HOI NGHI KHOA HOC COA HOI HOA SINH Y DL/QC H A NQI VA CAC T I N H PHIA BAC L A N THLT XVII
XAC DINH CAC PHAN TYP EBNA-1 CUA VIRUS EPSTEIN-BARR 0 CAC MAU BENH PHAM TLf BENH NHAN NPC TAI VIET NAM
^. i i . . i
Hoang Thi Minh Chau', Vu Thi Tien', Nguyen Dinh Phuc^ va Le Thanh Hoa'
TOM TAT
Phan tfch da hinh tai vj tri acid amin 487 ciia EBNA-1 cho phep xac djnh 5 phan typ la P-ala, P-thr, V-val, V-leu, V-pro (P: prototype;
V: Variant). Nam mu'di bay mau benh pham thu nhan tit benh nhan NPC nhap vien tai Ha Ngr(ky hieu HN va EB), Hai Phong (HP), Da Ning (MT) va TP Ho Chi Minh (S), du'dc tach DNA, thi/c hien PCR, thu nhan doan gen EBNA-1 (~0,76 kb); va phan tfch amino acid vj tri 487 de xac djnh phan typ. Ket qua cho thay, tong sd 57 mau NPC cua Viet Nam du'dc phat hien cd 3 phan typ: P-ala, P-thr va V-val, chu'a phat hien du'dc V-leu va V-pro. Ha Ngi cd 36 mau nghien ciiu (29 mau V-val; 5 mau P- thr); Hai Phong cd 6 mau (4 mau V-val, 1 mau P-ala, 1 mau P-thr); Da Nang cd 8 mau (7 mau V-val, 1 mau P-thr); TP Ho Chf Minh cd 9 mau (5 mau V-val, 2 mau P-ala, 2 mau P-thr). Nhu' vay, ba phan typ P-ala, P-thr va V-val deu ton tai trong sd 57 mlu NPC cua Viet Nam, ngoai trif P-ala chu'a phat hien d Ha Ngi, 3 vung con lai deu chiia ca 3 phan typ. Phan typ V-val noi trgi trong tat ca benh pham, du'dc xac nhan cd vai tro trong tien trien ung thu' (tumorigenesis) ciia NPC. Sang Igc phan t i i xac djnh phan typ EBNA-1 de dmh hu'dng phong chong NPC la can thiet.
Tiy khoa: EBV, NPC, EBNA-1, PCR, phan typ, P-ala, P-thr, V-val, V-leu, V-pro.
SUMMARY
DETERMINATION OF EBNA-1 SUBTYPES OF EPSTEIN-BARR VIRUS ISOLATED
FROM NPC PATIENTS I N VIETNAM
' yi?n Cong nghe sinh hoc 'Trumg Dai hoc Y Hd Noi
Analysis of polymorphic variation at amino acid 487 in polypeptide EBNA-1 of Epstein-Barr virus (EBV) allows us to determine 5 subtypes as P-ala P- thr, V-val, V-leu and V-pro (P: prototype; V:
Variant). Fifty seven EBV isolates collected from NPC patients admitted in hospitals in Ha Noi (designated as HN and EB), Hai Phong (HP), Da Nang (MT) and Ho Chi Minh city (S) were subjected to DNA extraction, PCR for obtaining of EBNA-1 fragment (0.76 kb); sequencing; comparative analysis of amino acids; and reconition of a partiicular amino acid at position 487 for subtyping.
As results, there are three subtypes detected in 57 isolates: P-ala, P-thr and V-val in the Vietnamese patients; but V-leu and V-pro not to be found yet.
Subtypes are: 29 V-val and 5 P-thr in 36 samples of Ha Noi; 4 V-val, 1 P-ala and 1 P-thr in 6 samples of Hai Phong; 7 V-val and 1 P-thr in 8 samples of Da Nang; and 5 V-val, 2 P-ala, and 2 P-thr in 9 samples of Ho Chi Minh city. Three common subtypes (V-val, P-ala, and P-thr) are detected in 57 NPC samples of Viet Nam; except for P-ala not found in Ha Noi; samples of other 3 localities contain 3 subtypes. The V-valsubtype was dominant in Vietnam, referred to as the role of playing primary role in tumorigenesis of NPC. Molecular screening for subtyping of EBNA-1 is essential in prevention of the NPC in our country.
Key words: EEN, NPC, EBNA-1, PCR, subtype, P-ala, P-thr, V-val, V-leu, V-pro.
I. DAT VAN DE
Epstein-Barr virus (EBV) gay nhiem phan Idn cgng dong, nhung chi mgt sd phat hien thanh ung thu vdm miii hgng (NPC,
Y HOC VIET NAIVI T H A N G 8 - SO 2/2011
nasopharyngeal carcinoma) (Young, Rickinson, 2004). Protein EBNA-1 (Epstein-Barr Nuclear Antigen 1) la loai cd vai tro quan trgng nhdt anh hudng dkn sir nhan len ciia he gen, sao chep va phan chia te bao (Aiyar et al., 1998; Gruhne et ai, 2009). Polypeptide EBNA-1 chua 641 acid amin (aa), chia lam 2 viing churc nang chinh, cd mgt viing gom nhieu motif lap glycine/alanine (GAR) tir aa 90 den aa 327 (Mai et al, 2010) va dau tan ciing C trung gian cho DNA bam dinh va hinh thanh phan til kep (Wu et al., 2002). Da hinh tai vi tri acid amin 487 cho phep xac dinh 5 phan typ P-ala P-thr, V-val, V-leu, V-pro (P: prototype; V: variant), lien quan den luu hanh EBV trong mo, mau va dich hgng (Bhatia et al., 1996). Theo cac nghien cuu gkn day, EBV phan lap d vimg Nam Trung Qudc chii y6u la V-val (Zhang et al., 2004), ddng vai trd quan trgng tang cudfng sao chep ddi vdi EBNA-1 (Mai et al, 2010).
EBV phan lap tai Viet Nam cQng da dugc nghien curu ve dac diem sinh hgc phan tii, trong do cd phan tich gen EBNA-
1 cung vdi nhilu nghien cuu lam sang, bgnh tich va tiln triln benh (Do et al, 2008). Tuy nhien, viec xac dinh phan typ (subtyping) dua tren bien ddi acid amin 487 con rdt it nghien curu tai Viet Nam, gkn day mdi dugrc chiing toi dk cap den (Le Thanh Hoa e/a/., 2010).
Trong bai bao nay, chung toi phan tich da hinh tai vi tri acid amin 487 ciia EBNA-
1 trong tdng s6 57 chung EBV d cac mlu bgnh phdm tir benh nhan NPC, nhap vien tai Hai Phong, Ha Ngi, Da NEng va TP H6 Chi Minh.
I I . NGUYEN LIEU VA PH^QNG PHAP NGHIEN CU'U
2.1. Mau benh pham va xir ly benh pham cho nghien cihi
MIU mo tuoi thu bang sinh thiet tir benh nhan chan doan la NPC, bao quan d -20°C, chu yeu tir 4 viing: i) nhap vien tai Ha Ngi, ky hieu:
HN va EB; ii) nhap vien tai Hai Phong, ky hieu HP; iii) nhap vien tai TP H6 Chi Minh, ky hieu S; iv) nhap vien tai Da NSng (miln Trung), ky hieu: MT. Tong so co 57 mau da dugc su dung trong nghien curu ciia chiing toi (Bang 1).
DNA tdng s6 dugc tach chilt su dung bg kit cua hang BIONEER (Han Qu6c). Qua trinh xu ly mlu va tach chilt DNA da dugc mo ta tai Le Thanh Hoa era/. (2010).
2.2. Thuc hien PCR, giai trinh tu va phan tich so lieu
Doan gen EBNA-1 (~ 0,76 kb) thu dugc bang PCR vdi khuon la DNA tdng s6 tach chiet tir cac mlu benh phdm, kilm tra, tinh sach true tilp, giai trinh tu, thu nhan chuoi nucleotide;
phan tich xu ly, khao sat amino acid tai vi tri 487 xac dinh phan typ theo Bhatia et al. (1996) nhu mo ta trudc day (Le Thanh Hoa et al., 2010).
I I I . KET QUA VA BAN LUAN
3.L Thu nhan chudi nucleotide va khao sat vi tri aa 487 xac djnh phan typ
Vj tri amino acid 487 dugc svr dung de so sanh ddi chiiu EBNA-1 cua 57 mau EBV thu nhan tir benh nhan NPC nhap vien t^i cac viing.
Mgt so chiing qu6c tl do la chung c6 diln B95- 8 gdm B95-8 (1), B95-8(2) va B95-8(3), dugc su dung dl phan biet prototype; chung C18 va GDI (Trung Qudc) dai dien Nam Trung Qudc ciia chau A va chung AG876(1) va AG876 (2) cd nguon gdc chau Phi (Ghana) dai dien EBV typ 1 ciia chau Phi.
HQI NGH! KHOA HQC COA HQI HOA SINH Y DJOC HA N 6 | VA CAC TJNH PHIA BAC LAN THifXVII
Kit qua cho thdy, tai vi tri 487, 57 chung EBV cua Viet Nam chura 3 loai amino acid, do la V (valine), A (alanine) va T (threonine), tir do dugrc xac dinh thugc 3 loai phan typ: P-thr (Prototype threonine); P-ala (Prototype alanine) va V- val (Variant valine). Khong tim thay cac biln thi phan typ V-leu (Variant-leucine) va V-proline (Variant-proline). Cac biln thi phan ty p V-leu va V-pro thong thuong cd ngudn gdc chau Phi, nhu vay, chua thdy xuat hien tai Viet Nam.
3.2. Ket qua tong hop xac dinh phan typ EBNA-1 tai Viet Nam
Ket qua xac dinh phan typ cua 57 chiing ciia Viet Nam va 7 chiing tham
chiiu cua thi gidi, dugrc liet ke a Bang 1. Bang 1 cho thay chi tiet:
Trong tdng s6 34 mau EBV cua Ha N^i (ky hieu EB va HN) cd 29 mlu thugc V-val, con lai 5 mau thugc P-thr (EB12, EB102, EB108,HNIlc4vaHNll);
Trong 6 mlu ciia Hai Phong ky hieu HP, cd 4 mlu thugc V-val, 1 mlu thugc P-thr (HP4) va 1 mlu thugc P-ala (HP6);
- Trong 8 mlu ciia mien trung ky hieu MT tir benh nhan nhap vien Da Nlng, cd 7 mlu thugc V-val, I mlu thugc P-thr (MT7);
Trong 9 mlu TP H6 Chi Minh, cd 5 mau thugc V-val, 2 mlu thugc P-ala (S18 va S20) va 2 mau thugc P-thr (S7 va S27).
Bang 1. Tdng kit kit qua xac dinh phan typ EBNA-I ciia cac chiing EBV Viet Nam qua phan tich amino acid tai vi tri 487.
TT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Chung
EBl EB3 EB4 EB5 EB7 EBB EB9 EBIO EB10C2 EB12 EB13 EB14 EB17 EB18 EB19 EB20 EB61 EBIOO
Amino acid d vj
tri 487 V V V V V V V V V T V V V V V V V V
Xac djnh phan typ
V-val V-val V-val V-val V-val V-val V-val V-val V-val P-thr V-val V-val V-val V-val V-val V-val V-val V-val
TT
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Chung
H P l HP2 HP3 HP4 HP5 HP6 MT7 MT9 MTIO M T l l MT12 MT13 MT14 MT20 S I S5 S7 S l l
Amino acid d vi
tri 487 V V V T V A T V V V V V V V V V T V
Xac djnh phan typ
V-val V-val V-val P-thr V-val P-ala P-thr V-val V-val V-val V-val V-val V-val V-val V-val V-val P-thr V-val 1
Y HQC VlgT NAM THANG 8 - SO 2/2011
19 EB102 P-thr 53 S16 V-val
20 EB103 V-val 54 S17 V-val
21 EB106 V-val 55 S18 P-ala
22 EB107 V-val 56 S20 P-ala
23 EB108 P-thr 57 S27 P-thr
24 E B l l l V-val 58 AG876-1 V-leu
25 HN2 V-val 59 AG876-2 V-leu
26 HN2cl V-val 60 B958-1 P-ala
27 HN9 V-val 61 B958-2 P-ala
28 HN11C4 P-thr 62 B958-3 P-ala
29 H N l l P-thr 63 C18 P-thr
30 HN12 V-val 64 GDI V-val
31 HN14 V-val
32 HN17 V-val
33 HN24 V-val
34 HN26 V-val
Ghi chii: Trong so 57 chiing cua Viet Nam, cac chiing thugc cac phan typ ngoai V-val dugc bdi d$m.
Nhu vSy, kit qua phan tich amino acid cua 57 chiing EBV Viet Nam cho thiy cd tdn tai 3 phan typ (V-val, P-thr va P-ala);
chilm d^ida. sd (46 chdng) thugc v l V-val.
S6 con lai (11 chung) dugc phan chia gdm P-thr va P-ala, trong dd tit ca cac chiing d Ha Ngi chi cd P-thr; con d Hai Phong, Da Ning va TP Ho Chi Minh con cd them P- ala. EBV cd dac tinh da hinh biln ddi giiia cac chiing phan l^p 6 cac vung dia ly thugc Nam va Ddng Nam chau A (Zhang et al., 2004; Wang et al., 2010) va cac chung EBV thugc V-val d vung Nam Trung Qudc dugc chumg minh cd kha nang tang cudmg sao chep va cd vai tro quan trgng trong tien hiln NPC (Mai et al., 2007; 2010). Viec
\Ac dinh dugrc 3 phan typ (V-val; P-thr va P-ala) trong tong so 57 mau EBV cua Viet Nam trong nghien curu cua chiing toi, khang dinh tinh da hinh phan typ ciia EBV vk Cling cho thiy EBV Vigt Nam, v l mat djch te hgc, cd bi anh hudmg ciia vet dich
te EBV tir Quang Ddng Nam Trung Quoc tdi Dong Nam chau A. Sir tuomg quan giira cac chung thugc cac phan typ khac nhau vdi dich te va benh ly benh cimg nhu tiln trien ung thu NPC nhu thi nao d Viet Nam la mgt ITnh vuc rkt can nghien cuu.
IV. KET LUAN
Trong sd 57 mlu NPC ciia Viet Nam, chi cd 3 phan typ P-ala, P-thr va V-val dugc phat hien, frong dd ndi trgi la V-val chilm ddng nhlt; ngoai trir P-ala chua phat hien d cac mlu Ha Ngi, cac mlu d 3 vung con lai (Hai Phdng, Da Ning, TP H6 Chi Minh) dIu chua ca 3 phan typ. Phan typ V-val ndi trgi trong tat ca cac mlu, duoc xac nhan cd vai trd trong tien trien ung thu (tumorigenesis) cua NPC va phan bd dia ly viing Nam va Pong Nam A, cd thi, cd anh hudng din NPC tai Viet Nam.
LCJI CAM ON: Chiing toi cam an syr hd tra kinh phi cua dk tdi nhanh do PGS.TS Le Thanh Hoa chu nhiem, thuoc de tdi
HQI NGH! KHOA HQC CCiA HQI HOA SINH Y D JQC HA NQI vA CAC TINH PHIA BAC LAN THlf XVII
KClO.31/06-10 cua Bo Khoa hoc vd Cong nghe do PGS.TS Nguyen Dinh Phiic chu nhiem. Cam an Phong Thi nghiem trong diem Cong nghe gen (Vien Cong nghe sinh
hoc) bao dam trang thiet bi de thuc hien 6.
cong trinh nay.
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