Thông tin tài liệu
KHOA HQC KY THUAT THU Y TAP XXV SO - 2018 O A N H Gift K H A N A N G G A Y D A P UfNG M l i N DWH C H U O T C U A P R O T E IN DUNG HtfP O Q C T O D U O N G R U O T T tf V IK H U A N E N T E R O T O X IG E N IC ESCHERICHIA COLI Le Dinh Hai1, Doan Van That1, Dang Van Tuan1, Vo Thanh Thin1 TOM TAT Vi khuan enterotoxigenic E coli (ETEC) la mot nhung tac nhan chinh gay tieu chay d Ion trade va sau cai sua, lam thiet hai Ion ve kinh te chan nuoi Ion Do vay, viec phong benh tieu chay cho dan Ion la het sue quan Muc tieu cua nghien cuu la san xuat duqc protein dimg hop doc to tinh khiet va bude dau danh gia kha nang gay dap ung mien dich cua protein tren chuot, lam co so cho viec san xuat vacxin phong tieu chay ETEC gay tren lorn Ket qua nghien cuu cho thay protein dung hop doc td vdi ham luqng protein la l-l,5m g/m l da duoc tinh sach bSng he thong AKTA pure 25M Protein dung hop doc to khong gay tich nude dudng tieu hoa cua chuot so sinh (khong doc) va co kha nang kich thich dap ung m ien dich tren chuot nhat trang tiem vdi lieu 200pg bang dudng tiem dudi da hoac bap thit Tit khoa: enterotoxigenic Escherichia coli, protein dung hop doc to, dap ung mien dich Evaluating immune responses in mice of enterotoxin fusion protein from Enterotoxigenic Escherichia coli Le Dinh Hai, Doan Van Thai, Dang Van Tuan, Vo Thanh Thin SUMMARY Enterotoxigenic E coli (ETEC) is an important bacteria causes diarrhea in the sucking and post-weaning piglets, resulting in significant economic losses in pig farming Therefore, prevention of this disease for the piglets is very necessary The objective of this study aimed at producing the pure enterotoxin fusion protein and to evaluate the ability of inducing immune response of this protein in mice The results of this study showed that the enterotoxin fusion protein was successfully purified by AKTA pure 25M system with to 1.5 mg per ml protein concent The purified enterotoxin fusion protein did not induce significant fluid accumulation in the intestinal tract of the suckling mouse, and could induce the immune response in mice significantly with a dose of 200pg by subcutaneous or intramuscular injection Keywords: enterotoxigenic Escherichia coli (ETEC), enterotoxin fusion protein, immune response I DAT VAN DE Vi khuan enterotoxigenic E coli (ETEC) mang khang nguyen bam dinh va tiet doc t6 dudng ruot la mot nhung nguyen nhan chu yeu gay tieu chay d Ion trude va sau cai sua va ket qua la lam giam hieu qua chan '■Phan vien Thu y mien Trong Hoc vien cao hoc, Dai hoc Nong Lam Hud nuoi (Dubreuil va cs., 2016, Nagy va Fekete, 2005) Do vay, viec phong benh tieu chay vi khuan bang vacxin la mot nhung bien phap hieu qua nhat (Melkebeek va cs., 2013, Zhang, 2014) Trong nhieu nam trade day, cac nghien cuu san xuat vacxin phong benh tieu chay vi khuan ETEC d lpn chu yeu la su dung cac yeu to bam dinh de lam khang nguyen san xuat vacxin 13 KHOA HOC KY THUAT THU Y TAP XXV S6 - 2018 Tuy nhien, viec su dung yeu to bam dinh lam khang nguyen thudng khong mang lai hieu qua phong benh vi su da dang cua chung Ngoai ra, mot so chung khong mang khang nguyen bam dmh van co the gay benh tieu chay (Zhang va cs., 2010).Trong do, chinh cac doc to ducmg m ot mdi la nguyen nhan true tiep gay nen hien tugng roi loan trao doi muoi va c h it dien giai ducmg ruot, ket qua la Ion bi tieu chay (Gyles va cs., 2011; Nagy va cs., 1992) Do vay, doc to duong m ot can phai dupe su dung nhu la khang nguyen vacxin phong benh Doc to vi khuan ETEC tiet dupe chia nhdrn la doc to chiu nhiet (STs) va doc to khong chiu nhiet (LT) Trong doc to chiu nhiet STs dupe chia loai, la STa va STb Doc to STa, STb va LT dong vai tro quan gay tieu chay d Ion va khong co kha nang gay mien dich cheo cho Do vay, de phong benh hieu qua, ca loai doc to STa, STb va LT can phai co phan vacxin (Fleckenstein va cs., 2014, Melkebeek va cs., 2013, Zhang va Sack, 2015) Nhieu nghien cuu da chung minh doc to STs co lupng phan tu thap nen kha nang gay dap ung m ien dich cung thap Chinh vi vay ma khong the su dung doc to STs d dang don le de san xuat vacxin Tuy nhien, kha nang gay dap ung mien dich cua doc to se dupe tang len ket hop vdi nhung loai protein mang khac (Frantz va Robertson, 1981; Klipstein va cs., 1982; Sanchez va cs., 1988) Doc to khong chiu nhiet LT chua tieu phan A va B, tieu phan B chi la tieu phan bam dmh khong doc Ben canh co vai tro nhu la mot khang nguyen thi chung dong vai tro nhu la mot chat bo trp, lam tang dap ung m ien dich cua vacxin (Rosales-M endoza & cs., 2011) Chinh vi vay ma hien nay, mot nghien cuu da tao nhung protein dung hop cac loai doc to dudng ruot cua vi khuan ETEC Nhung protein co kha nang kich thich dap ung mien dich cao, cung nhu co kha nang trung hoa cac loai doc to vi khuan ETEC tiet Theo nghien cuu cua You va cs (2011), noi cac doan gen ma hoa doc 14 to STa, LTB va STb mot chuoi gen ma hoa cho mot protein dung hop loai doc to va dupe bieu hien b ln g te bao E coli BL21 vdi he thong bieu hien pET30 Chuot bach sau dupe tiem loai protein dung hop nay, co kha nang bao ho cong cudng doc vdi chung vi khuan ETEC Cung nghien cuu tuong tu nam 2016, Rausch va cs da gay m iln dich cho lpn nai bang chuoi protein dung hop cac loai doc td gom LT, STb, Stx2e va STa (LTR192GSTb-Stx2e-3xSTaP) Lpn sinh tu lpn me dupe tiem vacxin thi cung co kha nang chong lai vi khuan ETEC gay tieu chay Tai Viet Nam, mot so nghien cuu san xuat vacxin phong tieu chay vi khuan ETEC da dupe tien hanh, nhien cac nghien cuu chi su dung dang vacxin chet toan khuan (Dang Xuan Binh va Do Van Trung, 2008; Nguyen Van Thang va cs., 2018) Mac du vay thi phong tieu chay d lpn vi khuan ETEC bang vacxin chet da dupe chung minh la khong hieu qua Trong nghien cuu nay, chung toi da tao chung E coli co kha nang san xuat protein dung hop doc td bao gom loai doc td STa, LTB va STb Trong do, doc td STa da dupe dot bien mot nucleotide nham giam tinh doc cua doc td Ben canh do, protein dung hop doc td dupe bieu hien bang he thong vector bieu hien pET32a nen protein tao se dupe dung hop vdi trinh tu gom amino acid histidine (Vo Thanh Thin va cs., 2018) Trinh tu His-tag se giup protein tai td hop ket bam vao cot sac ky cac protein khac se bi loai bo bang dung dich rira Dieu cho phep viec tinh sach doc td dupe thuan lpi bang he thong sac ky su dung cot sac ky luc Ni-NTA.Trong nghien cuu nay, chung toi tien hanh tinh sach, kiem tra tinh doc cung nhu kha nang gay dap ung mien dich cua protein dung hop loai doc td II NOI DUNG, NGUYEN LIEU VA PHlTONG PHAP NGHIEN CIJ'U 2.1 Noi dung nghien curu - Tinh sach va xac dinh tinh doc cua protein dung hop doc td dudng ruot tai td hop KHOA HOC KY THUAT THU Y TAP XXV s - 2018 - Xac dinh li6u, chat bo trg va dudng tiem gay dap ung mien dich cua protein dung hop doc to 2.2 Nguyen vat lieu - Chung vi khuan: chung vi khuan B L 21 E coli BL21 [ FompT, hsd SB (rB, mB), dcm, gal, k (DE3), pLysS, Cmr] mang vector pET32a co gan chuoi gen tai to hop ma hoa protein dung hop loai doc to ducmg m ot STa-LTB-STb vdi kich thudc 21 kDal (chung SLS321) (Vo Thanh Thin va cs., 2018), chung vi khuan EcoPV-173 co mang gen ma hoa dong thdi loai doc to dudng m ot (ma so GenBank: MF990202.1, KY 581592.1, KY581591.1) (Le Dinh Hai va cs., 2017) - Cac loai moi trirdug nudi cay vi khuan: LB Broth, LB agar - C&c loai hoa chat: IPTG (ThermoFiser - My, ma so R0392); Ampicillin sodium salt (Sigma-Aldrich - My, ma so: A9518); khang the IgG khang chuot (Invitrogen - My, ma so: 31430; khang the khang doc to LTB (Novus Biologicals - My, ma so: N B P1-05446); dung dich ABTS (Invitrogen - My, ma so: 002024); bo kit BCAThermoFiser - My, m a so: 23225); lysozyme (Sigma-Aldrich - My, ma so: L6876G) - Cac loai dem dung de tinh sach doc td + Dem ly giai te bao (lysis buffer): 100 ml PBS, 1mM PMSF, 1OmM beta-Mercaptoethanol, 10 mM Imidazole + Dem lien ket (binding buffer): 20 mM sodium phosphate, 0.5M NaCl, mM imidazole, 6M Urea + Dem phan tach (elute buffer): 20 mM sodium phosphate, 0.5M NaCl, 500mM imidazole, 6M Urea, pH 7,4 2.3 Phirong phap nghien cuu 2.3.1 Tinh sach protein dung hop doc to - Nuoi cay va pha vote bao: chung vi khuan SLS321 (Vo Thanh Thin va cs ,2018) dupe nuoi cay tren moi trucmg LB broth, sau gia tri OD cua canh khuln b ln g 0,6 thi tien hanh bo simg IPTG cho nong cuoi cung la ImM va nuoi cay d 30°C qua dem Vi khuan sau thu hoach bang each ly tarn 4.000 vong/phut 30 phut Xac vi khuan dupe hoan nguyen 30ml lysis buffer co bo sung lysozyme (lm g/m l) va u 30 phut a nhiet dp phong Sau tidn hanh pha hoan toan te bao blng may pha sonicator 10 phut a t6c dp 25.000 vong/phut Dich noi co chua protein dupe thu hoach bang each ly tarn 12.000 vong/phut 20 phut - Tinh sach protein: Protein dung hop doc to tai to hop (SLS) dupe tinh sach bang he thong sac ky AKTA pure 25M voi cot sSc ky Histrap HP theo hudng d in cua nha san xuat Protein dung hop doc to sau dupe tham tich dung dich PBS nham loai bo cac muoi Nong protein dupe xac dinh bang phan ling BCA, tinh sach cua protein dupe kiem tra bang phuorng phap SDS-PAGE 2.3.2 Kiem tra tinh doc cua protein dung hyp doc t(> (SLS) Tinh doc cua protein dung hop doc to dupe kiem tra tren chuot nhat so sinh theo mo ta cua Giannella (1976) Chuot nhat trang so sinh (2-3 tuoi) dupe chia nhom, moi nhom Nhom cho uong 0,2 ml protein SLS (20pg/con), nhom cho uong 0,2 ml dich noi sau nuoi cay 24 gid chung vi khuan EcoPV-173 mang gen ma hoa loai doc to STa, STb va LT Nhom doi chung cho uong 0,2ml dung dich PBS Sau gid, tien hanh gay me, mo va xac dinh ty le lupng cua dudng ruot/ lupng co the lai (G/C) Neu ty le G/C ldn hon 0,09 thi xem la doc to hoat dong 2.3.3 Kiem tra kha nang gay dap im g mien dich cua protein - Xac dinh lieu gay d&p ung mien d|ch: Chuot nhat trang co lupng tir 18-22g, khong co khang the khang lai doc to STa, STb va LTB cua vi khuan E coli, dupe chia nhom, moi nhom 10 Nhom 1, 2, 3, dupe tiem protein SLS vdi cac lieu: 100, 200, 15 KHOA HQC KY THUAT THU Y TAP XXV SO - 2018 300 va 400pg Nhom dugc gay mibn dich canh khuan chung EcoPV-173 dugc bit hoat bing 0,5% formaline Nhom doi chung dugc tiem bang dung dich PBS Sau 21 tiem, chuot dugc lay mau, thu huyet va kiem tra hieu gia khang the bang phuong phap ELISA - Xac dinh dirbng gay dap ung mien djch: kha nang gay dap ung mien dich cua protein dung hop doc to dugc kiem tra bang ba dubng tiem khac la dubi da, bap thit va xoang phuc mac Sau 21 tiem, lay mau kiem tra hieu gia khang the bang phuong phap ELISA - So sanh sir dung chat bo tru: hai chit bb trg la 20% nhom hydroxyt va 10% Montanide gel 01 da dugc su dung de so sanh kha nang kich thich dap ung mien djch tren chuot cua protein dung hgp doc to Sau 21 tiem, lay mau kiem tra hieu gia khang the bang phan ung ELISA - Phuong phap ELISA xac dinh hieu gia khang the khang lai protein doc to dung hop: khang the dac hieu khang lai protein dung hgp doc to dugc xac djnh theo phuong phap cua Zhang va cs (2018) Dia 96 gieng sau dugc phu bang protein dung hgp doc to da tinh sach (10 pg/gieng) dugc u qua dem Sau dia dugc rua lan bang PBS co bo sung 0,5% Tween 20 va block cac gieng bang 2% skim milk PBS Sau loai bo skim milk bang each rua dia lan, lOOjul huyet chuot mien dich, chuot doi chung da pha loang a cac nong khac cung nhu doi chung duong va doi chung am dugc cho vao moi gieng va u 1-2 gib b nhiet phong Sau do, dla dugc rua lan va bo sung lOOpl conjugate (khang the khang IgG cua chuot co gan men HRP) vao moi gieng Sau u 1-2 gib va rua lan, 150pl co chat ABTS dugc bo sung vao moi gieng Ket qua ELISA dugc xac dinh sau gib u b nhiet phong Trong phan ung ELISA, doi chung am la huyet chuot khong co khang the khang lai protein dung hgp doc to, doi chung duong la khang the khang lai doc to LTB III KET QUA VA THAO LUAN 3.1 Ket qua tinh sach protein dung hop doc to duong ruot Protein dung hgp doc to dubng ruot la mot dang protein khong hoa tan (insoluble protein) Sau pha vb te bao, protein dugc tach chiet va tinh sach tren he thong AKTA pure 25M su dung cot HisTrap HP Sau tinh sach, protein tai to hop dugc tham tich de loai bo muoi va kiem tra nong protein bang phuong phap BCA Bo tinh sach dugc kiem tra bang phuong phap dien di SDS-PAGE Ket qua tach chiet va tinh sach dugc bieu thi b hinh Hinh Ket qua tinh sach va kiem sach protein dung hggc) (Le Dinh Hai va cs., 2017b) Theo nghien cuu cua You va cs (2011), lieu toi thieu de doc to STa gay doc d chuot nhat trang so sinh la 9ng Trong nghien cuu nay, chung toi cho chuot uong vdi lieu 20 pg protein dung hgp doc td (khoang 2pg doc 17 KHOA HOC KY THUAT THU Y TAP XXV SO - 2018 Kha nang gay dap ung mien dich tot nhat cua khang nguyen phu thuoc vao nhieu yeu to khac nhu duong tiem, ham luong khang nguyen va chat bo trg Vi vay, nghien cuu nay, chung toi kiem tra kha nang gay dap ung mien djch cua protein dung hop doc to tren chuot nhat trang vdi cac lieu khang nguyen khac nhau, ducmg tiem khac va cac chat bo trg khac Ket qua nghien cuu dupe the hien b hinh to STa) nhung khong gay doc cho chuot Vai tro cua doc to STb doi vai benh tieu chay van chua that ro rang Cac nghien cuu gan day cho thay doc to STb van dong vai tro benh tieu chay a Ion con, nhimg khong quan nhu la doc to STa va LT hoac la mot minh STb khong co kha nang gay benh (Berberov va cs., 2004; Chapman va cs., 2006; Zhang va cs., 2006) Tu ket qua cho thay, protein dung hop doc to khong gay doc cho chuot so sinh nen co the su dung de lam khang nguyen san xuat vacxin Tu hinh a cho thay vai cac lieu tiem tir 100 den 400pg vao duai da, protein dung hpp doc to duang ruot deu co kha nang gay dap ung mien dich tren chuot nhat trang Ham lupng khang the IgG khang lai protein dung hop doc to a nhom mien dich cao han nhom doi chung 3-3,31ogl0 Tuy nhien, lieu phu hpp de gay mien djch tot nhat la 200pg/chuot Hinh Kiem tra tinh doc cua doc to tren chuot nhat trang s c sinh 3.3 Xac djnh kha nang gay dap u-ng mien djch cua protein B cichim f Nghien cuu cua chung toi cung cho thay hai chat bo trp vacxin la nhom hydroxit (20%) va montanide gel 01 (10%) (hinh 3b) deu la nhung chat bo trp phu hpp cho vacxin tieu phan tai to hpp doc to ducmg ruot Khi phoi tron vai protein dung hap doc to, chung gay kich thich dap ung mien dich cao han nhom doi chung la 3,45 log 10 Trong cac duang tiem da thir nghiem (hinh c), chung toi nhan thay duang tiem duai da va bap gay kich thich dap ung mien djeh cao hem nhom doi chung la 3,05 loglO Trong Nhcm Ivbntanids hy&rcodt Chat bo trtf k 01 D uong tiem Hinh Ket qua xac dinh kha nang gay dap (mg mien djch cua protein dung h
Ngày đăng: 03/08/2022, 22:32
Xem thêm:
