s dng d dng, vic sn xut cc khng th 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
6.5.2 ng dng k thut di truyn trong chun on v iu tr gen. Trong
chn on bnh mc phn t, thnh tu ni bt nht l vo nm 1981, ln u tin bnh
thiu mu hng cu hnh lim c chn on trc sinh mc gene nh phn tch DNA bng
enzyme gii hn. T y m ra hai hng chnh trong chn on gene l: chn on cc
bt thng bm sinh v chn on cc bt thng DNA soma. Mt s thnh tu khc t c
theo hng u gm c: bnh hemophilia A, ri lon dng c Duchenne, hi chng
X-fragile, bnh retinoblastoma - mt dng ung th vng mc...Theo hng
sau, ngi ta p dng i vi mt s dng ung th mu nh cc lympho Burkitt,
lympho nang, bnh bch cu...ng k l gn y, ngi ta xc nh c nhiu gene gy
bnh quan trng ngi, nh: gene gy bnh ha x nang (cystic fibrosis) nm
1990, gene gy bnh Huntington nm 1993... Liu php gene l mt phng thc
sn xut v iu tr mi bng cc phn t tr liu. y l hng c nhiu trin vng nht
nhng cn kh thc hin.6.5.3 K thut di truyn vi hnh php hc...K thut di
truyn cn c ng dng rt hiu qutrong cc ngnh hnh php hc, chng hn bng
cch s dng k thut 'du vn DNA' (DNA fingerprinting) thay cho 'du vn
tay' trc y cho php cc ngnh cnh st hnh s c th xc nh chnh xc cc ti
phm; cctrung tm phn tch DNA v t vn di truyn c th gip xc nh quan h
huyt thng gia cc anh em hay gia cha/m vi con ci trong nhng trng hp
cn thit; hoc nhn dng xc nn nhn trong chin tranh hoc do tai nn gy ra
v.vHnh 6.9
