Tinh chế, nghiên cứu một số tính chất của các Lectin từ một số loài thuộc họ đậu (Fabaceae), họ đậu tằm (Moraceae) và khả năng ứng dụng trong y học : Luận án PTS. Sinh học: 1 05 10

CAC C H C VlfiT TAT TRONG LUAN AN BSA (Bovine Serum Albumin) : Albumin huyet bo ConA (Concanavaline A) : Lectin tir hat dau rira (Canavalia ensiformis L,) DC Da : Dalton DEAE (Dietilaminoetil) : Nhom chiic trao doi anion HAA (Hemagglutininating activity) : Hoat d6 ngung ket hong cau HDTS : Hoat t6ng so, HDR : Hoat d6 rieng Ig (Immuno globuline): globulin mi6n dich kDa : kilo Dalton k.x.d : Kh6ng xac dinh LM : Lectin tijr hat mit mit Mr : Kh6'i luong phan tir CD (Optical density) : M^it d6 quang hoc PAGE : (Polyacrylamide Gel Electrophoresis) Dien di tren gel polyacrilamit PBS (Phosphate buffered Saline) : Dem phot phat mu6'i pr: Protein SDS : Sodium dodecyl sulfate SDS - PAGE : Dien di tren gel polyacrilamit c6 SDS KY HIEU QUOC TE CAC AXIT AMIN A R N D C Q E G H I L K M F P S T W Y V Ala Arg Asn Asp Cys Gin Glu Gly His He Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Alanine Arginine Asparagine Axit aspartic Cysteine Glutamine Axit glutamic Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophane Tyrosine Valine MUC LUC Trang Ma dau Chuang : TONG QUAN TAI LIEU 12 1.1 Luge sir tinh hinh nghien ciiu lectin tren the gidi va a Viet nam 12 1.1 L Luge sir ve lectin 12 1.1.2 Tinh hinh nghien ciiu lectin tren the gi6i va a Viet nam 15 1.2 Su phan bo lectin gidi sinh vat 20 1.2.1 Su phan b6' lectin gi6i thuc vat 20 1.2.2 Su phan b6' lectin gi6i dong v^t 21 1.2.3 Lectin c6 ngubn goc vi sinh vat 22 1.2.4 Sir dinh khu cua lectin te bao va co the sinh vat 23 1.3.Cac tinh chat hoa hoc, sinh hoc, m i i n dich hoc cua lectin va ling dung 24 1.3.1 Kh6'i lugng phan tir lectin 24 1.3.2 Cau tao phan tir lectin 26 1.3.3 Tinh chat ly, hoa hoc cua lectin 30 1.3.3.1 Tinh tan va ket tiia 30 1.3.3.2 Thanh phan hoa hoc cua lectin 30 1.3.3.3 Su tuang tac cua lectin vdi cac loai duang 31 1.3.3.4 Anh hucfng cua m6t so tac nhan moi tru&ng den hoat lectin „ 34 1.3.4 Tinh chat sinh hoc va mien dich hoc cua lectin 35 1.3.4.1 Kha nang kich thich phan bao va ngung ket te bao 35 1.3.4.2 Vai tro ciaa lectin li6n ket vi khuan c6 dinh dam 41 1.3.4.3 Vai tro cua lectin doi vdi vi6c bao vt ca the sinh vat 42 1.3.4.4 Vai tro ciia lectin la cha't du trii va mang dac tinh enzim 43 1.3.5 Uhg dung lectin d5i song 44 Chuang : NGUYEN LIEU VA PHUONG P H A P NGHIEN CUU 47 Nguyen li6u 47 2.1.1 Ho dau (Fabaceae) 47 2.1.1.1 Loai dau cove {Phaseolus vulgaris L.) 2.1.1.2 Loai dau guam {Canavalia 2.1.1.3 Loai dau rua {Canavalia gladiata jacq.) DC ensiformi 2.1.1.4 Loai dau van {Lablab purpureus 2.1.2 Ho dau tam {Moraceac) Loai mit mat {Artocarpus 47 s L.) DC L Sweet) 49 49 50 : heterophyllus Lamk) 51 2.2 Phuang phap nghien ciJu 53 2.2.1 Xtr ly va bao quan mSu thi nghiem 53 2.2.2 Phuang phap tach lectin 54 2.2.3 Xac djnh ham lugng protein 54 2.2.4.Phuang phap xac dinh hoat d6 lectin 55 2.2.5 Tinh chd' lectin 56 2.2.5.1 Sac ky loc gel Sephadex G - 75 va G - 100 56 2.2.5.2 Sac ky trao d6i ion DEAE - Xenluloza va DEAE - Trysacryl 2.2.5.3 Sac ky luc 56 56 2.2.6 ICiem tra tinh sach va xac dinh khoi lugng phan tir lectin 58 2.2.7 Xac dinh mot s6' tinh chat sinh hoa cua lectin 59 2.2.8 Phuang phap xac dinh ph'an axit amin 62 2.2.9 Phan tich trinh tu axit amin tan ciing d'au N 63 2.2.10 Phuang phap tinh che IgAi 64 Chuang : KET QUA VA BAN LUAN 66 3.1 Tinh che lectin iix giong trbng loai dau cove {Phaseolus vulgaris L,) va nghien cihi tinh cha't ciia chiing 66 3.1.1 Tinh ch6' lectin tir giong trbng loai dau cove 66 3.1.2 Nghien ciiu so sanh m6t s6' tmh cha't sinh hoa cua lectin tir hat cua gi6'ng trbng loai dau cove 71 3.1.2.1 Anh huang cua pH den hoat lectin 71 3.1.2.2 Anh hucfng cua nhidt d6 m6i tru5ng 71 3.1.2.3 Cac yeu t6 kich thich va kim ham hoat lectin 72 3.1.2.4 Su tuang tac dac hieu dudng 74 3.1.2.5 Khbi lugng phan tu 76 3.1.3 Nhan xet chung 78 3.2 Tinh che cac lectin tir hai loai dau ciing chi, dau guam {Canavalia gladiata jacq DC) va dau rua (Canavalia ensiformis L DC) va nghien ciiu so sanh m6t s6' tinh cha't ciia chung 3.2.1 Tinh ch€ lectin tir hat dau guam va dau rua 79 80 3.2.2 Nghien ciiu so sanh mot s6' tinh cha't sinh hoa ciia lectin dau guam va dau rua 83 3.2.2.1 Anh huang cua pH moi trucrng 83 3.2.2.2 Su tuang tac vcfi cac loai dudng 84 3.2.2.3 So sanh cau tao phan tu 85 3.2.3 Nhan xet chung 90 3.3 So sanh tinh cha't sinh hoa va ca'u tao phan tu cua lectin tir hai loai dau khac chi : Loai dau cove {Phaseolus va loai dau guam (Canavalia vulgaris L gladiata jacq ) 91 3.3.1 So sanh m6t s6' tinh chat sinh hoa 91 3.3.1.1 Anh hucfng cua nhiet d6 91 3.3.1.2 Anh hudng cua pH m6i trudng 92 3.3.1.3 Tuang tac dac hi6u ducfng 92 3.3.1.4 So sanh cau tao phan tii 92 3.3.2 Nhan xet chung 95 3.4 Nghidn ciiu d6ng thai lectin qua trinh sinh truang va phat trien cua cay dau van den {Lablab purpureus hat cay mit mat {Artocarpus heterophyllus L Sweet) va tir Lamk) 95 3.4.1 Nghien ciiu d6ng thai lectin tir cay dau van den 96 3.4.1.1 Sir tich liiy lectin cac b6 phan ciia cay dau van den cac giai doan sinh truang va phat trien 96 3.4.1.2 Xac dinh m6t s6' dac diem sinh hoa ciia lectin cay dau van den 97 3.4.1.3 Nhan xet 98 3.4.2 Nghien ciiu dong thai lectin qua trinh sinh truang cua hat cay mit mat 98 3.4.2.1 Tinh che lectin qua cac giai doan sinh truang cua hat mit mat 99 3.4.2.2 M6t so tinh chat sinh hoa ciia lectin 104 3.4.3 Nhan xet chung 106 3.5 Uhg dung lectin mit mat : (Artocarpus heterophyllus Lamk) nghi6n ciiu chdn doan bdnh bazadd va benh vi^m gan sieu vi trung a ngu6i 3.5.1 Dieu che c6t Sepharose - 4B - LM 107 107 3.5.2 Tinh che IgAi huyet ngucfi binh thucrng va benh ly 108 3.5.3 Nhan xet chung 112 Chuang : KET LUAN VA KHUYENNGHI 117 4.1 Ket luan 117 4.2 Khuy^'n nghi 11^ Tai lieu tham khao 12Q Phu luc 135 MO DAU Tren 100 nam trudc day (vao nam 1888) lectin da dugc phat hien va nghien ciiu Ngay nay, cac nha khoa hoc da khang dinh ban cha't hoa hoc cua lectin chinh la nhOng protein va chu yeu la glycoprotein c6 mat a nhieu ca the dong vat, thuc vat, vi sinh vat va ca d ca the ngu5i Nhieu dac tinh hoa sinh cua lectin da dugc m6 ta Cac lectin da dugc xac nhan la nhung cha't CO vai tro du tru eho te bao va ca the, c6 kha nang bao v6 va lam ngung ket te bao m6t each chon loc v.v (Bourillon, 1973, Sharon, 1979) Viec kham pha kha nang tuang tac va nhan biet cac loai te bao cua lectin da md nhieu trien vong to Idn M6t s6 dang lectin da dugc su dung nghien ciiu chan doan cac bdnh truyi^n nhilm vi chiing c6 kha nang tuang tac dac hieu vdfi mot s6 dang vi khuan va vinit (Sharon, 1979 ; Doyle, 1994) Nhieu lectin c6 kha nang gay ngung ket tinh trung ngu5i nen da dugc nghien ciiu de sir dung chiing nhu m6t loai thuoc tranh thai (Chulavanatratol, 1987) M6t s6 dang lectin khac lai c6 kha nang kim ham su phat tri^n cua cac t^' bao di dang vi vay chiing da dugc sir dung d^ nghien ciiu chan doan benh ung thu (Bourillon, 1973 ; Marrink et al, 1990) hoac chan doan va phong ngira benh suy giam mi6n dich AIDS (Pineau et al, 1990) Nghien ciiu lectin d Viet nam da dugc ti^'n hanh hang chuc nam chuang trinh hgp tac nghien ciiu khoa hoc vdi trung tam khoa hoc quoc gia Phap (CNRS) va trudng dai hoc Paris Nhimg ket qua nghien ciiu cua cac nha khoa hoc Viet nam da khang dinh : lectin rat bien cac ca th^ d6ng vat va thuc vat cua nu6c ta Trong sb cac lectin c6 ngubn gbc thuc vat, dang chii y nha't la lectin tii cac loai thu6c ho dau {Fabaceae) va ho dau tam {Moraceae) Mot so cong trinh khoa hoc ve lectin thu6c hai ho da dugc c6ng bb (Nguy§n Qubc Khang va c6ng su, 1983, 1989 ; D6 Ngoc Lien va cong su, 1987^1991, 1992, 1993, 1994, 1995) Lectin tir cac loai thu6c ho Fabaceae va Moraceae c6 nhieu tiem nang Idn thuc tiln n^n chiing toi da tien hanh cac nghien ciiu sau han v'lS chiing Do vay du6i su hudfng dan khoa hoc cua thay D6 Ngoc Lien, PGS PTS khoa hoc Sinh hoc, chung t6i da tie'n hanh tfe tai : 'Tinh che, nghien cihi m6t sb tinh chat ciia cac lectin tir mot sb loai thuoc ho dau {Fabaceae), ho dau tam {Moraceae) va kha nang ling dung y hoc" Muc dich dat eho luan an la : tien hanh nghien ciiu sau ve cau tnic phan tir va mot sb tinh chat sinh hoa cua cac dang lectin ke ca dong thai hoc cua chiing, trdn ca sd tha'y dugc sir bien d6i cua lectin qua trinh sinh truang va phat trien cua ca the va qua trinh tien hoa cua loai Tir dinh hudng eho qua trinh khai thac lectin va su dung chiing nghien ciiu y hoc, sinh hoc va mitn dich Mat khac sii dung mot vai dang lectin da tinh sach theo ti^u chuan qubc te dugc thuc hien dieu kien phong thi nghiem cua nude ta de nghien ciiu ling dung y hoc, luan an mubn dong gop nhung dan lieu khoa hoc ve tiem nang to Ibn cua cac lectin CO ngubn gbc noi dia Viet Nam viec bao v6 siic khoe cong dong dan t6c, van de c'an thi^'t dang dugc toan xa hCi quan tam De dat dugc muc dich do, nhi6m vu ca ban dat eho luan an la : - Tach, tinh chS' va nghien ciiu m6t sb tinh chat hoa sinh va cau tao phan tu cua cac dang lectin tii mbt sb loai cay ho dau {Fabaceae) va ho dau tam {Moraceae), 10 - Nghien ciiu d6ng thai lectin qua trinh sinh truang va phat trien ca the va cua b6 phan tir m6t vai loai thu6c hai ho thuc vat noi tren - Nghien curu so sanh cau tnic phan tir va dac diem sinh hoa cua cac dang lectin di tha'y dugc su bien d6i cua lectin pham vi m6t loai va giiia cac loai khac - Sir dung lectin tir hat cay mit mat {Artocarpus heterophyllus Lamk) de nghien ciiu ung dung y hoc dua vao dac tinh mi^n dich cua no Vdi nhiSm vu ca ban tr6n luan an nham gop phan vao viec mo ta sau hem, day du han ve cau tao phan tir va mot sb dac tinh sinh hoa cua cac dang lectin tii m6t s6' loai thuoc ho dau {Fabaceae) va ho dau tam {Moraceae), Nhiing dong gop mdi cua luan an la : Nhiing dan lieu khoa hoc vt cau tao phan tir, vt cac dac tinh sinh hoa va mi6n dich, ve dong thai hoc cua cac dang lectin tir cac loai dau cove {Phaseolus vulgaris L.), dau van {Lablab purpureus L Sweet), dau guam {Canavalia gladiata Jacq.) va cay mit mat {Artocarpus heterophyllus Lamk) la nhung dan lieu khoa hoc dhu tien cua nu6c ta ve van de Lan d^u tien, mot sb lectin da dugc tinh che tai Vi6t Nam c6 d6 tinh sach theo tieu chuan qu6'c te c6 the dung eho nghien ciiu cau tnic bac nhat va ling dung y hoc Luan an ciing da dua nhung nhan xet ban dau mang tinh qui luat ve su bien doi cua lectin pham vi m6t loai va giira cac loai khac No la ca sd khoa hoc d^u tien eho vi6c sir dung ca'u tao phan tuf lectin phan loai thuc vat Ciing la lan dau tien d nude ta iitm nang to Icfn nghien ciiu y hoc cua lectin , cu the la lectin mit mat {Artocarpus heterophyllus Lamk) dang dugc thir nghi6m c6 ket qua Chiing t6i hy vong rang luan an se gop phan nham lam d^y du han, sau han ca Vt ly thuyet va thuc ti§n vi6c m6 ta cac lectin tir m6t sb 24 American Society for Biochemmistry and Molecular Biology Vol 269 (33), 1994, p p : 20807-20810 32 Blumberg chromatography P of M and penicillin Strominger binding J L - Covalcnl compcments from affinity bacterial membranes Methods in Enzymology XXXIV 1974, p : 401 - 405 33 Bunn - Moreno M and Campos - Neto A - Lectin extracted from seeds of Artocarpus integrifolia (jack fruit) : Potent and selective stimulator of distinct human T and B cell function Immunol J 127, 1981, p : 427 - 429 34 Chulavanatratol M - Spermagglutinination and immubilization strategics for vagianal contractive Proceeding of the first congress of the Asian and Oceanian physiological society (Thailand) 1987, p ; 241 - 294 35 Chatelain C , Lemoine A., Dugoujon J.M., Blanc M and Rouge P.Interaction of polyclonal and monoclonal human IgG with various lectins (ConA, pea lectins) Lectins - Biology Vol III 1982, pp : 460-465 36 Corbeau P., Haran M., Binz H and Devaux C - Jacalin, a lectin with anti - HIV - I properties and HIV - I gp 120 envelope prolein interact with distinct regions of the CD4 molecule Molecular immunology Vol 31 (8) 1994, p p : - 37 Cunningham B A., Hemperiy J., Hopp T P and Edelman G M Structure of ConA Proc Nath Acad Sci USA 76, 1975 p : 3218-3222 38 Davis B J., - 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Hoat lectin dbi chiing la 256 d.v HAAb pH 7,4 , nhiet 25''C Nhiet "C Hoat lectin Ihco nhiet va thoi gian thf nghiem 24 gia 48 gia 72gicy 96 gia 144giaf 256 256 256 256 256 256 256 256 256 256 20 256 256 256 256 256 30 256 256 256 256 256 35 256 256 256 256 64 40 64 64 0 60 0 k.x.d k.x.d k.x.d 136 Phu luc Tinh che IgAi huyei ngubi binh thirbng bang sac ky lire qua c6t Sepharose-4B-LM Chiai rut bang a - Metylgalaetozit 0,05 M Thu 18 phan doan, mbi phan doan ml Tbc d6 chay 15 ml/gib MAu BTI MAu BT2 M^u BT3 M^u BT4 MAu BT5 Mau BI Xoi) s'rr Ol) srr OD STT on STV Ol) sr\ Ol) SIT Ol) srr Ol) I 0.020 I 0,000 0,000 I 0,000 0,015 I 0.020 I 0.009 O.I 00 0,052 0.060 0,110 0,060 0,070 0.075 0,130 0.180 0,150 0,140 0,190 0.140 0.150 O.I 10 0,090 0,090 0.120 0,080 0,110 0,100 0.060 0.080 0,070 0,070 0.060 0,080 0,070 0,050 0,050 0,050 0,050 0,(W0 0,060 0,050 O.fUO 0,(U0 0,050 0.050 0,(U0 0,(U0 0.040 0,{W0 0,035 0,(W0 0,040 0.030 0,030 0.025 0,027 0,030 0,(U0 0.030 0,030 0,030 0,030 10 0.030 10 0,030 10 0,035 10 0.025 10 0,020 10 0,020 10 0.025 11 0.027 II 0,025 11 0,030 11 0,025 11 0,020 It 0.025 11 0,025 12 0.026 12 0.025 12 0,030 12 0.020 12 0,025 12 0.030 12 0,026 13 0,020 13 0.020 13 0,025 13 0,020 13 0,020 13 0.030 13 0.020 14 0.020 14 0,020 14 0.020 14 0,010 14 0,015 14 0,030 14 0.020 15 0,020 15 0,025 15 0.020 15 0,015 15 0,010 15 0,015 15 0.017 16 0,010 16 0,020 16 0,020 16 0,010 16 0,010 16 0,010 16 0,013 17 0,000 17 0.000 17 0,020 17 0,010 17 0,000 17 0,010 17 0,007 18 0,000 18 0,000 18 0,000 18 0,000 18 0,000 18 0.000 18 0,000 137 Phu luc Tinh che IgAi huydi ngubi benh viem gan bang sdc ky lire qua cot Sepharose-4B-LM Chiei riit bang a - Metylgalaetozit 0,05 M Thu 18 phan doan, mfii phan doan ml Tbc d6 chay 15 ml/gib MAu VfJI MAu vc;2 sn MAu V(i3 MA u VG4 sn MAu VC]5 MAu V(;6 MAu VG7 Xoi) Ol) sr\ OD SI'I Ol) s'l-r Ol) srr Ol) I 0,010 I 0,025 I 0.020 0,020 0,020 0,120 O.I 00 0,080 0.070 0,090 0,090 0,130 0,140 0,135 0.140 0,135 0,013 0.095 0,080 0,100 0,100 0.090 0,090 0,090 0,085 0,080 0,075 0,100 0,090 0.080 0,086 0.080 0,080 0,070 0,075 0.090 0,090 0.080 0,080 0.085 0,080 0,070 0,070 0.090 0,080 0,070 0,077 0,085 0,080 0.075 0.080 0,090 0.080 0,070 0.080 0,090 0,075 0,070 0,070 0,090 0,080 0,070 0.077 10 0,070 10 0,075 10 0.080 10 0,095 10 0,080 10 0,060 10 0.070 It 0,070 It 0,070 II 0,070 11 0,060 II 0,095 11 0,075 II 0,060 11 0,070 12 0,085 12 0.070 12 0,075 12 0,070 12 0,080 12 0,075 12 0,050 12 0.070 13 0.085 13 0.070 13 0,075 13 0,065 13 0,080 13 0.070 13 0,050 13 0,070 14 0,075 14 0,075 14 0,060 14 0,060 14 0,080 14 0,060 14 O.CMO 14 0,050 15 0.060 15 0,065 15 0,065 15 0,060 15 0.060 15 0,060 15 0.(U0 15 0,060 16 0,050 16 0,060 16 0.060 16 0,050 16 O.CWO 16 0.050 16 o.cuo 16 0,050 17 0,050 17 0,050 17 0,040 17 0,030 17 0,020 17 0.030 17 0,020 17 0.030 18 0,020 18 0.030 IS 0,020 * 18 0.020 18 0,010 18 0,020 18 0,010 18 0,020 Ol) sri Ol) 0,020 0,010 0.095 0,085 0.120 0,140 0,095 0.090 ST\ Ol) 0.(M0 10 0,090 138 Phu luc Tinh che IgAi huyei ngubi benh bazotdo bang sac ky lire qua cot Sepharose-4B-LM, Chiei rut bang a - Metylgalaetozit 0,05 M Thu 18 phan doan, mbi phan doan ml Tbc chay 15 ml/gia Man MBI Mau MB2 Mau MB3 Xoi) sT'r Ol) srr on S'l'r Ol) sir Ol) 0.030 0,020 0,000 0,015 0.060 0.075 0,070 0.065 0,130 0,150 0,120 0.133 0,100 0,085 0,080 0,090 0,075 0.050 0,070 0,065 0,050 0.(U0 0,050 0,046 0,(W0 0,(45 0,(M0 O.CMO 0,(U0 0,035 o,mo O.CUO 0,035 0.035 0,030 0,033 to 0,035 10 0.035 10 0.020 10 0,026 11 0,030 11 0,030 II 0.025 It 0.030 12 0,030 12 0.025 12 0.020 12 0,025 13 0.030 13 0,025 13 0,025 13 0,025 14 0.025 14 0,020 14 0.020 14 0,020 15 0,020 15 0,025 15 0,020 15 0.020 16 0,020 16 0,025 16 0.010 16 0.020 17 0,015 17 0,000 17 0,000 17 0,005 18 0,000 18 0,000 18 0,000 18 0,000