Luận án Nghiên cứu tạo hạt giả virus lở mồm long móng type o trong hệ biểu hiện baculovirus tế bào côn trùng định hướng sản xuất vaccine

Để khắc phục tất cả những yếu tố ảnh hưởng tới quá trình biểu hiện gen cấu trúc của virus LMLM chúng tôi đã đi theo hướng tách biệt các gen VP0, VP1 và VP3 để việc phân tách của chúng dễ dàng, và cũng loại bỏ bớt ảnh hưởng của protease 3C, enzyme này làm giảm quá trình trình hình thành lớp vỏ của virus LMLM hay cũng là để không ảnh hưởng tới quá trình tạo thành hạt VLPs. Với hướng đi này của chúng tôi đã khắc phục được hầu hết những yếu tố có ảnh hưởng tới quá trình tạo thành hạt VLPs. Các nghiên cứu trước đây đã chỉ ra rằng việc lắp ráp thành lớp vỏ virus LMLM trong các tế bào chủ đòi hỏi phải phân tách đượcpolyprotein thành protein riêng lẻ. Mỗi đơn vị cấu trúc được hình thành từ một bản sao của mỗi protein cấu trúc VP0, VP1 và VP3; trong đó VP0 là tiền thân của VP2 vàVP4.VP1, VP3, và VP0 tự tạo thành 5Sprotomer, sau đó lắp ráp thành tiểu đơn vị 12Spentameric, lần lượt tập hợp thành các procosid icosahedral 75S tạo thành hạt VLP hoàn chỉnh (Belsham., 1993; Abramset và cs., 1995).

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SQ SEQUENCE 867 BP; 220 A; 271 C; 208 G; 168 T; ATGAACACCG GGAGCATCAT CAACAACTAC TACATGCAGC AGTACCAGAA TTCTATGGAC ACACAACTTG GTGACAACGC TATCAGCGGA GGCTCCAACG AGGGGTCCAC AGACACAACC TCCACCCACA CAACCAACAC CCAGAACAAT GACTGGTTTT CAAAGCTGGC TAGCTCTGCT TTTAGCGGTC TGTTCGGCGC CCTCCTCGCC GACAAGAAAA CCGAGGAAAC TACCCTCCTC GAGGACCGCA TCCTCACCAC CCGAAACGGG CACACCACCT CGACAACCCA GTCGAGCGTT GGCGTTACGT ACGGGTATGC AACAGCTGAG GACTTTGTGA GCGGGCCAAA CACCTCTGGT CTCGAGACCA GGGTTGTTCA GGCAGAACGG TTTTTCAAAA CCCACTTGTT CGACTGGGTC ACCAGCGACC CGTTCGGACG GTGCCACCTC CTGGAACTCC CGACTGACCA CAAAGGTGTC TACGGCGGCC TGACCGACTC ATATGCCTAC ATGAGGAATG GCTGGGACGT CGAAGTTACC GCTGTGGGGA ACCAGTTCAA CGGGGGCTGC CTATTGGTGG CCATGGTGCC TGAGCTTTGT TCCATCCAAA AGAGAGAGCT GTACCAACTC ACGCTCTTCC CCCATCAGTT CATCAACCCT CGGACGAACA TGACAGCCCA CATCAAGGTG CCCTTTGTTG GCGTCAACCG CTATGACCAG TACAAGGTAC ACAAACCTTG GACCCTTGTG GTTATGGTTG TAGCCCCCCT GACTGTCAAC ACCGAAGGCG CTCCACAGAT CAAGGTGTAC GCCAACATCG CACCTACCGA CGTACACGTT GCAGGCGAGT TCCCTTCCAA AGAGTAA // 2. Trình tự gen VP0 của virus LMLM type O ở Viêt Nam sau tối ưu ID VP0_S_Tu gan them Restriction Sites PRELIMINARY; DNA; 887 BP. SQ SEQUENCE 887 BP; 232 A; 250 C; 219 G; 186 T; NheI NcoI GCTAGCCCAT GGACACAGGC TCAATTATCA ACAACTACTA CATGCAGCAA TACCAGAACT CAATGGACAC GCAACTCGGA GACAACGCGA TTTCGGGTGG CAGTAACGAG GGCTCGACAG ATACCACTAG TACGCACACA ACGAACACCC AGAACAACGA CTGGTTCTCT AAGCTCGCAT CCAGCGCGTT CTCAGGCTTG TTCGGTGCTC TGCTGGCTGA TAAGAAAACT GAGGAAACCA CTTTGCTGGA GGACCGCATC CTGACAACGC GTAACGGACA CACCACTAGT ACAACGCAGT CTTCAGTCGG AGTTACTTAC GGTTACGCTA CAGCCGAAGA CTTCGTCTCC GGTCCTAACA CCTCCGGACT GGAGACCAGG GTTGTGCAAG CTGAAAGATT CTTCAAGACG CACCTCTTCG ACTGGGTGAC CTCCGATCCA TTCGGTAGAT GCCATCTGCT GGAGCTGCCG ACTGACCATA AAGGTGTCTA CGGCGGTTTG ACAGATAGCT ACGCATACAT GCGCAACGGT TGGGACGTGG AAGTCACTGC TGTGGGCAAC CAATTCAACG GAGGTTGCCT GCTCGTGGCT ATGGTCCCTG AGCTGTGTAG CATTCAAAAG CGCGAACTCT ACCAGTTGAC GCTGTTCCCA CACCAATTCA 2 TCAACCCGCG TACAAACATG ACGGCCCATA TTAAGGTGCC ATTCGTTGGC GTGAACCGTT ACGACCAGTA CAAGGTCCAC AAACCGTGGA CATTGGTGGT CATGGTTGTG GCTCCTCTGA CCGTTAACAC TGAAGGAGCC CCCCAAATCA AGGTGTACGC AAACATTGCG CCTACCGACG TCCATGTTGC TGGAGAGTTC CCCTCAAAAG AATAAGCATG CGGTACC SphI KpnI // ===02-SEP-2016====================PALIGN====================PC/GENE=== Done on DNA sequence VP0_Sau_TU+Res sites, total number of bases: 887. - The sequence was considered to be: LINEAR. - The analysis was done on bases 1 to 887 3. Kết quả so sánh trình tự protein suy diễn của gen VP0 0 trước và sau khi tối ưu codon *************************************** * ALIGNMENT OF TWO PROTEIN SEQUENCES. * *************************************** The two sequences to be aligned are: VP0_S_Pro (Protein sau tối ưu) Total number of residues: 288. VP0_T_Pro (Protein trước tối ưu) Total number of residues: 288. Comparison matrix : Structure-genetic matrix. Open gap cost : 7 Unit gap cost : 1 The character to show that two aligned residues are identical is '|' VP0_S_PRO- MNTGSIINNYYMQQYQNSMDTQLGDNAISGGSNEGSTDTTSTHTTNTQNN -50 |||||||||||||||||||||||||||||||||||||||||||||||||| VP0_T_PRO- MNTGSIINNYYMQQYQNSMDTQLGDNAISGGSNEGSTDTTSTHTTNTQNN -50 VP0_S_PRO- DWFSKLASSAFSGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSV -100 |||||||||||||||||||||||||||||||||||||||||||||||||| VP0_T_PRO- DWFSKLASSAFSGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSV -100 3 VP0_S_PRO- GVTYGYATAEDFVSGPNTSGLETRVVQAERFFKTHLFDWVTSDPFGRCHL -150 |||||||||||||||||||||||||||||||||||||||||||||||||| VP0_T_PRO- GVTYGYATAEDFVSGPNTSGLETRVVQAERFFKTHLFDWVTSDPFGRCHL -150 VP0_S_PRO- LELPTDHKGVYGGLTDSYAYMRNGWDVEVTAVGNQFNGGCLLVAMVPELC -200 |||||||||||||||||||||||||||||||||||||||||||||||||| VP0_T_PRO- LELPTDHKGVYGGLTDSYAYMRNGWDVEVTAVGNQFNGGCLLVAMVPELC -200 VP0_S_PRO- SIQKRELYQLTLFPHQFINPRTNMTAHIKVPFVGVNRYDQYKVHKPWTLV -250 |||||||||||||||||||||||||||||||||||||||||||||||||| VP0_T_PRO- SIQKRELYQLTLFPHQFINPRTNMTAHIKVPFVGVNRYDQYKVHKPWTLV -250 VP0_S_PRO- VMVVAPLTVNTEGAPQIKVYANIAPTDVHVAGEFPSKE -288 |||||||||||||||||||||||||||||||||||||| VP0_T_PRO- VMVVAPLTVNTEGAPQIKVYANIAPTDVHVAGEFPSKE -288 Identity : 288 (100.00%) Number of gaps inserted in VP0_S_PRO: 0 Number of gaps inserted in VP0_T_PRO: 0 ===02-SEP-2016================PALIGN===========================PC/GENE=== ===02-SEP-2016=========RESTRI==================================PC/GENE=== 4. Danh sách kiểm tra trình tự enzyme cắt trong gen VP0 sau tối ưu ***************************** * RESTRICTION-SITE ANALYSIS * ***************************** List of cuts by enzyme ====================== AatII : 841 Acc65I : 882 AccI : 487 AciI : 254 493 666 880 AcyI : 838 AeuI : 377 AflIII : 267 AluI : 173 390 464 508 602 4 AsuI : 251 351 593 683 734 AvaII : 251 351 593 734 BbvI : 56 451 BbvII : 292 343 BetI : 348 363 BinI : 429 BsiYI : 458 584 692 705 731 809 BsmFI : 546 579 BsmI : 610 BsmAI : 73 350 366 BspMII : 363 BsrI : 165 373 426 632 725 BsrDI : 823 BssKI : 375 BstEII : 425 BstXI : 245 849 Csp6I : 130 289 670 728 815 883 DdeI : 169 191 229 598 DpnI : 436 DrdI : 540 DsaI : 7 744 Eco31I : 366 Eco57I : 286 811 EcoRII : 375 Esp3I : 350 Fnu4HI : 45 465 FnuDII : 87 186 269 622 668 FokI : 164 243 GsuI : 267 390 480 871 HaeIII : 684 HgaI : 416 647 HgiAI : 209 HgiCI : 695 882 HgiJII : 114 800 HhaI : 186 523 622 830 Hin6I : 184 521 620 828 HindII : 637 785 5 HinfI : 397 HpaI : 785 HpaII : 349 364 HphI : 437 KpnI : 886 Ksp632I : 420 MaeI : 2 127 286 MaeII : 535 838 MaeIII : 102 270 311 321 425 542 717 MboI : 434 MboII : 292 348 392 407 MluI : 267 MnlI : 101 224 242 371 423 440 564 784 872 MseI : 691 784 MslI : 481 549 765 MstI : 522 MwoI : 183 213 464 825 NcoI : 7 NheI : 1 NlaIII : 11 44 521 680 764 846 880 NlaIV : 595 697 772 797 884 NspI : 44 521 880 PflMI : 458 705 RsaI : 131 290 671 729 816 884 ScrFI : 377 SduI : 114 209 800 SecI : 7 376 744 SfaNI : 186 265 438 SfeI : 328 SpeI : 126 285 SphI : 880 StyI : 7 TaqI : 114 418 TfiI : 397 Tsp509I : 22 561 654 Tth111I : 342 Total number of cuts is : 195. 6 --------------------------------------- Sorted list of enzymes by number of cuts ======================================== +------------------+------------------+------------------+--------------- ---+ | MnlI : 9 | SduI : 3 | HindII : 2 | BsmI : 1 | | MaeIII : 7 | MslI : 3 | DsaI : 2 | AatII : 1 | | NlaIII : 7 | Tsp509I : 3 | BbvII : 2 | NcoI : 1 | | Csp6I : 6 | SfaNI : 3 | BsmFI : 2 | BssKI : 1 | | BsiYI : 6 | SecI : 3 | Ksp632I : 1 | Eco31I : 1 | | RsaI : 6 | NspI : 3 | HphI : 1 | MstI : 1 | | NlaIV : 5 | HgiJII : 2 | BspMII : 1 | AeuI : 1 | | AluI : 5 | TaqI : 2 | HgiAI : 1 | SfeI : 1 | | BsrI : 5 | SpeI : 2 | Esp3I : 1 | BsrDI : 1 | | AsuI : 5 | BstXI : 2 | NheI : 1 | HpaI : 1 | | FnuDII : 5 | HgiCI : 2 | HaeIII : 1 | Tth111I : 1 | | AvaII : 4 | Fnu4HI : 2 | EcoRII : 1 | TfiI : 1 | | MboII : 4 | MseI : 2 | StyI : 1 | BinI : 1 | | AciI : 4 | HpaII : 2 | Acc65I : 1 | MboI : 1 | | GsuI : 4 | BetI : 2 | KpnI : 1 | SphI : 1 | | MwoI : 4 | HgaI : 2 | ScrFI : 1 | DpnI : 1 | 7 | DdeI : 4 | Eco57I : 2 | HinfI : 1 | AcyI : 1 | | HhaI : 4 | BbvI : 2 | AflIII : 1 | AccI : 1 | | Hin6I : 4 | MaeII : 2 | MluI : 1 | | | MaeI : 3 | PflMI : 2 | DrdI : 1 | | | BsmAI : 3 | FokI : 2 | BstEII : 1 | | +------------------+------------------+--------------+------------------+ List of non cutting selected enzymes ==================================== AclI , AflII , AgeI , AhaIII , AhyI , AlwNI , ApaI , ApaLI , ApoI , AscI , AsuII , AvaI , AvrII , BalI , BamHI , BbeI , BcgI , BclI , BglI , BglII , BsaAI , BsaBI , BsgI , Bsp120I , Bsp1407I, BspHI , BspM90I , BspMI , BsrBI , BssHII , CauII , CfrI , Cfr10I , ClaI , DraII , DraIII , Eam1105I, Ecl136II, Eco47III, Eco56I , Eco78I , EcoNI , EcoRI , EcoRV , EcoT22I , EspI , FseI , HaeII , HindIII , KasI , McrI , MfeI , NaeI , NarI , NdeI , NotI , NruI , NspBII , PacI , PleI , PmaCI , PmeI , Ppu10I , PpuMI , PshAI , PstI , PvuI , PvuII , RsrII , SacI , SacII , SalI , SapI , SauI , ScaI , SexAI , SfiI , SgfI , SgrAI , SmaI , SnaBI , SplI , SrfI , Sse8387I, SspI , StuI , SwaI , VspI , XbaI , XcmI , XhoI , XhoII , XmaIII , XmnI Total number of selected enzymes which do not cut: 94 --------------------------------------- Sorted list of cuts =================== +------------------+------------------+------------------+--------------- ---+ | 1 NheI U | 270 MaeIII | 438 SfaNI | 695 HgiCI D | 8 | 2 MaeI | 285 SpeI D | 440 MnlI | 697 NlaIV | | 7 DsaI D | 286 MaeI | 451 BbvI D | 705 BsiYI | | 7 NcoI U | 286 Eco57I D | 458 BsiYI | 705 PflMI D | | 7 SecI | 289 Csp6I | 458 PflMI D | 717 MaeIII | | 7 StyI U | 290 RsaI | 464 AluI | 725 BsrI | | 11 NlaIII | 292 BbvII D | 464 MwoI | 728 Csp6I | | 22 Tsp509I | 292 MboII | 465 Fnu4HI D | 729 RsaI | | 44 NlaIII | 311 MaeIII | 480 GsuI | 731 BsiYI | | 44 NspI | 321 MaeIII | 481 MslI | 734 AsuI | | 45 Fnu4HI D | 328 SfeI U | 487 AccI U | 734 AvaII | | 56 BbvI D | 342 Tth111I U | 493 AciI | 744 SecI | | 73 BsmAI | 343 BbvII D | 508 AluI | 744 DsaI D | | 87 FnuDII | 348 MboII | 521 NlaIII | 764 NlaIII | | 101 MnlI | 348 BetI D | 521 Hin6I | 765 MslI | | 102 MaeIII | 349 HpaII D | 521 NspI | 772 NlaIV | | 114 TaqI D | 350 BsmAI | 522 MstI U | 784 MseI D | | 114 SduI | 350 Esp3I U | 523 HhaI | 784 MnlI | | 114 HgiJII D | 351 AsuI | 535 MaeII D | 785 HindII D | 9 | 126 SpeI D | 351 AvaII | 540 DrdI U | 785 HpaI U | | 127 MaeI | 363 BetI D | 542 MaeIII | 797 NlaIV | | 130 Csp6I | 363 BspMII U | 546 BsmFI D | 800 SduI | | 131 RsaI | 364 HpaII D | 549 MslI | 800 HgiJII D | | 164 FokI D | 366 Eco31I U | 561 Tsp509I | 809 BsiYI | | 165 BsrI | 366 BsmAI | 564 MnlI | 811 Eco57I D | | 169 DdeI | 371 MnlI | 579 BsmFI D | 815 Csp6I | | 173 AluI | 373 BsrI | 584 BsiYI | 816 RsaI | | 183 MwoI | 375 BssKI U | 593 AvaII | 823 BsrDI U | | 184 Hin6I | 375 EcoRII U | 593 AsuI | 825 MwoI | | 186 SfaNI | 376 SecI | 595 NlaIV | 828 Hin6I | | 186 FnuDII | 377 AeuI U | 598 DdeI | 830 HhaI | | 186 HhaI | 377 ScrFI U | 602 AluI | 838 AcyI U | | 191 DdeI | 390 GsuI | 610 BsmI U | 838 MaeII D | | 209 SduI | 390 AluI | 620 Hin6I | 841 AatII U | | 209 HgiAI U | 392 MboII | 622 FnuDII | 846 NlaIII | | 213 MwoI | 397 HinfI U | 622 HhaI | 849 BstXI D | | 224 MnlI | 397 TfiI U | 632 BsrI | 871 GsuI | 10 | 229 DdeI | 407 MboII | 637 HindII D | 872 MnlI | | 242 MnlI | 416 HgaI D | 647 HgaI D | 880 NspI | | 243 FokI D | 418 TaqI D | 654 Tsp509I | 880 NlaIII | | 245 BstXI D | 420 Ksp632I U | 666 AciI | 880 AciI | | 251 AsuI | 423 MnlI | 668 FnuDII | 880 SphI U | | 251 AvaII | 425 BstEII U | 670 Csp6I | 882 HgiCI D | | 254 AciI | 425 MaeIII | 671 RsaI | 882 Acc65I U | | 265 SfaNI | 426 BsrI | 680 NlaIII | 883 Csp6I | | 267 MluI U | 429 BinI U | 683 AsuI | 884 RsaI | | 267 GsuI | 434 MboI U | 684 HaeIII U | 884 NlaIV | | 267 AflIII U | 436 DpnI U | 691 MseI D | 886 KpnI U | | 269 FnuDII | 437 HphI U | 692 BsiYI | | +------------------+------------------+------------------+--------------- ---+ U: Unique restriction site for this enzyme (on the complete sequence). D: Enzyme with two restriction sites (on the complete sequence). ===02-SEP- 2016====================RESTRI===========================PC/GENE=== 5. Trình tự gốc gen VP1-2A-VP3 của virus LMLM type O ở Việt Nam ID VP1-2A-VP3 goc PRELIMINARY; DNA; 1356 BP. SQ SEQUENCE 1356 BP; 312 A; 406 C; 356 G; 282 T; ATGACCACTT CGACGGGCGA GTCAGCCGAC CCCGTGACCG CTACCGTTGA GAACTACGGT GGCGAGACAC AGGTCCAGAG GCGTCACCAC ACAGACGTCT CATTTATATT GGACAGATTT GTGAAAGTCA CACCACAAGA CCAAATTAAT GTTTTGGACC TGATGCAGAC CCCCCCCCAC 11 ACTCTGGTGG GAGCGCTCCT TCGTACTGCC ACTTACTACT TTGCTGATCT AGAGGTGGCA GTGAAACACG AGGGGGATCT CACCTGGGTA CCAAATGGAG CACCTGAGGC AGCCTTGGGT AATACCACCA ACCCAACGGC ATACCACAAA GCGCCACTCA CTCGGCTTGC ACTGCCTTAC ACGGCACCAC ACCGTGTTCT GGCTACCGTT TACAACGGGA ACTGCAAATA CGCTGGGGGT CCACTGACCA ACGTGAGAGG CGATCTCCAG GTGCTGGCTC AGAAGGCGGC GAGGCCGCTG CCTACCTCCT TCAACTATGG TGCCATCAAA GCCACCCGGG TGACAGAACT GCTGTACCGC ATGAAGAGGG CCGAGACGTA TTGCCCGCGG CCTCTTTTGG CCGTCCACCC GGATCAAGCT AGACACAAAC AGAAAATTGT GGCACCTGTG AAACAGTCCT GGAAGTTTGA CCTGCTCAAG TTGGCGGGAG ACGTTGAGTC CAACCCCGGG CCAGGGATTT TCCCTGTGGC ATGTAGCGAC GGTTACGGCG GTTTGGTGAC CACTGACCCA AAGACGGCTG ACCCCGTCTA CGGCAAGGTG TTTAACCCCC CCCGTAACAT GTTGCCGGGG CGGTTCACCA ACTTCTTGGA TGTGGCCGAG GCGTGCCCCA CGTTTCTGCA CTTCGAAGGT GACGTGCCAT ACGTGACCAC GAAGACGGAC TCAGACAGGA TTCTCGCGCA ATTTGACTTG TCTCTGGCAG CAAAACACAT GTCAAACACC TTTCTTGCAG GTCTTGCCCA GTACTACACG CAGTACAGTG GTACGATCAA CCTGCACTTC ATGTTCACAG GTCCCACTGA CGCGAAAGCG CGTTACATGA TTGCATACGC CCCACCGGGT ATGGAGCCGC CTCGCACGCC TGAGGCCGCT GCCCATTGCA TTCATGCTGA GTGGGACACG GGTTTGAATT CAAAGTTCAC CTTTTCCATC CCATATCTCT CAGCAGCTGA CTACGCATAC ACCGCGTCTG ACACTGCCGA GACCACAAAT GTTCAGGGAT GGGTCTGCTT GTTCCAGATA ACACACGGGA AAGCTGACGG CGACGCTCTT GTCGTGCTGG CCAGCGCTGG TAAAGACTTT GAGCTGCGCT TGCCTGTGGA CGCCCGCCAA CAGTAG // 6. Trình tự gen VP1-2A-VP3 sau khi tối ưu của virus LMLM type O ở Việt Nam ID Vp1-2A-VP3-S Sau toi uu va gan Res Sites PRELIMINARY; DNA; 1382 BP. SQ SEQUENCE 1382 BP; 336 A; 428 C; 340 G; 278 T; EcoRIBamHI GAATTCGGAT CCATGACGAC CAGCACAGGA GAATCAGCAG ATCCGGTTAC AGCGACGGTT GAAAACTACG GTGGAGAGAC GCAAGTCCAA AGACGCCACC ACACCGACGT GTCCTTCATC CTCGATAGGT TCGTTAAAGT GACTCCTCAA GACCAGATTA ACGTGCTCGA TTTGATGCAA ACCCCACCAC ACACTCTGGT CGGCGCGCTG CTCAGAACCG CTACTTACTA CTTCGCCGAC TTGGAGGTCG CCGTTAAGCA TGAAGGCGAT CTGACATGGG TCCCAAACGG AGCCCCGGAG GCAGCACTGG GTAACACCAC TAACCCTACC GCGTACCACA AGGCTCCCCT CACACGCCTG GCACTCCCTT ACACGGCACC ACATCGTGTG TTGGCAACTG TCTACAACGG TAACTGCAAG TACGCCGGTG GTCCACTGAC TAACGTCAGA GGTGACCTGC AAGTGCTCGC TCAAAAGGCT GCCCGCCCGC TGCCGACGTC TTTCAACTAC GGAGCCATCA AGGCAACCAG GGTCACTGAG TTGCTGTACA GGATGAAAAG AGCTGAAACC TACTGTCCAC GTCCGCTCTT GGCTGTGCAC 12 CCAGATCAAG CCCGCCACAA GCAGAAAATT GTTGCTCCGG TGAAGCAAAG TTGGAAGTTC GACTTGCTGA AATTGGCCGG TGACGTCGAA AGTAACCCTG GCCCCGGAAT TTTCCCAGTC GCCTGCTCCG ACGGTTACGG AGGCCTGGTT ACAACGGACC CAAAGACCGC TGACCCTGTT TACGGAAAAG TTTTCAACCC TCCCCGCAAC ATGCTGCCAG GTCGTTTCAC GAACTTCCTC GACGTCGCCG AGGCATGTCC TACATTCCTG CACTTCGAGG GAGACGTCCC CTACGTTACC ACTAAGACAG ACTCGGATCG TATCCTCGCC CAGTTCGATT TGTCGCTGGC AGCGAAACAC ATGTCCAACA CCTTCTTGGC CGGACTGGCA CAGTACTACA CCCAATACTC CGGTACTATC AACCTGCATT TCATGTTCAC AGGCCCAACG GACGCGAAGG CTAGGTACAT GATTGCCTAC GCACCACCAG GAATGGAGCC TCCAAGGACC CCTGAAGCTG CCGCACACTG CATCCATGCT GAGTGGGACA CCGGACTCAA CTCTAAGTTC ACTTTCTCAA TTCCTTACTT GAGTGCGGCT GACTACGCGT ACACAGCTTC CGATACGGCC GAAACAACGA ACGTTCAGGG TTGGGTGTGT CTGTTCCAAA TCACTCACGG AAAGGCCGAC GGTGACGCAC TCGTGGTCTT GGCGTCTGCT GGCAAAGACT TCGAACTCAG GTTGCCCGTG GATGCTAGAC AACAATAAAA GCTTGCGGCC GCHindIIINotI // ===31-AUG-2016================NALIGN=======================PC/GENE=== 7. Kết quả sao sánh giữa hai gen VP1-2A-VP3 trước và sau tối ưu codon The two sequences to be aligned are: VP1-2A-VP3-S. Total number of bases: 1356. VP1-2A-VP3-T. Total number of bases: 1356. Open gap cost : 10 Unit gap cost : 10 The character to show that two aligned residues are identical is '|' VP1-2A-VP3-S - ATGACGACCAGCACAGGAGAATCAGCAGATCCGGTTACAGCGACGGTTGA -50 ||||| || || || || ||||| || || || || || || ||||| VP1-2A-VP3-T - ATGACCACTTCGACGGGCGAGTCAGCCGACCCCGTGACCGCTACCGTTGA -50 VP1-2A-VP3-S - AAACTACGGTGGAGAGACGCAAGTCCAAAGACGCCACCACACCGACGTGT -100 ||||||||||| ||||| || ||||| || || |||||||| ||||| | VP1-2A-VP3-T - GAACTACGGTGGCGAGACACAGGTCCAGAGGCGTCACCACACAGACGTCT -100 VP1-2A-VP3-S - CCTTCATCCTCGATAGGTTCGTTAAAGTGACTCCTCAAGACCAGATTAAC -150 13 | || || | || || || || ||||| || || |||||||| ||||| VP1-2A-VP3-T - CATTTATATTGGACAGATTTGTGAAAGTCACACCACAAGACCAAATTAAT -150 VP1-2A-VP3-S - GTGCTCGATTTGATGCAAACCCCACCACACACTCTGGTCGGCGCGCTGCT -200 || | || ||||||| ||||| || ||||||||||| || ||||| || VP1-2A-VP3-T - GTTTTGGACCTGATGCAGACCCCCCCCCACACTCTGGTGGGAGCGCTCCT -200 VP1-2A-VP3-S - CAGAACCGCTACTTACTACTTCGCCGACTTGGAGGTCGCCGTTAAGCATG -250 | || || ||||||||||| || || | ||||| || || || || | VP1-2A-VP3-T - TCGTACTGCCACTTACTACTTTGCTGATCTAGAGGTGGCAGTGAAACACG -250 VP1-2A-VP3-S - AAGGCGATCTGACATGGGTCCCAAACGGAGCCCCGGAGGCAGCACTGGGT -300 | || ||||| || ||||| ||||| ||||| || |||||||| ||||| VP1-2A-VP3-T - AGGGGGATCTCACCTGGGTACCAAATGGAGCACCTGAGGCAGCCTTGGGT -300 VP1-2A-VP3-S - AACACCACTAACCCTACCGCGTACCACAAGGCTCCCCTCACACGCCTGGC -350 || ||||| ||||| || || |||||||| || || ||||| || || || VP1-2A-VP3-T - AATACCACCAACCCAACGGCATACCACAAAGCGCCACTCACTCGGCTTGC -350 VP1-2A-VP3-S - ACTCCCTTACACGGCACCACATCGTGTGTTGGCAACTGTCTACAACGGTA -400 ||| ||||||||||||||||| ||||| |||| || || |||||||| | VP1-2A-VP3-T - ACTGCCTTACACGGCACCACACCGTGTTCTGGCTACCGTTTACAACGGGA -400 VP1-2A-VP3-S - ACTGCAAGTACGCCGGTGGTCCACTGACTAACGTCAGAGGTGACCTGCAA -450 ||||||| ||||| || ||||||||||| ||||| ||||| || || || VP1-2A-VP3-T - ACTGCAAATACGCTGGGGGTCCACTGACCAACGTGAGAGGCGATCTCCAG -450 ===02-SEP-2016===========RESTRI===========================PC/GENE=== ***************************** * RESTRICTION-SITE ANALYSIS * ***************************** Done on DNA sequence VP1-2A-VP3-Sau TU+Res sites, total number of bases: 1382. - The sequence was considered to be: LINEAR. - The analysis was done on bases 1 to 1382. 14 --------------------------------------- 8. Danh sách trình tự enzyme cắt trong gen VP1-2A-VP3 List of cuts by enzyme ====================== AatII : 499 686 845 887 AccI : 401 AciI : 218 329 483 487 583 612 767 804 1121 1195 1375 1379 AclI : 1241 AcyI : 93 496 683 842 884 1312 AeuI : 358 528 698 745 818 1088 AflIII : 107 959 1205 AluI : 562 1117 1216 1371 ApaLI : 595 ApoI : 1 707 AsuI : 279 430 700 756 1042 1106 AsuII : 1331 AvaII : 279 430 756 1106 Bam HI : 7 BbvI : 194 313 465 476 800 961 1104 BcgI : 1345 BetI : 42 636 1009 1150 BglI : 298 520 985 BinI : 2 15 34 923 BsgI : 853 BsiYI : 372 703 781 BsmFI : 265 871 1159 BsmAI : 70 875 Bsp1407I : 545 BspMI : 464 1031 BsrI : 311 715 930 989 BsrBI : 585 BssHII : 203 BssKI : 293 356 526 696 702 743 816 1086 BstEII : 451 CauII : 295 704 15 CfrI : 674 977 1226 1376 Cfr10I : 424 676 Csp6I : 333 420 546 993 1013 1065 1209 DdeI : 211 536 902 1139 1163 1336 DpnI : 9 41 269 605 917 DraII : 279 1106 DraIII : 385 DrdI : 244 DsaI : 1346 Eco57I : 1133 Eco RI : 1 EcoRII : 356 526 696 743 816 1086 Esp3I : 70 875 Fnu4HI : 208 302 479 490 814 950 1118 1121 1196 1376 1379 FnuDII : 205 331 1054 1207 FokI : 103 564 1117 1363 HaeIII : 676 701 743 979 1043 1228 1285 1378 HgaI : 87 101 1060 1301 1303 HgiAI : 167 467 599 HgiCI : 375 HgiJII : 294 HhaI : 205 207 Hin6I : 203 205 Hind III : 1369 HinfI : 31 141 910 1154 HpaII : 43 295 425 637 677 704 980 1010 1151 HphI : 463 651 691 1303 MaeI : 1061 1355 MaeII : 107 161 443 496 579 683 842 884 893 1241 MaeIII : 45 138 310 409 451 531 679 691 733 747 894 1291 MboI : 7 39 267 603 915 McrI : 1229 1379 MluI : 1205 16 MnlI : 130 155 237 291 358 442 733 809 843 847 870 934 1109 MseI : 134 158 254 MslI : 106 MwoI : 298 475 520 985 1204 1213 NlaIII : 15 262 278 813 857 963 1035 1071 1138 NlaIV : 9 280 281 291 344 377 513 702 758 1097 1108 NotI : 1376 NspI : 813 857 963 NspBII : 489 769 PleI : 135 904 1148 PpuMI : 279 1106 PshAI : 82 277 442 RsaI : 334 421 547 994 1014 1066 1210 ScaI : 994 ScrFI : 295 358 528 698 704 745 818 1088 SduI : 167 294 467 599 SecI : 293 527 696 702 848 1102 1346 SfaNI : 164 1139 1341 SgrAI : 424 StuI : 743 StyI : 1102 TaqI : 122 167 659 686 839 875 935 1331 TfiI : 31 Tsp509I : 1 626 670 707 1178 XcmI : 972 XhoII : 7 39 XmaIII : 1226 1376 Total number of cuts is : 339. --------------------------------------- Sorted list of enzymes by number of cuts ======================================== +----------------+----------------+------------------+------------------+ 17 | MnlI : 13 | MboI : 5 | HgiAI : 3 | BcgI : 1 | | MaeIII : 12 | Tsp509I : 5 | BsmFI : 3 | Eco57I : 1 | | AciI : 12 | HgaI : 5 | XmaIII : 2 | AccI : 1 | | Fnu4HI : 11 | BetI : 4 | BspMI : 2 | HgiCI : 1 | | NlaIV : 11 | BinI : 4 | Cfr10I : 2 | AsuII : 1 | | MaeII : 10 | BsrI : 4 | PpuMI : 2 | DraIII : 1 | | HpaII : 9 | AatII : 4 | Hin6I : 2 | Bsp1407I : 1 | | NlaIII : 9 | SduI : 4 | CauII : 2 | StuI : 1 | | HaeIII : 8 | CfrI : 4 | Esp3I : 2 | MluI : 1 | | ScrFI : 8 | AvaII : 4 | XhoII : 2 | NotI : 1 | | BssKI : 8 | AluI : 4 | HhaI : 2 | BsrBI : 1 | | TaqI : 8 | FokI : 4 | MaeI : 2 | ScaI : 1 | | BbvI : 7 | FnuDII : 4 | BsmAI : 2 | AclI : 1 | | SecI : 7 | HphI : 4 | DraII : 2 | BstEII : 1 | | Csp6I : 7 | HinfI : 4 | ApoI : 2 | EcoRI : 1 | | RsaI : 7 | PshAI : 3 | NspBII : 2 | XcmI : 1 | | AsuI : 6 | NspI : 3 | McrI : 2 | BsgI : 1 | | EcoRII : 6 | PleI : 3 | StyI : 1 | SgrAI : 1 | 18 | MwoI : 6 | AflIII : 3 | DsaI : 1 | HindIII : 1 | | AeuI : 6 | BglI : 3 | BssHII : 1 | DrdI : 1 | | AcyI : 6 | MseI : 3 | MslI : 1 | ApaLI : 1 | | DdeI : 6 | SfaNI : 3 | BamHI : 1 | TfiI : 1 | | DpnI : 5 | BsiYI : 3 | HgiJII : 1 | | +----------------+----------------+------------------+------------------+ --------------------------------------- List of non cutting selected enzymes ==================================== Acc65I , AflII , AgeI , AhaIII , AhyI , AlwNI , ApaI , AscI , AvaI , AvrII , BalI , BbeI , BbvII , BclI , BglII , BsaAI , BsaBI , BsmI , Bsp120I , BspHI , BspM90I , BspMII , BsrDI , BstXI , ClaI , Eam1105I, Ecl136II, Eco31I , Eco47III, Eco56I , Eco78I , EcoNI , EcoRV , EcoT22I , EspI , FseI , GsuI , HaeII , HindII , HpaI , KasI , KpnI , Ksp632I , MboII , MfeI , MstI , NaeI , NarI , NcoI , NdeI , NheI , NruI , PacI , PflMI , PmaCI , PmeI , Ppu10I , PstI , PvuI , PvuII , RsrII , SacI , SacII , SalI , SapI , SauI , SexAI , SfeI , SfiI , SgfI , SmaI , SnaBI , SpeI , SphI , SplI , SrfI , Sse8387I, SspI , SwaI , Tth111I , VspI , XbaI , XhoI , XmnI Total number of selected enzymes which do not cut: 84 --------------------------------------- Sorted list of cuts =================== +----------------+----------------+------------------+------------------+ | 1 ApoI D | 333 Csp6I | 698 AeuI | 1013 Csp6I | | 1 Tsp509I | 334 RsaI | 698 ScrFI | 1014 RsaI | 19 | 1 EcoRI U | 344 NlaIV | 700 AsuI | 1031 BspMI D | | 2 BinI | 356 EcoRII | 701 HaeIII | 1035 NlaIII | | 7 MboI | 356 BssKI | 702 BssKI | 1042 AsuI | | 7 BamHI U | 358 MnlI | 702 SecI | 1043 HaeIII | | 7 XhoII D | 358 ScrFI | 702 NlaIV | 1054 FnuDII | | 9 NlaIV | 358 AeuI | 703 BsiYI | 1060 HgaI | | 9 DpnI | 372 BsiYI | 704 CauII D | 1061 MaeI D | | 15 NlaIII | 375 HgiCI U | 704 HpaII | 1065 Csp6I | | 15 BinI | 377 NlaIV | 704 ScrFI | 1066 RsaI | | 31 TfiI U | 385 DraIII U | 707 Tsp509I | 1071 NlaIII | | 31 HinfI | 401 AccI U | 707 ApoI D | 1086 BssKI | | 34 BinI | 409 MaeIII | 715 BsrI | 1086 EcoRII | | 39 XhoII D | 420 Csp6I | 733 MnlI | 1088 AeuI | | 39 MboI | 421 RsaI | 733 MaeIII | 1088 ScrFI | | 41 DpnI | 424 Cfr10I D | 743 StuI U | 1097 NlaIV | | 42 BetI | 424 SgrAI U | 743 BssKI | 1102 StyI U | | 43 HpaII | 425 HpaII | 743 HaeIII | 1102 SecI | | 45 MaeIII | 430 AsuI | 743 EcoRII | 1104 BbvI | 20 | 70 Esp3I D | 430 AvaII | 745 AeuI | 1106 PpuMI D | | 70 BsmAI D | 442 MnlI | 745 ScrFI | 1106 DraII D | | 82 PshAI | 442 PshAI | 747 MaeIII | 1106 AsuI | | 87 HgaI | 443 MaeII | 756 AsuI | 1106 AvaII | | 93 AcyI | 451 MaeIII | 756 AvaII | 1108 NlaIV | | 101 HgaI | 451 BstEII U | 758 NlaIV | 1109 MnlI | | 103 FokI | 463 HphI | 767 AciI | 1117 AluI | | 106 MslI U | 464 BspMI D | 769 NspBII D | 1117 FokI | | 107 AflIII | 465 BbvI | 781 BsiYI | 1118 Fnu4HI | | 107 MaeII | 467 HgiAI | 800 BbvI | 1121 Fnu4HI | | 122 TaqI | 467 SduI | 804 AciI | 1121 AciI | | 130 MnlI | 475 MwoI | 809 MnlI | 1133 Eco57I U | | 134 MseI | 476 BbvI | 813 NlaIII | 1138 NlaIII | | 135 PleI | 479 Fnu4HI | 813 NspI | 1139 DdeI | | 138 MaeIII | 483 AciI | 814 Fnu4HI | 1139 SfaNI | | 141 HinfI | 487 AciI | 816 EcoRII | 1148 PleI | | 155 MnlI | 489 NspBII D | 816 BssKI | 1150 BetI | | 158 MseI | 490 Fnu4HI | 818 AeuI | 1151 HpaII | 21 | 161 MaeII | 496 AcyI | 818 ScrFI | 1154 HinfI | | 164 SfaNI | 496 MaeII | 839 TaqI | 1159 BsmFI | | 167 SduI | 499 AatII | 842 MaeII | 1163 DdeI | | 167 TaqI | 513 NlaIV | 842 AcyI | 1178 Tsp509I | | 167 HgiAI | 520 MwoI | 843 MnlI | 1195 AciI | | 194 BbvI | 520 BglI | 845 AatII | 1196 Fnu4HI | | 203 Hin6I D | 526 BssKI | 847 MnlI | 1204 MwoI | | 203 BssHII U | 526 EcoRII | 848 SecI | 1205 MluI U | | 205 FnuDII | 527 SecI | 853 BsgI U | 1205 AflIII | | 205 HhaI D | 528 AeuI | 857 NspI | 1207 FnuDII | | 205 Hin6I D | 528 ScrFI | 857 NlaIII | 1209 Csp6I | | 207 HhaI D | 531 MaeIII | 870 MnlI | 1210 RsaI | | 208 Fnu4HI | 536 DdeI | 871 BsmFI | 1213 MwoI | | 211 DdeI | 545 Bsp1407I U | 875 TaqI | 1216 AluI | | 218 AciI | 546 Csp6I | 875 BsmAI D | 1226 XmaIII D | | 237 MnlI | 547 RsaI | 875 Esp3I D | 1226 CfrI | | 244 DrdI U | 562 AluI | 884 MaeII | 1228 HaeIII | | 254 MseI | 564 FokI | 884 AcyI | 1229 McrI D | 22 | 262 NlaIII | 579 MaeII | 887 AatII | 1241 AclI U | | 265 BsmFI | 583 AciI | 893 MaeII | 1241 MaeII | | 267 MboI | 585 BsrBI U | 894 MaeIII | 1285 HaeIII | | 269 DpnI | 595 ApaLI U | 902 DdeI | 1291 MaeIII | | 277 PshAI | 599 HgiAI | 904 PleI | 1301 HgaI | | 278 NlaIII | 599 SduI | 910 HinfI | 1303 HgaI | | 279 PpuMI D | 603 MboI | 915 MboI | 1303 HphI | | 279 AvaII | 605 DpnI | 917 DpnI | 1312 AcyI | | 279 AsuI | 612 AciI | 923 BinI | 1331 TaqI | | 279 DraII D | 626 Tsp509I | 930 BsrI | 1331 AsuII U | | 280 NlaIV | 636 BetI | 934 MnlI | 1336 DdeI | | 281 NlaIV | 637 HpaII | 935 TaqI | 1341 SfaNI | | 291 NlaIV | 651 HphI | 950 Fnu4HI | 1345 BcgI U | | 291 MnlI | 659 TaqI | 959 AflIII | 1346 DsaI U | | 293 BssKI | 670 Tsp509I | 961 BbvI | 1346 SecI | | 293 SecI | 674 CfrI | 963 NlaIII | 1355 MaeI D | | 294 HgiJII U | 676 HaeIII | 963 NspI | 1363 FokI | | 294 SduI | 676 Cfr10I D | 972 XcmI U | 1369 HindIII U | 23 | 295 ScrFI | 677 HpaII | 977 CfrI | 1371 AluI | | 295 HpaII | 679 MaeIII | 979 HaeIII | 1375 AciI | | 295 CauII D | 683 MaeII | 980 HpaII | 1376 XmaIII D | | 298 BglI | 683 AcyI | 985 BglI | 1376 Fnu4HI | | 298 MwoI | 686 TaqI | 985 MwoI | 1376 CfrI | | 302 Fnu4HI | 686 AatII | 989 BsrI | 1376 NotI U | | 310 MaeIII | 691 HphI | 993 Csp6I | 1378 HaeIII | | 311 BsrI | 691 MaeIII | 994 ScaI U | 1379 McrI D | | 313 BbvI | 696 BssKI | 994 RsaI | 1379 AciI | 329 AciI | 696 SecI | 1009 BetI | 1379 Fnu4HI | 331 FnuDII | 696 EcoRII | 1010 HpaII | +----------------+-----------------+-----------------+------------------+ U: Unique restriction site for this enzyme (on the complete sequence). D: Enzyme with two restriction sites (on the complete sequence). ===02-SEP-2016==================RESTRI=========================PC/GENE=== ===31-AUG-2016==================PALIGN=========================PC/GENE=== *************************************** 9. Kết quả so sánh trình tự protein suy diền của gen VP1-2A-VP3 trước và sau khi tối ưu codon *************************************** The two sequences to be aligned are: VP1-2A-VP3-S. Total number of residues: 451. VP1-2A-VP3-T. Total number of residues: 451. 24 Comparison matrix : Structure-genetic matrix. Open gap cost : 7 Unit gap cost : 1 The character to show that two aligned residues are identical is '|' VP1-2A-VP3-S - MTTSTGESADPVTATVENYGGETQVQRRHHTDVSFILDRFVKVTPQDQIN -50 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - MTTSTGESADPVTATVENYGGETQVQRRHHTDVSFILDRFVKVTPQDQIN -50 VP1-2A-VP3-S - VLDLMQTPPHTLVGALLRTATYYFADLEVAVKHEGDLTWVPNGAPEAALG -100 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - VLDLMQTPPHTLVGALLRTATYYFADLEVAVKHEGDLTWVPNGAPEAALG -100 VP1-2A-VP3-S - NTTNPTAYHKAPLTRLALPYTAPHRVLATVYNGNCKYAGGPLTNVRGDLQ -150 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - NTTNPTAYHKAPLTRLALPYTAPHRVLATVYNGNCKYAGGPLTNVRGDLQ -150 VP1-2A-VP3-S - VLAQKAARPLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAVHPDQA -200 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - VLAQKAARPLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAVHPDQA -200 VP1-2A-VP3-S - RHKQKIVAPVKQSWKFDLLKLAGDVESNPGPGIFPVACSDGYGGLVTTDP -250 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - RHKQKIVAPVKQSWKFDLLKLAGDVESNPGPGIFPVACSDGYGGLVTTDP -250 VP1-2A-VP3-S - KTADPVYGKVFNPPRNMLPGRFTNFLDVAEACPTFLHFEGDVPYVTTKTD -300 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - KTADPVYGKVFNPPRNMLPGRFTNFLDVAEACPTFLHFEGDVPYVTTKTD -300 VP1-2A-VP3-S - SDRILAQFDLSLAAKHMSNTFLAGLAQYYTQYSGTINLHFMFTGPTDAKA -350 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - SDRILAQFDLSLAAKHMSNTFLAGLAQYYTQYSGTINLHFMFTGPTDAKA -350 VP1-2A-VP3-S - RYMIAYAPPGMEPPRTPEAAAHCIHAEWDTGLNSKFTFSIPYLSAADYAY -400 |||||||||||||||||||||||||||||||||||||||||||||||||| 25 VP1-2A-VP3-T - RYMIAYAPPGMEPPRTPEAAAHCIHAEWDTGLNSKFTFSIPYLSAADYAY -400 VP1-2A-VP3-S - TASDTAETTNVQGWVCLFQITHGKADGDALVVLASAGKDFELRLPVDARQ -450 |||||||||||||||||||||||||||||||||||||||||||||||||| VP1-2A-VP3-T - TASDTAETTNVQGWVCLFQITHGKADGDALVVLASAGKDFELRLPVDARQ -450 VP1-2A-VP3-S - Q -451 | VP1-2A-VP3-T - Q -451 Identity : 451 (100.00%) Number of gaps inserted in VP1-2A-VP3-S: 0 Number of gaps inserted in VP1-2A-VP3-T: 0 ===31-AUG-2016==================PALIGN=========================PC/GENE===

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