Nghiên cứu biểu hiện gen kháng thể tái tổ hợp đặc hiệu kháng nguyên ung thư vú her2 trong tảo chlamydomonas reinhardtii

B GIO DC V O TO TRNG I HC BCH KHOA H NI - TRN TH HUYN TRANG NGHIấN CU BIU HIN GEN KHNG TH TI T HP C HIU KHNG NGUYấN UNG TH V HER2 TRONG TO CHLAMYDOMONAS REINHARDTII Chuyờn ngnh : Cụng ngh sinh hc LUN VN THC S KHOA HC NGI HNG DN KHOA HC : PGS TS Lấ QUANG HUN H Ni 2010 Trần Thị Huyền Trang LI CAM OAN Tụi xin cam oan õy l cụng trỡnh nghiờn cu m bn thõn tụi ó trc tip thc hin Cỏc s liu v kt qu trỡnh by lun l trung thc, khỏch quan v cha tng c cụng b bt k cụng trỡnh no khỏc H Ni, ngy 25 thỏng 10 nm 2010 Tỏc gi lun Trn Th Huyn Trang CNSHK810 Trần Thị Huyền Trang LI CM N Trc tiờn, tụi xin by t lũng cm n sõu sc nht ti PGS TS Lờ Quang Hun- Trng phũng cụng ngh T bo ng vt, Vin Cụng ngh Sinh hc, Vin Khoa hc v Cụng ngh Vit Nam, l ngi thy trc tip hng dn tụi sut quỏ trỡnh lm khoỏ lun ny Trong quỏ trỡnh thc v thc hin ti ti phũng thớ nghim, tụi ó nhn c s giỳp nhit tỡnh ca KS ng Th Minh La v ton th cỏn b nhõn viờn phũng Cụng ngh T bo ng vt, nhõn dp ny tụi mun gi li cm n chõn thnh nht vỡ s giỳp quý bỏu ú hon thnh khoỏ lun ny khụng th khụng núi ti cụng lao ca cỏc thy, cụ giỏo Vin Cụng ngh sinh hc v cụng ngh thc phm, Vin o to Sau i hc - i hc Bỏch Khoa - H Ni ó ging dy, truyn t cho tụi nhng kin thc v to iu kin, tn tỡnh giỳp tụi sut thi gian hc ti trng Cui cựng, tụi xin gi li cm n chõn thnh ti gia ỡnh, bn bố, ng nghip c quan cụng tỏc Chi cc Bo v Bo v thc vt H Ni ó luụn bờn cnh ng viờn, chia s nhng khú khn giỳp tụi hon thnh khoỏ hc v thc hin tt khoỏ lun ny Mt ln na cho tụi xin cm n v mi s giỳp quý bỏu ú! H Ni, ngy 25 thỏng 10 nm 2010 Hc viờn Trn th Huyn Trang CNSHK810 Trần Thị Huyền Trang MC LC Trang M U Chng TNG QUAN 1.1 Tng quan v bnh ung th vỳ 1.1.1 Khỏi nim ung th vỳ (UTV) 1.1.2 Phõn loi 1.1.3 Nguyờn nhõn gõy UTV 1.1.4 Cỏc phng phỏp iu tr ung th vỳ 1.2 Khỏng nguyờn HER2 c hiu t bo ung th vỳ. 1.2.1 Cu trỳc ca gen HER2 1.2.2 Cu trỳc khỏng nguyờn HER2 1.2.3 C ch hot ng ca th th HER2 11 1.3 Ung th vỳ dng tớnh HER2 11 1.4 Khỏng th tỏi t hp c hiu khỏng nguyờn HER2 13 1.4.1 Khỏi nim 13 1.4.2 Khỏng th n dũng v ng dng iu tr ung th vỳ 16 1.5 Cỏc phng phỏp sn xut khỏng th n dũng 18 1.5.1 Phng phỏp to dũng t bo lai hybridoma 18 1.5.2 Phng phỏp tỏi t hp 19 1.6 Cỏc h thng biu hin gen truyn thng 20 1.6.1 H thng biu hin t bo vi khun 20 1.6.2 H thng biu hin t bo nm men 21 1.6.3 H thng biu hin t bo ng vt cú vỳ 21 1.6.4 H thng biu hin t bo thc vt 21 1.6.5 H thng biu hin vi to 22 1.7 H biu hin to Chlamydomonas reinhardtii 23 1.7.1 c im sinh hc 23 1.7.2 ng dng ca to Chlamydomonas reinhardtii cụng ngh gen 25 CNSHK810 Trần Thị Huyền Trang 1.8.Phng phỏp chuyn gen vo to Chlamydomonas reinhardti thụng qua vi khun Agrobacterium tumefaciens 27 1.8.1 Vi khun Agrobacterium tumefaciens 28 1.8.2 C ch chuyn gen ca Agrobacterium tumefaciens 29 1.8.3 H thng vector dựng chuyn gen 29 Chng VT LIU V PHNG PHP 31 2.1 VT LIU 31 2.1.1 Sinh phm 31 2.1.2 Hoỏ cht v mụi trng 31 2.1.3 Thit b mỏy múc 33 2.2 PHNG PHP NGHIấN CU 33 2.2.1 Phng phỏp tỏch plasmid t vi khun E coli 33 2.2.2 Phng phỏp khuch i gen bng PCR 35 2.2.3 Phng phỏp in di trờn gel agarose 36 2.2.4 Phng phỏp thu DNA t gel agarose 36 2.2.5 Phng phỏp ct, gn AND 37 2.2.6 Phng phỏp bin np plasmid vo E coli 38 2.2.7 Phng phỏp xỏc nh trỡnh t nucleotide ca DNA 39 2.2.8 Bin np vector chuyn gen tỏi t hp vo Agrobacterium tumefaciens bng phng phỏp xung in 39 2.2.9 Phng phỏp chuyn gen vo to C reinhardtii nh A Tumefaciens 41 2.2.10 Phng phỏp m t bo 42 2.2.11 Phng phỏp kim tra biu hin gen GUS bng nhum hoỏ mụ t bo 42 2.2.12 Phng phỏp tỏch ADN tng s ca to 42 2.2.13 Phng phỏp tỏch protein tng s t to 43 2.2.14 Phng phỏp in di protein trờn gel polyacrylamide 43 Chng KT QU V BN LUN 45 3.1 Khuch i on gen mó hoỏ khỏng th tỏi t hp t vector pStrepHis1525/antiHER2 CNSHK810 45 Trần Thị Huyền Trang 3.2 Thit k vector chuyn gen mang gen mó húa khỏng th c hiu khỏng nguyờn HER2 51 3.3 Bin np vector chuyn gen tỏi t hp vo vi khun A tumefaciens 58 3.4 Chuyn gen vo to Chlamydomonas reinhardtii 60 3.4.1 Phng phỏp v iu kin chuyn gen vo to C reinhardtii thụng qua A tumefaciens 60 3.4 Kim tra s chuyn gen antiHER2 vo to C reinhardtii bng phn ng PCR 63 3.5 Kim tra s biu hin gen GUS 66 3.6 Kim tra s biu hin ca gen antiHER2 to C reinhardtii 68 KT LUN V KIN NGH 71 Kt lun 71 Kin ngh 72 TI LIU THAM KHO 73 PH LC CNSHK810 80 Trần Thị Huyền Trang DANH MC CC Kí HIU, CC CH VIT TT A tumefaciens DNA RNA AS bp BRCA C reinhardtii CBB CBD CTAB ddNTP dNTP E coli EDTA EGFR ER EtBr Fab Fc Fv GF GUS HER2 Ig Kb kDa LB MAbs MAPK mARN Agrobacterium tumefaciens Deoxyribonucleic Acid Ribonucleic Acid Acetosyringone Base pair Breast cancer = ung th vỳ Chlamydomonas reinhardtii Coomasie Brilliant Blue Chitin Binding Domain Cetyl Trimethyl Ammonium Bromide Dideoxyonucleotit triphosphat Deoxyonucleotit triphosphat Escherichia coli Ethylenediaminetetraacetic acid Epidermal Growth Factor Receptor = Th th cỏc yu t tng trng biu bỡ Estrogen Ethidium bromide Fragment antigen binding Fragment cristallisable Fragment constant Growth Factor -glucuronidase Human Epidermal Growth Factor Receptor Immunoglobulin Kilobases kiloDaltons Left Border Monoclonal antibody = Khỏng th n dũng Mitogen-activated protein kinases Messenger acid ribonucleic CNSHK810 Trần Thị Huyền Trang Nos NPTII PCR PI3K PR RB scFv SDS SDS-Page Taq T-DNA TAE TAP TE Ti-plasmid TNM UTV v/ph VH Vir VL X- Glu Nospaline synthease Neomycin phosphotransferase gene Polymerase chain reaction Phosphoinositide 3-kinases Progesterone receptor Right border single chain Fv Sodium Dodecyl Sulphate SDS- Polyacrylamide gel electrophoresis Thermus aquaticus polymerase Transfer- DNA Tris acid acetic EDTA Tris-Acetate-Phosphate Tris- EDTA Tumor inducing plasmid = plasmid gõy u Tumor- nodes - metastasis Ung th vỳ Vũng/phỳt Variable region of heavy chain = vựng bin i ca chui nng Virulence region = vựng gõy c Variable region of light chain = vựng bin i ca chui nh 5-bromo-4-chloro-3-indolyl glucuronide CNSHK810 Trần Thị Huyền Trang DANH MC CC BNG Bng Tờn bng Trang 2.1 Mt s mnh khỏng th n chui 16 3.1 Thnh phn phn ng PCR khuych i gen AntiHER 47 3.2 3.3 3.4 3.5 Thnh phn phn ng ct plasmid pBI101 v pJet1.2 mang gen antiHER2 bng BamH I v Sac I Thnh phn phn ng ghộp ni gia gen antiHER2 vo pBI101 Thnh phn phn ng PCR khuych i on AntiHER2 t ADN tng s ca to C reinhardtii T l chuyn gen GUS 53 55 64 67 CNSHK810 Trần Thị Huyền Trang DANH MC CC HèNH Hỡnh Tờn Hỡnh Trang 1.1 Cỏc dng ung th vỳ 1.2 Cu trỳc ca protein HER2 10 1.3 C ch gõy ung th biu hin quỏ mc HER2 12 1.4 S cu to ca khỏng th 14 1.5 S cu to ca mnh khỏng th n chui 16 1.6 Cu to t bo to Chlamydomonas reinhardtii 24 1.7 Vũng i ca Chlamydomonas 25 3.1 Cu trỳc khỏng th tỏi t hp c hiu HER2 46 3.2 in di sn phm PCR vi cp mi AntiHER2F /AntiHER2R 48 3.3 S vector PJET1.2/blunt 48 3.4 Vi khun E coli mang vector tỏi t hp mc trờn mụi trng cú 49 cha Amppicllin sau 15 gi nuụi cy 370C 3.5 Sn phm ct pJet1.2 tỏi t hp bng Enzyme gii hn BamH I, 50 Sac I 3.6 on T- DNA ca vector chuyn gen pBI101 52 3.7 Sn phm ct loi b gen GUS ca pBI101 bng enzyme 54 BamH I, Sac I 3.8 Kt qu tỏch plasmid t E coli TG1 ó bin np pBI101 tỏi t hp 55 3.9 Kt qu sn phm PCR t plasmid tỏch t khun lc E coli TG1 56 sau bin np 3.10 Mt on Chromatograph ca gen antiHER2 57 3.11 Kt qu bin np vector pBI101 tỏi t hp vo A tumefaciens 59 sau 24 gi nuụi cy nhit 28oC 3.12 Kt qu sn phm PCR t khun lc A tumefaciens bin np 59 3.13 Hỡnh thỏi t bo to C reinhardtii trờn mụi trng TAP sau 61 ngy nuụi cy CNSHK810 Trần Thị Huyền Trang Bng 3.5 T l chuyn gen GUS Tng s t bo T bo dng tớnh GUS T l (%) 32 37 24 46 65 9,3 10,8 8,8 10,9 12,3 10,42 % Trung bỡnh T l chuyn gen GUS = Tng s t bo nhum xanh/ tng s t bo to x100% Theo ú hiu sut chuyn gen cn c vo hot tớnh biu hin gen GUS t c l 10,42 % Theo mt s cụng b v chuyn gen ngoi lai vo C reinhardtii nh Agrobacterium thnh cụng thỡ hiu sut chuyn gen da vo hot tớnh biu hin gen GUS l 8- 10% v 11% [41,42] Nh vy, bc u chỳng tụi ó kim tra c s biu hin ca gen GUS nh l mt ch th ỏnh giỏ kh nng chuyn gen thnh cụng ca chng C58C1 (pGV226) v vector chuyn gen pBI101 Ngoi ra, quỏ trỡnh nhim chng A tumefaciens mang vector pBI101 mang gen GUS luụn c thc hin song song, cựng iu kin vi quỏ trỡnh nhim chng A tumefaciens mang vector pBI101- antiHER2 nờn kh nng gen antiHER2 cng c chuyn thnh cụng gn vo b gen ca to, chộp v biu hin vi t l phn trm tng ng t l 10,42% dng tớnh vi s biu hin gen GUS Tuy nhiờn cn phi cú thờm mt s thớ nghim tip theo mi cú th khng nh c iu ny 3.6 Kim tra s biu hin ca gen antiHER2 to C reinhardtii bc u kim tra s biu hin ca gen antiHER2 to C reinhardtii, chỳng tụi ó tin hnh tỏch protein tng s ca dch nuụi cy to ó c chuyn gen bng dung dch m tỏch chit protein tng s, in di trờn gel polyacrylamide 12,5% 67 CNSHK810 Trần Thị Huyền Trang Hỡnh 3.18 Kt qu in di kim tra protein tng s ca to C reinhardtii ng chy M: Thang protein chun ng chỏy s 1: Mu to khụng c chuyn gen antiHER2 ng chy s 2,3; Mu to ó c chuyn gen antiHER2 Nhỡn hỡnh 3.18, ng chy s v xut hin bng khỏc bit so vi ng chy s l dũng to khụng c chuyn gen, v trớ ca bng protein ny nm di bng 66,2 kDa v trờn bng 45 kDa ca thang protein chun, ú kớch thc lý thuyt ca on protein tỏi t hp ca chỳng tụi l khong 59,5 kDa Cựng vi kt qu kim tra kh nng biu hin ca gen GUS, cú th s b ỏnh giỏ h promoter Nos v trỡnh t polyadenyl ca vector nh th pBI101 la chn ó thc hin c chc nng ca chỳng cỏc t bo to C reinhardtii iu ny giỳp chỳng tụi cú th bc u nhn nh kh nng biu hin thnh cụng khỏng th tỏi t hp khỏng khỏng nguyờn ung th vỳ HER2 to C reinhardtii Hn mt thp k qua, liu phỏp cha bnh da vo cỏc loi protein (khỏng th) cú nhiu thnh cụng nht nh Nh phõn tớch phn tng quan ti liu da vo cỏc u im so vi cỏc h biu hin truyn thng, trờn th gii, cỏc nh khoa hc ó bt u phỏt trin cỏc h thng biu hin cỏc protein tỏi t hp loi to lc n bo, chlamydomonas reinhardtii, ú cú biu hin thnh cụng 68 CNSHK810 Trần Thị Huyền Trang mt s protein tỏi t hp nh khỏng th IgA n chui hay cu trỳc protein VP1 ca virus gõy l mm long múng ó c ni vi tiu phn B c t gõy bnh t ó c biu hin t lp th v quỏ trỡnh gp np chớnh xỏc ó c chng minh[49,61] H biu hin to C reinhardtii ó t l mt h biu hin mang nhiu u im Cỏc nh nghiờn cu ó nm gi nhng thun li ca hn 80 nm phỏt trin ca ngnh cụng ngh gen v hn 30 nm ca ngnh cụng ngh sinh hc phõn t bt u cỏc k thut biu hin hiu qu cỏc protein cha bnh quan trng C reinhardtii l mt ng c viờn hp dn biu hin cỏc protein cha bnh cho ngi c bit l cỏc loi khỏng th n dũng da vo c im sinh vt hc cú ca sinh vt, d dng nuụi cy, quy mụ phỏt trin v tc ca cỏc dũng chuyn gen cú th c tỏi sinh nhanh ch cn vi tun m t nhng t bo chuyn gen ban u c nuụi tng sinh lờn ti quy mụ th tớch ln Gen ngoi lai cú th c chuyn vo c b gen nhõn v lp th ca to m c hi rt ln cú th biu hin nhiu loi protein khỏc mt h biu hin n gin c bit l cỏc protein phc nh khỏng th tỏi t hp Ngoi ra, so vi h biu hin ng võt hay thc vt bc cao thỡ chi phớ to cỏc sn phm protein tỏi t hp to l rt thp Vỡ vy, nghiờn cu ny ca chỳng tụi phn no m c hi biu hin thnh cụng khỏng th n dũng núi chung, khỏng th tỏi t hp c hiu khỏng nguyờn ung th vỳ HER2 núi riờng nhm gúp phn to cỏc ch phm mi iu tr bnh ca ngi va hiu qu, va an ton v c bit l chi phớ thp 69 CNSHK810 Trần Thị Huyền Trang KT LUN V KIN NGH Kt lun ó thit k thnh cụng vector chuyn gen pBI101-antiHER2 ó bin np thnh cụng vector pBI101 tỏi t hp vo chng vi khun Agrobacterium tumefaciens chng C58C1 (pGV2260) ó chuyn thnh cụng gen khỏng th tỏi t hp khỏng khỏng nguyờn HER2 vo to Chlamydomonas reinhardtii nh Agrobacterium tumefaciens bng phng phỏp ng nuụi cy Bc u kim tra s biu hin khỏng th tỏi t hp c hiu khỏng nguyờn HER2 to Chlamydomonas reinhardtii 70 CNSHK810 Trần Thị Huyền Trang Kin ngh Trờn õy ch l nhng kt qu bc u v kh nng biu hin khỏng th tỏi t hp c hiu khỏng nguyờn ung th vỳ HER2 to Chlamydomonas reinhardtii Do vy, cn cú nhng nghiờn cu tip theo mt cỏch ton din hn v: - Ti u quỏ trỡnh biu hin gen khỏng th c hiu khỏng nguyờn HER2 vo to Chlamydomonas reinhardtii - Nghiờn cu iu kin tinh sch khỏng th tỏi t hp thu c - Nghiờn cu hot tớnh ca khỏng th tỏi t hp thu c 71 CNSHK810 Trần Thị Huyền Trang TI LIU THAM KHO Ting vit Lờ Trn Bỡnh (2008), Phỏt trin cõy trng chuyn gen Vit Nam, B sỏch chuyờn kho, NXB Khoa hc v cụng ngh Lờ Trn Bỡnh, Phan Vn Chi, Nụng Vn Hi, Trng Nam Hi, Lờ Quang Hun (2003), p dng cỏc k thut phõn t nghiờn cu ti nguyờn sinh vt Vit Nam, NXB Khoa hc v k thut, H Ni Nguyn Bỏ c, (2001), Bi ging ung th hc lõm sng, NXB Y hc, H Ni Lờ Quang Hun, Ló Th Huyn (2009), Khỏng th tỏi t hp v ng dng, Nxb Khoa hc t nhiờn v cụng ngh, H Ni Khut Hu Thanh (2006), K thut gen nguyờn lý v ng dng, NXB Khoa hc v k thut, H Ni 72 CNSHK810 Trần Thị Huyền Trang Lờ Duy Thnh (2001), C s di truyn chn ging thc vt, NXB Khoa hc v k thut, H Ni Nguyn Quang Thch, Nguyn Th Lý Anh (2006), Giỏo trỡnh cụng ngh sinh hc nụng nghip, NXB Nụng nghip Ting Anh Apt, K.E Kroth-Pancic, P.G., and Grossman, A.R (1996), Stable nuclear transformationof the diatom Phaeodactylum tricornutum Mol Gen Genet 252, 572579.6 Berrington de Gonzalez, A., and Darby, S (11/28/2007), Risk of cancer from diagnosticX-rays, http://info.cancerresearchuk.org/cancerstats/types/breast/riskfactors/ (9 of 11).17 10 Bianco AR, (2004), Targeting c-erb2 and other receptors of the c-erB family: rationale and clinical applications, J Chemother; 16:52-54.18 11 Biscardi, J.S., Ishizawar, R.C., Silva, C.M and Parsons, S.J (2000), Tyrosine kinase signalling in breast cancer: epidermal growth factor receptor and c-Src interactions in breast cancer, Breast Cancer Res 2, 203-10.21 12 Borg, J.-P., Marchetto, S., Le, Bivic, A., Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Adộlaùde, J., Margolis, B., and Birnbaum, D, (2000), ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor, Nat Cell Biol 2, 407-14 23 13 Cereghino, J L and Cregg, J M (2000), Heterologousprotein expression in the methylotrophic yeast Pichia pastoris FEMS Microbiol Lett 24, 4566 14 Chirstoph Griesbeck, Iris Kobl, Markus Heizer (2006), Chlamydomonas reinhardtii A protein expression system for pharmaceutical and biotechnological proteins, Molecular biotechnology review, Vol 34, 213221 73 CNSHK810 Trần Thị Huyền Trang 15 Cho, H S, Mason, K., Ramyar, K X., Stanley, A M., Gabelli, S B., Denney, D W., Jr., & Leahy, D J, (2003), Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab, Nature 421, 756-60 30 16 Cho, H-S, Leahy DJ, (2002), Structure of the extracellular region of HER3 reveals an interdomain tether, Science 297, 1330-3 29 17 Christos Sotirou and Lajos Pusztai (2008), Molecular origin of cancer: gene-expression signatures in breast cancer, , N Engl J Med 360:790 18 David F Stern, (2000), Tyrosine kinase signalling in breast cancer ErbB family receptor tyrosine kinases, Breast Cancer Res, 2:176183 39 19 Debuchy, R., Purton, S., and Rochaix, J D (1989), The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus EMBO J 8, 28032809 40 20 Ding Z S., Zhao M., Jing Y.X., Li L B and Kuyang T Y (2006), "Efficient Agrobacterium - Mediated transformation of Rice by Phosphomanose Isomerase/Mannose selection", Plant Molecular Biology Reporter, (24), pp.295-303 21 Ditt R F., Nester E W and Comai L (2005), "The plant cell defense and Agrobacterium tumefaciens", FEMS Microbiology letters, 247, pp.207213 22 Doyle, J.J and Doyle, J.L (1990) Isolation of plant DNA from fresh tissue Focus 12, 1315.18 23 Endogenous Hormones Breast Cancer Collaborative Group (2003), Body Mass Index, Serum Sex Hormones, and Breast Cancer Risk in Postmenopausal Women, JNCI Cancer Spectrum, 95; 16; 1218-1226 41 24 European Patent Application EP0108580 Method for introducing foreign genes into green algae utilizing T-DNA of agrobacterium 74 CNSHK810 Trần Thị Huyền Trang 25 Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C., and Bowler, C (1999), Transformation of nonselectable reporter genes in marine diatoms Mar Biotechnol 1,239251.8 26 Fischer, R and Emans, N (2000), Molecular farming of pharmaceutical proteins Transgenic Res 9, 279299 13 27 Franklin, S E and Mayfield, S P (2004), Prospects for molecular farming in the green alga Chlamydomonas Curr Opin Plant Biol 7, 159165 10 28 Gao, B and Tsan, M F (2003), Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages.J Biol Chem 278, 174179 29 Gelvin S B (2003), "Agrobacterium - mediated plant transformation the biology behind the "Gene- jockeying" Tool", Microbiology and Molecular Biology Reviews, 67 (1), pp 16-37 30 Gelvin S.B (2000), Agrobacterium and plant genes involved in T-DNA transfer and integration, Ann Rev Plant Physiol Plant Mol Biol.51, 223 256 31 Harari D, Yarden Y, (2000), Molecular mechanisms underlying ErbB2/HER2 action in breast cancer, Oncogene; 19:6102-6113 50 32 Harris, E H (2001), Chlamydomonas as a model organism Annu Rev Plant Physiol Plant Mol Biol 52, 363406.29 33 Harris, E.H (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use Academic Press, New York.2 34 Hendriks, B S, Opresko, L K., Wiley, H S., Lauffenburger, D, (2003), Quantitative analysis of HER2-mediated effects on HER2 and epidermal growth factor receptor endocytosis: distribution of homo- and heterodimers depends on relative HER2 levels, J Biol Chem 278, 2334351.51 75 CNSHK810 Trần Thị Huyền Trang 35 Jahanzeb M (August 2008) "Adjuvant trastuzumab therapy for HER2positive breast cancer" Clin Breast Cancer (4): 32433 36 Jefferson, R.A (1987), Assaying chimeric genes in plants: the uidA gene fusion system Plant Mol Biol Rep 5, 387405.17 37 Jeffrey S Ross and Jonathan A Fletcher, (1998), The HER-2/neu Oncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for Therapy, Stem Cells; 16; pp.413-428 58Kelsey, J.L., Gammon, M.D and John, E.M (1993), Reproductive factors and breast cancer Epidemiol Rev 15; 1; 36-47 63 38 Kindle, K.L (1990), High frequency nuclear transformation of Chlamydomonas reinhardtii Proc Natl Acad Sci USA 93, 13689 13693.11 39 Kindle, K.L., Schnell, R.A., Fernandez, E., and Lefebvre, P.A (1989), Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase J Cell Biol 109, 2589 2601.10 40 Kroes, H W., Growth interactions between Chlamydomonas globosa Snow and Chlorococcum ellipsoideum Deason and Bold: The role of extracellular substances Limnol Oceanogr., 1972, 17, 423432 11 41 Kumar, S.V., Misquitta, R.W., Reddy, V.S., Rao, B.J., and Rajam, M.V (2004), Genetic transformation of the green algaChlamydomonas reinhardtii by Agrobacterium tumefaciens, Plant Sci 166, 731738 42 Kumar, S V., Rajam, M V (2007), Induction of Agrobacterium tumefaciens vir genes by the green alga, Chlamydomonas reinhardtii, Current Science, Vol 12, No 12, 1727- 1729 43 Lacroix M (2006), "Significance, detection and markers of disseminated breast cancer cells", Endocr Relat Cancer, 13 (4): 1033-67 66 76 CNSHK810 Trần Thị Huyền Trang 44 LeỏnBaủares, R., Gonzỏlez-Ballester, D., Galvan, A and Fernỏndez, E (2004) Transgenic microalgae as green cell-factories Trends Biotech 22, 4552.1 45 Leon-Banares, R., Gonzalez-Ballester, D., Galvan, A., and Fernandez, E (2004), Transgenic microalgae as green cell-factories Trends Biotechnol 22, 4552 16 46 Malten, M., Barg, H., Martens, J.H., Biedendieck, R., Palme, O., Moser, Kỹnkel, A and Jahn, D, (2003), Bacillus megaterium a tool for biotechnology, VAAM-Jahrestagung, Berlin, 23 26/03 (Poster) 74 47 Manchikatla V Rajam and S Vinod Kumar, Green Alga (Chlamydomonas reinhardtii) Methods in Molecular Biology, vol 344: Agrobacterium Protocols, 2/e, volume 2, pp 421-433 48 Mayfield, S P and Franklin, S E (2005), Expression of human antibodies in eukaryotic micro-algae Vaccine 23, 18281832 19 49 Mayfield, S P., Franklin, S E., and Lerner, R A (2003), Expression and assembly of a fully active antibody in algae Proc Natl Acad Sci USA 100, 438442 24 50 Merck Manual, Professional Edition, Ch 253, Breast Cancer 51 Meric-Bernstam and Mien-Chie Hung, (2006), Advances in Targeting Human Epidermal Growth Factor Receptor-2 Signaling for CancerTherapy, Clin Cancer Res; 12(21).77 52 Moasser MM (2007) The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis Oncogene , 26(45):6469-87 53 Muthuswamy SK, Gilman M, Brugge JS (1999), Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers, Mol Cell Biol 19, 6845-57 80 54 Muyldermans, S (2001), Single domain camel antibodies: current status, J Biotechnol 74, 277.81 77 CNSHK810 Trần Thị Huyền Trang 55 Muyldermans, S., Lauwereys, M (1999), Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies, J Mol Recognit 12:131-140.82 56 Patent application US000006932980.69 57 Reichert J M, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz (2005), Monoclonal antibody successes in the clinic, Nature Biotechnology, 23, PP 1073 1078 janice 40 hien 58 Romond EH, Perez EA, Bryant J, et al (2005), Trastuzumab plus adjuvant chemotherapy for operable HER2+ breast cancer; 353:16731684 and supplementary appendix 59 Serenella Eppenberger-Castori, Willy Kueng, Christopher Benz, Rosmarie Caduff, Zsuzsanna Varga, Fridolin Bannwart, Daniel Fink, Holger Dieterich, Michael Hohl, Heinz Mỹller, Kaja Paris, Fabrice Schoumacher, and Urs Eppenberger, (2001) Prognostic and Predictive Significance of ErbB-2 Breast Tumor Levels Measured by Enzyme Immunoassay Journal of Clinical Oncology, Vol 19, pp: 645-656 60 Shimogawara, K., Fujiwara, S., Grossman, A., and Usuda, H (1998) High efficiency transformation of Chlamydomonas reinhardtii by electroporation Genetics 148, 18211828.9 61 Sun, M., Qian, K., Su, N., Chang, H., Liu, J., and Chen, G (2003), Footand-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast Biotechnol Lett 25,10871092 23 62 Walker, T L., Purton, S., Becker, D K., and Collet, C (2005), Microalgae as bioreactors Plant Cell Rep 24, 629641.18 63 WHO classification of tumours (2003), Tumours of the breast and female genital organs Ti liu t internet 78 CNSHK810 Trần Thị Huyền Trang 64 http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.52.1 363?cookieSet=1&journalCode=arplant.2 65 http://en.wikipedia.org/wiki/Breast_cancer 66 http://en.wikipedia.org/wiki/Chlamydomonas_reinhardtii 67 http://www.bioinfo.pte.hu/f2/pict_f2/pJETmap.pdf 68 http://www.macmillan.org.uk/Cancerinformation/Cancertypes/Breast/Sym ptomsdiagnosis/HormoneandHER2receptors/HER2andbreastcancer.aspx 69 http://www.molecularinfo.com/MTM/K/K2/K2-1/pBI101.pdf 70 http://www.lablife.org/p?a=vdb_view&id=g2.i6yV94UwtS9Qo6Xpg2VZ8 sVKtVw71 http://www.vja.org.vn/vi/detail.php?pid=6&catid=45&id=24322&dhnam e=Ung-thu-tang-chong-mat PH LC Ph lc S vector pBIN19 79 CNSHK810 Trần Thị Huyền Trang Ph lc Chu k nhit chy PCR khuych i gen AntiHER2 Nhit (C) Thi gian (phỳt) Tỏc dng S chu k 94 3:00 Bin tớnh 94 0:45 Bin tớnh 52 0:45 Gn mi 72 1:20 Tng hp 72 8:00 B sung Bo qun 30 Ph lc Dung dch s dng kim tra protein tng s 80 CNSHK810 Trần Thị Huyền Trang Dung dch Húa cht Nng m chy in di protein (1X Tris- Glycine pH 8,3) Tris base Glycine SDS H2O 3g 14,4 g 1g 1l m tra mu protein (Protein sample buffer 4X) Tris- HCl pH= 6,8 Glycerol SDS DTT Bromophenol Blue 0,5M 60% 20% 1M 0,2% Dung dch nhum protein (Comasive Brilliant Blue R250) Comasive R250 Metanol Acid acetic H2O 0,3 g 120 ml 30 ml 150 ml Dung dch ty gel protein Acid acetic 7% Metanol 20% H2O 35 ml 100 ml 365 ml Ph lc Thnh phn gel polyacrylamide Thnh phn Nng Gel cụ 4% (Stacking gel) Gel tỏch 10% (Separating gel) Tris- HCl, pH=6,8 0,5M 0,75 ml - Tris- HCl, pH= 8,8 1,5M - 2,25 ml SDS 10% 30 àl 90 àl Acrylamide 30% 0,4 ml 3,02 ml H2O - 1,8 ml 3,55 ml APS 10% 15 àl 45 àl - àl 4,5 àl ml ml TEMED Tng th tớch 81 CNSHK810 ... tài: Nghiên cứu biểu gen kháng thể tái tổ hợp đặc hiệu kháng nguyên ung thư vú HER2 tảo Chlamydomonas reinhardtii Mục tiêu đề tài: - Thiết kế vector chuyển gen mang gen mã hóa kháng thể đặc hiệu. .. kháng thể đặc hiệu kháng nguyên HER2 - Nghiên cứu chuyển gen kháng thể đặc hiệu kháng nguyên HER2 vào tảo Chlamydomonas reinhardtii - Nghiên cứu khả biểu kháng thể tảo Chlamydomonas reinhardtii CNSHK810... thể đơn dòng tái tổ hợp đặc hiệu với HER2 giúp cho chẩn đoán mang lại hiệu cao điều trị ung thư vú [52,59] Tuy nhiên, sau có gen tái tổ hợp việc biểu để thu nhận tinh kháng thể tái tổ hợp vấn đề