Xác định một số gen mã hóa yếu tố quyết định kháng nguyên của xoắn khuẩn leptospira interrogans gây bệnh leptospirosis tại việt nam

AI HOC THAI NGUYấN TRNG AI HOC KHOA HOC TRN TH THANH HU XC NH MT S GEN M HểA YU T QUYT NH KHNG NGUYấN CA XON KHUN Leptospira interrogans GY BNH LEPTOSPIROSIS TI VIT NAM LUN VN THC S SINH HC NG DNG THI NGUYấN - NM 2017 AI HOC THAI NGUYấN TRNG AI HOC KHOA HOC TRN TH THANH HU XC NH MT S GEN M HểA YU T QUYT NH KHNG NGUYấN CA XON KHUN Leptospira interrogans GY BNH LEPTOSPIROSIS TI VIT NAM CHUYấN NGNH: CễNG NGH SINH HC M S: 60.42.02.01 LUN VN THC S SINH HC NG DNG Ngi hng dn khoa hc: TS Vừ Th Bớch Thy THI NGUYấN, NM 2017 LI CM N hon thnh lun tt nghip ny, tỏc gi xin by t lũng bit n chõn thnh v sõu sc nht ca mỡnh ti TS.Vừ Th Bớch Thy phú trng phũng H gen hc vi sinh - Vin Nghiờn cu h gen - Vin Hn lõm Khoa hc v Cụng ngh Vit Nam, ngi ó tn tỡnh ch bo, hng dn quỏ trỡnh nghiờn cu v hon thnh lun Tỏc gi xin trõn trng cm n quý thy cụ Ban giỏm hiu Trng i hc Khoa hc Thỏi Nguyờn; Ban ch nhim khoa Cụng ngh Sinh hc - Trng i hc Khoa hc Thỏi Nguyờn; Quý thy, cụ trc tip ging dy, giỳp sut quỏ trỡnh hc tp, nghiờn cu khoa hc Tỏc gi xin chõn thnh cm n PGS TS Nghiờm Ngc Minh, KS Phm Thựy Linh v cỏc anh ch em, cỏn b phũng H gen hc vi sinh - Vin Nghiờn cu h gen - Vin Hn lõm Khoa hc v Cụng ngh Vit Nam, ó nhit tỡnh hng dn, giỳp v úng gúp nhiu ý kin quý bỏu tụi hon thnh lun ny Cui cựng, xin c by t lũng yờu thng v bit n sõu sc n gia ỡnh, ng nghip - nhng ngi ó to mi iu kin giỳp , ng viờn tỏc gi quỏ trỡnh hc v hon thnh lun Thỏi Nguyờn, thỏng nm 2017 Tỏc gi Trn Th Thanh Hu LI CAM OAN Tụi xin cam oan õy l cụng trỡnh nghiờn cu tụi trc tip thc hin vi s giỳp ca cỏn b, nhõn viờn phũng H gen hc vi sinh - Vin nghiờn cu h gen, Vin Hn lõm Khoa hc v Cụng ngh Vit Nam di s hng dn khoa hc ca TS Vừ Th Bớch Thy Cỏc t liu trớch dn u cú ngun gc rừ rng, cỏc kt qu lun l trung thc v cha tng c cụng b bt k cụng trỡnh no khỏc Tỏc gi lun Trn Th Thanh Hu i MC LC LI CM N LI CAM OAN DANH MC CAC Kí HIU, CAC CH VIT TT iii DANH MC BNG iv DANH MC HèNH v M U 1 t Mc tiờu nghiờn cu Ni dung nghiờn cu CHNG I TNG QUAN TI LIU 1.1 c im ca xon khun Leptospira 1.2 Tỡnh hỡnh nghiờn cu trờn th gii 1.2.1 Tỡnh hỡnh bnh Leptospirosis 1.2.2 Tỡnh hỡnh nghiờn cu vc xin phũng bnh Leptospirosis 10 1.3 Tỡnh hỡnh nghiờn cu nc 12 1.3.1 Tỡnh hỡnh bnh Leptospirosis 12 1.3.2 Tỡnh hỡnh nghiờn cu vc xin phũng bnh Leptospirosis 14 VT LIU V PHNG PHAP NGHIấN CU 15 2.1 Vt liu nghiờn cu 15 2.2 Húa cht, thit b 16 2.2.1 Húa cht 16 2.2.2 Thit b 17 2.3 a im nghiờn cu 17 2.4 Phng phỏp nghiờn cu 18 2.4.1 Phng phỏp tỏch chit DNA tng s 18 ii 2.4.2 nh lng v kim tra tinh sch ca DNA tng s 19 2.4.3 Thit k cỏc cp mi ca cỏc gen 16S rDNA, OmpL1, LipL32, LipL41, LipL21, LigA, LigB 20 2.4.4 K thut PCR 20 2.4.5 Tinh sch sn phm PCR ca gen 16S rDNA 21 2.4.6 Gii trỡnh t gen 16S rDNA ca chng xon khun 22 2.4.7 Phõn tớch v xõy dng s hỡnh cõy phõn loi chng Leptospira 23 CHNG III KT QU V THO LUN 24 3.1 Phõn loi hc phõn t chng Leptospira 24 3.1.1 Kim tra cht lng DNA tng s 24 3.1.2 Trỡnh t cỏc cp mi ca cỏc gen 16S rDNA, OmpL1, LipL32, LipL41, LipL21, LigA, LigB 26 3.1.3 Nhõn gen vi cỏc cp mi ca gen 16S rDNA 27 3.1.4 Tinh sch sn phm PCR gen 16S rDNA 28 3.1.5 Gii trỡnh t gen 16S rDNA ca chng xon khun 29 3.1.6 Phõn tớch v xõy dng cõy phỏt sinh chng loi ca chng xon khun 30 3.2 Xỏc nh s cú mt ca cỏc gen LigA, LipL32, LigB, OmpL1, LipL21, LipL41 34 KT LUN V NGH 41 Kt lun 41 ngh 41 TI LIU THAM KHO PH LC iii DANH MC CC Kí HIU, CC CH VIT TT bp Base pair dH2O Nc kh ion DNA Deoxyribonucleic acid dNTPs deoxyribonucleotide triphosphates EDTA Ethylene Diamine Tetraacetace Axit EMJH Ellinghausen - McCullough-Johnson-Harris EtBr Ethidium Bromide F- Forward Primer : Mi xuụi R- Reverse Primer: Mi ngc Kb Kilobase L Leptospira MLST Multilocus Sequence Typing OMP Protein ngoi mng (outer membrane protein) PCR Polymerase Chain Reaction RNA Ribonucleic acid SDS Sodium Dodecyl Sulfate TAE Tris-acetate-EDTA iv DANH MC BNG Bng Tờn bng Trang 2.1 Danh mc cỏc thit b ó s dng 17 2.2 Thnh phn phn ng nhõn gen 21 3.1 3.2 3.3 Giỏ tr mt quang ph hp th bc súng 260nm v 280nm ca chng Leptospira Trỡnh t cp mi dựng nhõn cỏc on gen Cỏc loi Leptospira quc t tham kho t c s d liu GenBank 25 27 30 v DANH MC HèNH Hỡnh Tờn hỡnh Trang Leptospira interrogans Chu kỡ nhit ca phn ng PCR nhõn gen 21 3.1 Hỡnh nh in di DNA tng s ca chng Leptospira 24 3.2 Hỡnh nh in di sn phm PCR nhõn gen mó húa rRNA 16S 28 3.3 3.4 Hỡnh nh in di sn phm PCR nhõn gen mó húa rRNA 16S tinh sch Hỡnh nh cõy phỏt sinh chng loi ca chng xon khun 29 32 3.5.a Hỡnh nh in di sn phm PCR nhõn gen LigA 35 3.5.b Hỡnh nh in di sn phm PCR nhõn gen LipL32 35 3.5.c Hỡnh nh in di sn phm PCR nhõn gen LigB 36 3.5.d Hỡnh nh in di sn phm PCR nhõn gen OmpL1 36 3.5.e Hỡnh nh in di sn phm PCR nhõn gen LipL21 37 Hỡnh nh in di sn phm PCR nhõn gen LipL41 37 3.5.f M U t Bnh Leptospirosis (bnh Xon khun) l mt nhng bnh truyn nhim cp tớnh, lan truyn t ng vt sang ngi ph bin trờn th gii, c T chc Sc khe ng vt Th gii (World Organisation for Animal HealthOIE) xp v trớ th nhúm bnh nguy him v c b sung vo nhúm bnh ngh nghip Vit Nam Nguyờn nhõn gõy bi mt loi xon khun Leptospira interrogans Leptospira interrogans l loi gõy bnh thng kớ sinh ngi v ng vt, bao gm hn 200 typ, c chia thnh 23 nhúm (trong ú Leptospira interrogans serovar Icterohaemorrhagiae thng gp nht) Xon khun ny cú kh nng gõy bnh trờn ng vt v cú th lõy sang ngi Bnh xon khun gõy cũn gi l bnh vng da, nú gõy nh hng n cỏc c quan v cú th gõy sy thai thỳ nuụi Bnh xon khun lõy nhim sang ngi qua niờm mc, da, mt hoc cỏc mng nhy tip xỳc trc tip hay giỏn tip vi nc tiu hoc mu mụ ng vt mc bnh Bnh phỏt trin khp mi ni trờn th gii, gõy thit hi ln cho ngnh chn nuụi v nh hng nghiờm trng n sc khe ca ngi Do tớnh a dng v triu chng nờn bnh Leptospirosis khú chn oỏn v t l t vong cao Vic iu tr bnh ny rt khú khn nờn ũi hi nhng bin phỏp phũng nga bnh cú hiu qu cao.Vỡ vy, vic sn xut vc xin phũng bnh xon khun Leptospira l mt yờu cu cp thit Trong quỏ trỡnh nghiờn cu ng dng, cỏc nh khoa hc ang trung vo cỏc loi vc xin cú kh nng phũng chng dch bnh mang tớnh dch t vựng, an ton, mc bo h cao v lõu di, giỏ thnh h, m bo sc khe cng ng v ng vt c bit quan tõm vi loi vc xin nhc c c thit k da trờn c s trỡnh t gen v cỏc yu t c lc ca cỏc tỏc nhõn gõy bnh ti u húa mc an ton ca cỏc loi vc xin õy l mt bc t 41 KT LUN V NGH Kt lun - Trỡnh t gen 16S rDNA ca chng xon khun v kt qu xõy dng s cõy phõn loi cho thy nhng mu chng c s dng nghiờn cu l cỏc chng Leptospira interrogans m khụng b nhm ln quỏ trỡnh thc nghim Cỏc chng Leptospira interrogans ca Vit Nam ó cú s khỏc bit so vi mt s chng Leptospira gõy bnh khỏc trờn th gii (mc gn gi v mt di truyn t 62 - 99 %, khong cỏch di truyn 0,00 - 0,82), cú th dch t hc a lý khỏc ó dn n s a dng v di truyn - chng xon khun Leptospira gõy bnh Leptospirosis, ang s dng ch vc xin vụ hot Vit Nam cú mt ca mt s gen mó húa khỏng nguyờn, bao gm: gen LipL32, LipL21 v gen LigA xut hin tt c cỏc chng kim tra, riờng gen OmpL1 ch xut hin chng (gm L interrogans serovar Bataviae, L interrogans serovar Canicola, L interrogans serovar Mitis v L interrogans serovar Pomona), gen LipL41 thỡ 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Bataviae GACCGGGGGGGGGACCTGAGCAGCGACGCCGCGTGAACGATGAA GGTCTTCGGATTGTAAAGTTCAGTAAGCAGGGAAAAATAAGCAGC AATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTGCC AGCAGCCGCGGTAATACGTATGGTGCAAGCGTTGTTCGGAATCAT TGGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGAAA ACTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTGGA GTTTGGGAGAGGCAAGTGGAATTCCAGGTGTAGCGGTGAAATGCG TAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGCCT AAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTA GATACCCCGGTAATCCACGCCCTAAAGGTTGTCTACCAGTTGTTGG GGGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACCGC CTGGGGACTATGCTCGCAAGAGTGAAACTCAATGAAATTTGACGG Trỡnh t gen 16S rDNA ca chng L interrogans serovar Mitis GGCCCAAGGGGGGGGACCTGAGCAGCGACGCCGCGTGAACGATGA AGGTCTTCGGATTGTAAAGTTCAGTAAGCMSGGAAAAATAAGCAG CAATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTGCC AGCAGCCGCGGTAATACGTATGGTGCAAGCGTTGTTCGGAATCATT GGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGAAAA CTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTGGAG TTTGGGAGAGGCAAGTGGAATTCCAGGTGTAGCGGTGAAATGCGT AGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGCCTA AAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTAA ATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCAGTTGTTGGG GGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACCGCCT GGGGACTATGCTCGCAAGAGTGAAACTCAAATGAATTGACGGA Trỡnh t gen 16S rDNA ca chng L interrogans serovar Pomona GGATGGCCGTCCCAGGGCGGTCTACTTAATCCGTTAGGTTCGTTAC TGAGGGTTAAAACCCCCAACAACTGGTAGACCACGTTTAGGGCGT GGATTACCGGGGTATCTAATCCCGTTCACTACCCACGCTTTCGTGC CTCAGCGTCAGTTTTAGGCCAGCAAGTCGCCTTCGCCACTGGTGTT CCTCCAGATATCTACGCATTTCACCGCTACACCTGGAATTCCACTT GCCTCTCCCAAACTCCAGACACATAGTTTCAAGTGCAGGCTGCGA GTTGAGCCCGCAGTTTTCACACCTGACTTACATGTCCGCCTACGCA CCCTTTACGCCCAATGATTCCGAACAACGCTTGCACCATACGTATT ACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTTAGGCAGGTAC CATCATCACATTGCTGCTTATTTTTCCCTGCTTACTGAACTTTACAA TCCGAAGACCTTCATCGTTCACGCGGCGTCGCTGCTTCAGGGTTCC CCCCAATATAGAATAATCCTAATCGGCCGTGCCCCATCAGCCAAG GCATTAATAACCCTGGCCAAGGGGGGGGAAACCCAAAAACCCCCC CCCCCGAGAAAGAAAAAATTTCCCCAATAGTAAAATTTTTCAAAA AGGGGGAAAAAAAAAACCCCCCGGGGAAAAAGGGTCCCCCCCCA AAGCCCCGAAAAAAGGGGGCCCCCCCGGGGAAAAAAATAAGGGG GGAAATGTTTTTAACAAAAAATTGCGGGCAAACAGGGGGGGGAG GGGGGGAAATAGATATTAGGTGGGATAAAAAACGAGACGCGCGG GGTGTTGCAAAAAAAACCTTTGCGCGTAGTGGTGAGAGAGAGAAG AGACCCCCTCTCCCGCGTGGGGCGCGGGGAAATATTATAATATTTT GGAAAGACACATCACGAGCAGACCAGCATTAAGGTATAACACTAG CTAAACTAGACTGAGCATACGA Trỡnh t gen 16S rDNA ca chng L interrogans serovar Canicola GGGCGTGGGGGGCAAATCTGACCACCCATGCCGCGTGAAGGATGA AGGTCCTCTGGATTGTAAACTTCTTTTATAGGGGGCGAAAAAAGG GAAATCTTTCTCACTTGACCGTACCCTATGAATAACACACCGGCTA ACTCCGTGCCAGCAAACCGCGGTACATACGGAGGGTGCAAGCGTT ATCCGGATTCACTGGGTTTAAAGGGTGCGTAGGCGGGCGTATAAG TCAAATGGTGAAATCCTGGAGCTTATCTCCAGAACTGCCATTGATA CTATATGTCTTGAATATGGTGGAGGTAACCGGAATATGTCATGTAG CGGTGAAATGCATAAATATGAACATAGAACACCTATTGCGAAGGC GGCTTACTACGCCTATTTTGACCCTAAGGCACGAAAGCGTGGGGA TCAAACAGGATTAGATACCCTGGTACTCCACGCCCTAAACCATGAT TACTCGACGTGTGCGATAAACGGTACGCGTCTGAGCGAAAGCATT AAGTAATCCACCTGGGAAGTACGACCGCAAGGTTGAAACTCAAAT GAAATTGACGG Trỡnh t gen 16S rDNA ca chng L interrogans serovar Grippotyphosa TTAACCTTGGGGGCACCTGTATCAGCGCAGGCCGCGTGAACGACG AAGGTCTTCGGATTGTAAAGTTCATTAGGCAGGGAAAAATAAGCA ACCTTGTGATGATGGTACCTGCCTAAAGCACCGGATAACTACCTGC CAGCAACCGCGGTAATACCTATGGGGCAAGCGTTGTTCGGAATCA TTGGGCGTAAAGGGTGCGTAAGCGGACATGTAATTCAAGTGTGAA AACTGCGGGCTCACCTCATAGCCTGCACTTGAAACTATGTGTCTGG AGTTTGGGAGAGGCAAGTGGAATTCCAGGTGTACCGGTGAAATGC GTAAATATCTGGAGGAACACCAGTGGCGAAGGCACTTGCTGGCCT AAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGATTA GATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCACTTGTTGG GGGTTTTAACCCTCATTAACGAACCTAACGGATTAACTAGACCGCC TGGGGACTATGCTCGCAAGAGTGAAACTCAAATGAATTGACGG Trỡnh t gen 16S rDNA ca chng L interrogans serovar Icteroheamorrhagiae GCACGGAGGGGGGGCACCTGAGCAGCGACGCCGCGTGAACGATG AAGGTCTTCGGATTGTAAAGTTCAGTAAGCAGGGAAAAATAAGCA GCAATGTGATGATGGTACCTGCCTAAAGCACCGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTATGGKGCAAGCGTTGTTCGGAATC ATTGGGCGTAAAGGGTGCGTAGGCGGACATGTAAGTCAGGTGTGA AAACTGCGGGCTCAACTCGCAGCCTGCACTTGAAACTATGTGTCTG GAGTTTGGGAGAGGCAAGTGGAATTCCRGGTGTAGCGGTGAAATG CGTAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTGCTGGC CTAAAACTGACGCTGAGGCACGAAAGCGTGGGTAGTGAACGGGAT TAGATACCCCGGTAATCCACGCCCTAAACGTTGTCTACCAGTTGTT GGGGGTTTTAACCCTCAGTAACGAACCTAACGGATTAAGTAGACC GCCTGGGGACTATGCTCGCAAGAGTGAAACTCAAATGAAATTGAC GGATAGCAAACCAAGAGTCTTGCTCACTGGGGGGAACCCTGAAGC ATTCACAACGCATGAGCCATCAACGTCGTCCAATTGTAAAAGTTCC CTAAGCAGGGAAGTATAARCAGCATGCTGAATGATTGTACCTGCC TAAAGGCACGGCTAATTACGTGCCGCRTCGGCCTAATACATTATGG GTGCAACGGGTTGTTTAGGAATCATTAGTAACGTAGAGAGGGCGA TATAGGGCGGAACATCGACAAGTCAGGATGTGAAAACTGGCGTGC TCACTACACTGCAATAGAAAAATAAACGTTATTCGAGGAGTTTCG GGAACAAGCAAATGATAATTCCCAGGATGTCACAAGGTGACAACC GTGGCTACAAAATRTCTCTCAGACAGGGA ... THÁI NGUYÊN TRƯỜNG ĐẠI HỌC KHOA HỌC TRẦN THỊ THANH HUỆ XÁC ĐỊNH MỘT SỐ GEN MÃ HÓA YẾU TỐ QUYẾT ĐỊNH KHÁNG NGUYÊN CỦA XOẮN KHUẨN Leptospira interrogans GÂY BỆNH LEPTOSPIROSIS TẠI VIỆT NAM CHUYÊN... phòng bệnh xoắn khuẩn Leptospira Xuất phát từ thực tiễn đó, tiến hành thực đề tài Xác định số gen mã hóa yếu tố định kháng nguyên xoắn khuẩn Leptospira interrogans gây bệnh Leptospirosis Việt Nam ... loại hình chủng xoắn khuẩn Leptospira gây bệnh Leptospirosis, sử dụng chế vắc xin vô hoạt Việt Nam - Xác định có mặt số gen mã hóa kháng nguyên chủng xoắn khuẩn Leptospira gây bệnh Leptospirosis,