i/TAO DOIMG V A BIEU Hl£l\l GIEIM M A HOA PROTEIIXI CpB3 TLT CRYPTOSPaSUDtUM PABVUM
Dmh Thi Bich LinS Phfmg Thing Long2, Nguydn Hoing LOc^
Trin Thtiy LanS Ld Qudc Vi^S Phan Vin Quydt^ Hoing T ^ Quing^
T6MTAT
Da tao ddng gien cp23 ma hda protein Cp23 cua Cryptosporidium parvum phSn lap tir cac miu phan bd d Thira Thidn - Hu^. Gien cp23 tir C. parvum phin lip a Thira TTudn - Hue cfl tiinh tu nucleotit tuong dong 99% va tiinh tu axit amin tuong dong 100% vfli gien cp23Az dupc cflng bd tidn Genhank (ma sd U34390).
Phan tich kdt qua didn di SDS va Western blot cho tiiay gien cp^tiong E. cofi"BL21 Star™ (DES) tai td hpp xuit hidn cac dogn polypeptit voi khdi lugng phin tu xIp xi 37 va 49 kDa. Diy co the la cac dang protein Cp23 dung hgp co ho§c khflng cfl gfycozit boa Protem Cp23 tai td hgp duoc tinh sgch bing sic ky ii luc GST (glutathione S-transferase) co nong do 3500 ug/mL cfl thd su dung lam khang nguyen d^ san xuit vie xin hoac khang till chdng lai C. parvum cung nhu san xuit kit chan doan bdnh do C. parvum giy ra.
T i khfla: Cryptosporidium parvum, gien cp23, khing nguy&n tii tShgp Cp23.
I.BATVJiNB^
Cryptosporidium parvum {C. parvum) Ii mdt ky smh trimg Iiy nhidm chung giua ngudi vi nhidu loii ddng vit. Ngtrdi vi gia sdc se bi nhidm bdnh khi nudt phai tning C. parvum trudng thanh trong thiic in, ntrde udng, sua, nudc tim, nudc trong bd boi...
BOnh do Cryptosporidium giy ra cd mit d nhidu noi ti-dn tiid gidi (O'Donoghue P. J., 1995). O Vidt Nam da cd mOt sd nghidn ciiu ve ty Id nhidm Cryptosporidium, trong dd cd C. parvum b ngudi (Ld Cdng Din et al., 1998; Tran Thi Kim Dung et al., 1997) vi gia siie (Dinh Thi Bich Lin et al., 2013;
Nguydn Thi Sim et al., 2007). Nghidn ciiu trudc ciia cbiing tdi ff)inh Thi Bich Lin et al., 2013) d i cho thiy ty 1$ nhidm C. parvum b bd nudi tai Vidt Nam Ii tiiong ddi cao (35,6% ). Yi vgy, bdnh do C. parvum giy ra ciing li mOt trong nhfing bdnh can dugc quan tim nghidn ciiu nhlm hgn ehe nhirng thidt hai cho nginh chin nudi ddng thai bio vg siie khde cho cOng ddng.
C. parvum cd nhidu loai khing nguyen bd mat cd gii tri cao trong chan doin vi sin xuat vie xin hoac khing tiid, ti-ong dd cd Cp23. Protein Cp23 li mdt dgng glycoprotein bd mit, cd khdi Itrgng phin ttr 23 kDa, cd tinh bao tdn cao giiia cic chiing C. parvum b cic vung dja ly khic nhau, Proteui Cp23 dtroc xic
Vi$n Cdng ngh? sinh hpc, D^ii hpc Hue
^ Tnrdng Dpi hge Nong Lam. Dgi hpc Hue ' Truong Dgi hpc Khoa hpc. D?i hpc Hue
dinh l i mOt khing nguydn mang epitope cd dO nhay cao trong phan iing trung hda. Vi viy, chiing dugc xem Ii mdt trong nhung ling vidn tidm ning dd phit tridn vac xin (Ebrahimzadeh et al., 2009) vi khing thd di loai chdng lai bdnh do C. parvum giy ra, hoic Iim nguydn Udu de phit tridn KIT chan doan (ELISA, que nhung) vi chiing cd the phit hidn ra C. parvum tir nhieu viing dia ly khic nhau (Ferryman et al., 1996).
Trong nghien ciiu niy, chiing tdi d i tao ddng v i bidu hidn gien m i hda khing nguydn Cp23 tir C.
parvum phin lip d Thira Tiiidn - Hud va d i sin xuit thinh cdng khing nguydn Cp23 tai td hgp. Tinh die hidu ciia khing nguydn tii td hgp Cp23 cung d i dugc kiem tra bang Western blot.
2. NM DUNG VA PHUDNG PIUP N G H H CUU 2.1. Xic dinh oocysts ciia C. parvum Miu phin bd dugc thu thgp tai cic tiang trgi 6 Thira Thidn - Hue. Qui trinh chuan hi miu dugc thuc hign theo md ta ciia Wang va ddng tic gia (2009).
Oocysts cua C. parvum dugc xic dinh theo phirong phip nhudm Ziehl-Neelsen cii tidn (MZN).
2.2. Tich chidt ADN vi phin lip gien cp23 ADN tdng sd ciia C. parvum dugc tich chiet bing QIAamp DNA Stool Mini Kit (Qiagen, Max- Vohner-Strafie, Hilden). ADN tdng sd sau dd dugc su dung lim khudn miu de phin Igp gien cp23vb\ cip mdi die hidu (Cp23F 5'- ACGGAlC£AAAAATGGGTTGTT-3' vi Cp23R: 5'-
NdNG NGHIEP VA PHAT TRI^N NdNG THdN - KY 2 - THANG 8/2014 59
ACGGATCCTAATTrAGGCATCA-3') dugc tiiiet kd dua tren tiinh tu gien cp23 dugc cdng bd trdn Genhank (ma sd U34390). Trinh tir GGATCC dugc thiet ke cho vi tri tao ddng cua BaniRl tuong ung tren vector bidu hidn pGEX 4T-3 (Inpankaew et al., 2009).
Thanh phdn phan ung PCR bao gdm 2 pi ADN khudn miu, 12,5 pi 2x GoTag PCR master mix (Promega, Madison, WI), 10 pmol mdi mdi loai, ntroc elt dugc bd sung den thd tich 25 pi. Phan ung PCR dugc thuc hien trdn he thdng iCycIer thermocyeler (Bio-Rad, Hercules, CA), bao gdm bidn tinh ADN tdng sd 3 phut d 94°C, tidp theo la 40 chu ky 1 phut bidn tinh d 94*'C, 45 giay gin mdi d 54''C v i 1 phut keo dai chudi d 72"'C. Cudi cung, nhiet dp dugc duy tri d 72''C tiong 7 phiit dd hoan thidn qua trinh tdng hpp. San phim PCR duoc tinh sach bing Quantum Prep™ Freeze N Squeeze DNA Gel Extraction Spm Columns Kit (Bio-Rad) sau khi dien di tren agaroza gel 2%.
2.3. Tao ddng gien cp23
San pham PCR (gien cp23) duac gin vao vector pGEM®-T Easy (Promega), hdn hpp phan img gan bao gdm 50 ng vector, 5 pi dem, 3 unit T4 ADN ligaza vi 25 ng san pham PCR, nudc cat dugc bd sung den the tich 10 pi. Phan iing gin dugc thuc hien d 4''C qua ddm. San pham gin dugc bidn nap vao te bio E. coi'DH5a k h i bien bing phuong phip shock nhidt. The bien nap duoc xic dinh bing ky thuat PCR vol cic didu kien nhu da md ta a trdn. The bien nap dugc nudi tiong 5 ml mdi trudng LB cd bd sung 50 pg/ml ampixilin qua ddm de thu sinh khdi. Vector tii td hpp (pGEM®-T Easy/cpi*^ dugc tiiu nhan tir E.
coh tai td hgp bing FureLmk"™ Quick Plasmid Miniprep Kit (Invitrogen, Carlsbad, CA). San pham PCR dugc phan tich tiinh tu bing phuong phap dideoxytermmator.
2.4. Bidu hidn protein Cp23 trong E. coll Gien cp23 dugc cit khdi vector pGEM®-T Y.d&ylcp23 bang enzym BarrMS., sau do duac tinh sach bang Wizard® SV Gel and PCR Clean-Up System Kit (Promega) vi gan vao vector bieu hien pGEX 4T-3 chiia gien ma hoa glutathion S- transferaza (GST) (GE Healthcare, Bjdrkgatan, Uppsala). Phan ung gan bao gdm 0,5 pg vector, 5 pi 2x ddm, 4 pg ADN, 3 don vi T4 ADN ligaza, nuac cat duoc bd sung ddn the tich 10 pi. Qua trinh gin duoc
thuc hidn qua ddm d 4"C.
Td bao E. C0ABL21™ (DES) mang vector p O J
^-Vcp23ti^ td hpp dugc nudi ciy trong 400 ml moi trudng LB cd bd sung 50 pg/ml ampixiUn, tdc dd lac 200 vdng/phut d 37°C ddn mat do td b i o ODSB = 0,a Qui trinh kich hoat bidu hien gien dugc thuc hien bang cich bd sung Isopropylthio-p-galactosit (IPTG) den ndng dO 1 mM, tidp tiic nudi trong 4 gio d- 25X, Sinh khdi td b i o dugc thu hing Iy tim lO.OOO vdng/phut d 4°C trong 10 phut, tii huyen phu td biio ti-ong 8 ml ddm TNE (50 mM Tris. HCI, pH 7,5; 100 mM NaCI, 2 mM EDTA) sau dd u trong d i khoang 1 gid-. Protein hda tan tdng sd dugc thu nhin sau khi sidu am 5 phut v i ly tam 10.000 vdng/phut d 4°C trong 5 phiit. Miic do bieu hidn cua protein Cp23 % td hgp dugc xac dmh bing didn di SDS tr&i polyacrylamit gel 12%. Trong vector pGEX 4T-3, duoi dung hgp GST co khdi lugng phan tu khoang 26 kDa (Zhang et al., 2006). Gel dugc nhudm blng Coomassie Blue R-250 v i phan tich bing phin mem Quality One (ver 4.1, Bio-Rad).
2.5. Westeni blot
Protein hda tan tdng sd dupc phan tich blng didn di SDS sau dd tham tich ldn mang nitroxenluloza. Western blot dugc thuc hidn blng WestemDot™ 625 Western Blot Kit (Invitiogen) theo chi din cua n h i sin xuat. Khing the dan dong Idling Cp23 do chung tdi ttr chd tgo (Bd mdn Miln dich hoc vi vie xin, Vien Cdng nghd sinh hoc, Dai hpc Hud). Hinh anh Western blot dugc thu nhan tren he thdng Transilluminator (DyNA Light, LabneL Edison, NJ) v i phan tich bing phan mdm Quality One (ver 4.1, Bio-Rad).
2.6. Tinh sach protem dtmg hgp
Protein Cp23 tii td hop dugc tinh sach bang s i ky lgc gel su dung he thdng Glutathione Sepharose™ 4B Purification (GE Healthcare) theo hudng din cua n h i san xuat. 8 ml dich ly giii te bao dugc bd sung vao 2 ml dung dich glutathion sepharoza 4B da chuan bi tir trudc, ii 30 phiit b nhiet do phong, sau dd duoc chuyen qua dng Falcon 15 ml Sau khi protein dung hgp gin Ien resin, glutathiOD sepharoza 4B dugc thu bing cicl> !y tim 500xg troiiil 5 phut, sau dd dugc riia bing 5 n i x p g g va tiep tnc ly tim 500xg ti'ong 5 phut, lap i^u,^ nia 3 Ik Protein dung hgp Cp23 duac thu - c^ch bd sung 1 ml ddm rtra (elution buffer), u \[ ^ nhidtdO
60 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANc 3/2014
phdng sau dd Iy tim 500xg trong 5 phut, budc nia giai dugc lap lgi 2 Ian. Protein dung hgp sau mdi lan nia giii dugc trOn chtmg vi sti dung true tidp dd chgy didn di SDS.
8. KET QUA VA THAU LUAN 3.1. Tgo ddng gien cp23
Theo Perryman vi ddng tic gii (1996), cADN ciia gien cp23 cd chidu dai 602 bp vdi doan ma hda dai 336 bp (tir nucleotit 111 den 446). Gien cp23 chiia hai vi tii cit ban chd BaitM\ b hai diu se duoc khuech dai bdi cip mdi die hieu d' trong nghidn ciiu niy. ADN tdng sd cua 10 oocyst duoc sir dung de phan lip gien cpi!? bing ky thuit PCR, kdt qua dugc trinh biy d hinh 1 cbo thay mdt bing ADN cd kich thudc khoang 360 bp xuat hidn d- tat ca cac miu nghidn cuu, kich thudc niy tuong duong vdi vung ma hda cua gien cp23. Trong cic mau nay, chung tdi chgn miu dirge khudeh dai tdt nhat (miu sd 3) de tidn hinh phin tich trinh tu vi tao ddng.
San pham PCR duac tgo ddng vao vector
pGEM--T Easy vi phin tich trinh tu, ket qua cho thiy sin pham PCR cd chieu dii 345 bp va co dO tuong ddng cao vdi gien cp23 da dtrpc cdng bd (99%).
Su khac nhau d' nucleotit 327 giiia gien cp23 cua chiing tdi va gien da dugc cdng bd khdng Iim thay ddi tiinh tir axit amin cua protem Cp23 (Hinh 2).
Hinh 1. San pham PCR vdi cap mdi die hidu eho gien cp23. M: tiiang chuin ADN (GeneRuler 100 bp DNA Ladder). 1-10: cic mlu C". j^arvumkhic nhau dugc
phin lip tgi TTiua Thidn - Hud.
cp23 ATGGGTTGTTCATCATCAAAGCCAGAAACTAAAGTTGCTGAAAATAAATCTGCAGCAGAT I I { M I I I I I 1 1 M I I M I I I I I I I M M I I I I i I I I I I I I I I I I I I I I I I I 1 I I I I I I I ATGGGTTGTTCATCATCAAAGCCAGAAACTAAAGTTGCTGAAAATAAATCTGCAGCAGAT
A N K Q R E L A E K K A Q L A K A V K N
cp23 61 GCTAACAAACAAAGAGAATTAGCTGAAAAGAAGGCTCAATTAGCCAAGGCTGTAAAGAAT 120
I II I I!I I I ! I I I I I M I I I I I I I M I I M I M I I I II I N I M I M I I M I I{ II I! I f U34390 61 GCTAACAAACAAAGAGAATTAGCTGAAAAGAAGGCTCAATTAGCCAAGGCTGTAAAGAAT 120
U34390 121
P A P I S N Q A Q Q K P E E P K K S E P
CCAGCTCCAATCAGCAACCAAGCTCAACAAAAGCCAGAAGAACCAAAGAAGTCCGAGCCT I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I M I M I I I I I 1 1 I I CCAGCTCCAATCAGCAACCAAGCTCAACAAAAGCCAGAAGAACCAAAGAAGTCCGAGCCT
N N D
cp2 3 181 GCTCCCAATAATCCTCCAGCTGCTGATGCACCAGCAGCCCAAGCTCCTGCTGCCCCTGCT 24 0 I 1 1 I I M I I I I I I I I I I I I I I I I I I I I I I I 1 1 I I I I I I I I I I I I I M M I I I I I I I I I I I U34390 181 GCTCCCAATAATCCTCCAGCTGCTGATGCACCAGCAGCCCAAGCTCCTGCTGCCCCTGCT 240
E P A P Q D K P A D A P A A E A P A A E
cp23 241 GAACCTGCTCCACAGGATAAGCCAGCTGATGCCCCAGCTGCTGAAGCTCCAGCTGCTGAA 300 I I I I I I I I I I I I I I I I H I I 1 1 I I I I I I I I I I I I I I I M I I I I I I I I I I M 1 1 I I I I I I I U34390 241 GAACCTGCTCCACAGGATAAGCCAGCTGATGCCCCAGCTGCTGAAGCTCCAGCTGCTGAA 300
P A A Q Q D K P A D A
Cp23 301 CCTGCTGCTCAACAAGACAAGCCAGCCGATGCCTAA 336 I I I I I I I I I M I I I I I I I I I I I I I I I I I I M II I I U 3 4 3 9 0 3 0 1 C C T G C T G C T C A A C A A G A C A A G C C A G C T G A T G C C T A A 3 3 6
Hinh 2. So sinh trinh tir nucleotit vimg ma hda ciia gien cp23 (ma sd U34390) vdi gien cp23 cua dd tai. Sg thay ddi d codon GCT thinh GCC khdng Iim thay ddi axit amin dugc mi hda (Alamin).
NdNG NGHlfP VA PHAT TRIEN NONG THON - KY 2 - THANG 8/2014 61
3.2. Bidu hi^n gien cp23
Gien cp23 dugc cit khdi vector pGEM®-T E3&y/cp23tA\ td hgp bing enzym BanMl v i gan vio vector bieu hien pGEX 4T-3. Vector tii td hgp pGEX 4T-3/cp23 sau do dugc bien nap vio te b i o E coh BL21 (DES) va cic khuan lac duong tinh dugc kiem tra bang ky thuat PCR Kdt qua nghidn ctru cho thay xuat hien bang ADN a vi tii mong mudn (Hinh 3), dieu nay cho thiy vector pGEX 4T-3/cp^^ da dupc bidn nap thanh cdng vao td bio E. coh.
cp23 gen
M NC PC 1 2
Huih 3. Xic dinh td b i o E cokBUl (DE3) mang gien cp23. M: thang chuan ADN, NC: ddi chiing i m (td bio E. cohBUl (DE3) khong bidn nap), PC: ddi chung duong (sin phim PCR tir vecti)r pGEM®-T
Easy/cp2^, 1-2: sin pham PCR tii td bio E. coh BL21 (DE3) mang vectijr pGEX AT-3/cp23 6 C. parvum. gien cp23 ma. hda chudi polypeptit cd chieu dat 111 axit amm (cd khdi lugng phan tu khoang 11.3 kDa) (Ferryman et al., 1996). Sau khi protein Cp23 dugc tdng hgp, qua trinh glycozit hda d dau N-terminal dugc thuc hidn de tao nen phin tii glycoprotein Cp23 cd khdi lugng xip xi 23 kDa (Wang et al., 2009). Trong nghien cuu nay, protein dung hpp Cp23 cd the da duoc glycozit hoa hoac khdng. Khi dugc glycozit hda, khdi lugng phan tu ciia protein dung hgp se la 49 kDa; khi khdng dugc glycozit hda, khdi luong phin tit cua protem dung hgp chi khoang 37 kDa (chudi polypeptit P23 cd khdi luong 11,3 kDa cdng vdi dudi dung hpp cd khdi luong 26 kDa).
O E. coli, qua trinh kich hoat bieu hien thudng dupc thuc hien d 25"C de tianh hien tuong hinh thanh the vin (Makrides, 1996). Trong nghien ciiu ciia chiing tdi, qui trinh bieu hien cua gien cp23s^
tao ra protein dung hop cd khdi luong phan tir khoang 49 kDa (protein P23 cd khdi lugng phan tu
23 kDa v i dudi dung hgp glutatiuoii S-ti-ansferaza (GST) cd khdi Iugng khoang 26 kDa). Ket qua phSn tieh hinh anh didn di SDS v i Western blot cho tiiay cd 2 bing proteui xuit hien vm khoi lugng phin tii khoang 49 v i 37 kDa (Hmh 4)
Hmh 4. Bieu hidn cua cic dang Cp23 dtmg hgp tiong td b i o E. coh'Bl21 (DE3). M: chuin khdi lugng phfln
tii protein, 14: Hinh anh didn di SDS cic dgng protein Cp23 dung hgp sau khi cam iing IPTG tu 2^
gid ti-ong td b i o E. coliBUl (DES), 5: Hinh inh Western blot sau 6 gid- cam iing bang IPTG.
Trong mdt sd nghidn ciru da cdng bd trudc d^y, glycoprotein Cp23 da duoc tao ddng v i bidu hi^n thanh cdng trong E coh'dua tren cac hd thdng vector bieu hien khac nhau nhu pET 30a(-H) (Liu et aL, 2010), pGEX 4T (Bonafonte et al., 2000), pGEX 4T-2 (Borad et al., 2012; Priest et al., 1999), hoac pGEX- 5X-2 vector (Shahbazi et al., 2009; Shayan et d.
2008). Cic nghien ciiu cho thiy protein Cp23 tai to hop cd khdi Iugng phin tu khac nhau nhu 43 kDa (Shahbazi et al, 2009) hoac 46 kDa (Bonafonte et al., 2000; Wang et al., 2009) (dang dung hgp), hoic 23 kDa (Inpankaew et al., 2009; Shahbazi et al., 2009;
Shirafuji et al., 2005) (dang khdng dung hop). Sy khac nhau v^ khdi luong phan tir d cac dang Cp23 tSi to hop cd the do chiing da dugc glycozit hda a ck dang khac nhau.
Dich ly giai te bao E. coli BL21 (DE3) mang vector pGEX-4T-Vcp23 sau khi kich hoat bi^u hien dugc tinh sach bing s i c ky ii luc GST sit dung phdi tii glutathion sepharoza 4B. Kdt qua dien di SDS cho thay cd 2 bang protein xuat hi^n k h i dim vdi khdi lugng phin ttr khoang 49 kDa va 37 kDa, tuong tir nhu kdt q u i phan tich Western blot (Hinh 5).
NdNG NGHIEP VA PHAT TRIEN NdNG THON - KY 2 - THANG 8/2014
Hinh 5. Tlnh sach protein dung hgp bang sic k^ ii Iuc GST. M: chuin khdi lugng protein, 1: cic dgng
Cp23 polypeptit 4.KfrLUAN
Gien cp23 ma hda cho khang nguyen Cp23 tir C.
parvum d i dugc tao ddng va cd trinh tu nucleotit tuong ddng 99% vdi tiinh tu gien cp23 da dugc cdng bd tidn ngin hang gien. Gien cp23 trong E. coli BL21(DE3) d i bieu hien ra cic chudi polypeptit cd khdi lupng phin tu khoang 37 va 49 kDa, dd cd the la cic dgng protein dung hop dtroc glycozit hda hoac khdng. Phin tich Western blot cho thay 2 dang protein duoc tao thinh deu cd dip iing midn dich vai khing thd don ddng khang Cp23. Tinh sach protein dung hop bing sic ky ii luc GST cho thay cd 2 bang protem bieu hien manh cd khdi lucmg phan tir khoang 37 va 49 kDa.
LOI CAM ON
Chiing tdi xin chan thanh cam on Bd Khoa hoc vi Cdngnghe da tii trg'kinh phi cho nghien cuu nay (thdng qua de tii ddc lap dp Nha nudc ma sd:
02/DTDL/2011-T/29/HD).
TAI UHJ THAM KHAO
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Parasite
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CLONING AND EXPRESSION OF GENE ENCODING Cp23 PROTEIN FROM Cryptosporidium parvum Dmh Thi Bich Lan, Phung Thang Long, Nguyen Hoang Loc Tran Thuy Lan, Le Quoc Viet, Phan Van Quyet, Hoang Tan Quang
Summary
We cloned the cp23 gene coding Cp23 (glyco) protein from Cryptospondium parvum (C. parvum) isolated irom cattle in Thua Thien - Hue province, Vietnam. The coding region of cp23gene from C. parvum is 99%
(nucleoUde) and 100% (amino acid) sunilanty with cpP23 gene deposited in NCBI (accession number:
U34390). SDSPAGE and Westeni blot analysis showed that the cp23 gene in K co/i BL21 Star™ (DES) produced polypeptides with molecular weights of approximately 37 and 49 kDa. These molecules may be non- or glycosylated P23 fusion polypeptides. Western blot result showed that both proteins had reaction with monoclonal antibody against Cp23 antigen. Recombinant Cp23 protein purified by GST (glutathione .v S-lransferase) affinity chromatography can be used as an antigen for vaccine or anUbody production as well as for development of diagnostic kit for C. parvum.
Keywords: Cp23gene, Cryptospondium parvum. Cp23 recombinant antigen.
Ngudi phan bien; PGS.TS. Nguydn Viet Khdng Ngiy nhin bai: 16/6/2014
Ngiy thdng qua phan hien: 16/7/2014 Ngay duydt ding: 23/7/2014
64 NONG NGHIEP VA PHAT TRIEN NdNG THdN - KY 2 - THAI. 8/2014
