CN103740736B - The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression, application - Google Patents
The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression, application Download PDFInfo
- Publication number
- CN103740736B CN103740736B CN201310751325.9A CN201310751325A CN103740736B CN 103740736 B CN103740736 B CN 103740736B CN 201310751325 A CN201310751325 A CN 201310751325A CN 103740736 B CN103740736 B CN 103740736B
- Authority
- CN
- China
- Prior art keywords
- ala
- pro
- ctg
- val
- cca
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000701074 Human alphaherpesvirus 2 Species 0.000 title claims abstract description 74
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 73
- 239000012634 fragment Substances 0.000 title claims abstract description 57
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 47
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 42
- 150000001413 amino acids Chemical class 0.000 claims abstract description 36
- 241000894006 Bacteria Species 0.000 claims abstract description 27
- 235000001014 amino acid Nutrition 0.000 claims abstract description 26
- 239000000427 antigen Substances 0.000 claims abstract description 23
- 102000036639 antigens Human genes 0.000 claims abstract description 23
- 108091007433 antigens Proteins 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 20
- 230000002068 genetic effect Effects 0.000 claims abstract description 15
- 238000011161 development Methods 0.000 claims abstract description 3
- 239000013612 plasmid Substances 0.000 claims description 49
- 238000000746 purification Methods 0.000 claims description 26
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 235000018102 proteins Nutrition 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- 230000029087 digestion Effects 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 claims description 11
- 241000699802 Cricetulus griseus Species 0.000 claims description 11
- 210000001672 ovary Anatomy 0.000 claims description 11
- 108020004414 DNA Proteins 0.000 claims description 10
- 238000001962 electrophoresis Methods 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 10
- 108010050475 glycyl-leucyl-tyrosine Proteins 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 238000012797 qualification Methods 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 238000001890 transfection Methods 0.000 claims description 7
- AFWYPMDMDYCKMD-KBPBESRZSA-N Gly-Leu-Tyr Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 AFWYPMDMDYCKMD-KBPBESRZSA-N 0.000 claims description 6
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 claims description 6
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 claims description 6
- LNLNHXIQPGKRJQ-SRVKXCTJSA-N Pro-Arg-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H]1CCCN1 LNLNHXIQPGKRJQ-SRVKXCTJSA-N 0.000 claims description 6
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 claims description 6
- 108010005233 alanylglutamic acid Proteins 0.000 claims description 6
- 108010087924 alanylproline Proteins 0.000 claims description 6
- 239000003636 conditioned culture medium Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 108020001507 fusion proteins Proteins 0.000 claims description 6
- 102000037865 fusion proteins Human genes 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 108010040030 histidinoalanine Proteins 0.000 claims description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 5
- 108091081024 Start codon Proteins 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 5
- 238000001262 western blot Methods 0.000 claims description 5
- 108010061982 DNA Ligases Proteins 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 4
- 239000000385 dialysis solution Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000013519 translation Methods 0.000 claims description 4
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 claims description 3
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 claims description 3
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-N 0.000 claims description 3
- FOWHQTWRLFTELJ-FXQIFTODSA-N Ala-Asp-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N FOWHQTWRLFTELJ-FXQIFTODSA-N 0.000 claims description 3
- ZDYNWWQXFRUOEO-XDTLVQLUSA-N Ala-Gln-Tyr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZDYNWWQXFRUOEO-XDTLVQLUSA-N 0.000 claims description 3
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 claims description 3
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-N 0.000 claims description 3
- VHEVVUZDDUCAKU-FXQIFTODSA-N Ala-Met-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O VHEVVUZDDUCAKU-FXQIFTODSA-N 0.000 claims description 3
- IPZQNYYAYVRKKK-FXQIFTODSA-N Ala-Pro-Ala Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IPZQNYYAYVRKKK-FXQIFTODSA-N 0.000 claims description 3
- IORKCNUBHNIMKY-CIUDSAMLSA-N Ala-Pro-Glu Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O IORKCNUBHNIMKY-CIUDSAMLSA-N 0.000 claims description 3
- OLVCTPPSXNRGKV-GUBZILKMSA-N Ala-Pro-Pro Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 OLVCTPPSXNRGKV-GUBZILKMSA-N 0.000 claims description 3
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 claims description 3
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 claims description 3
- AETQNIIFKCMVHP-UVBJJODRSA-N Ala-Trp-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AETQNIIFKCMVHP-UVBJJODRSA-N 0.000 claims description 3
- IYKVSFNGSWTTNZ-GUBZILKMSA-N Ala-Val-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IYKVSFNGSWTTNZ-GUBZILKMSA-N 0.000 claims description 3
- YFWTXMRJJDNTLM-LSJOCFKGSA-N Arg-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N YFWTXMRJJDNTLM-LSJOCFKGSA-N 0.000 claims description 3
- DBKNLHKEVPZVQC-LPEHRKFASA-N Arg-Ala-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O DBKNLHKEVPZVQC-LPEHRKFASA-N 0.000 claims description 3
- DCGLNNVKIZXQOJ-FXQIFTODSA-N Arg-Asn-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N DCGLNNVKIZXQOJ-FXQIFTODSA-N 0.000 claims description 3
- BBYTXXRNSFUOOX-IHRRRGAJSA-N Arg-Cys-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BBYTXXRNSFUOOX-IHRRRGAJSA-N 0.000 claims description 3
- LMPKCSXZJSXBBL-NHCYSSNCSA-N Arg-Gln-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O LMPKCSXZJSXBBL-NHCYSSNCSA-N 0.000 claims description 3
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 claims description 3
- FNXCAFKDGBROCU-STECZYCISA-N Arg-Ile-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FNXCAFKDGBROCU-STECZYCISA-N 0.000 claims description 3
- VIINVRPKMUZYOI-DCAQKATOSA-N Arg-Met-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VIINVRPKMUZYOI-DCAQKATOSA-N 0.000 claims description 3
- ATABBWFGOHKROJ-GUBZILKMSA-N Arg-Pro-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O ATABBWFGOHKROJ-GUBZILKMSA-N 0.000 claims description 3
- KXOPYFNQLVUOAQ-FXQIFTODSA-N Arg-Ser-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KXOPYFNQLVUOAQ-FXQIFTODSA-N 0.000 claims description 3
- INOIAEUXVVNJKA-XGEHTFHBSA-N Arg-Thr-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O INOIAEUXVVNJKA-XGEHTFHBSA-N 0.000 claims description 3
- ZPWMEWYQBWSGAO-ZJDVBMNYSA-N Arg-Thr-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZPWMEWYQBWSGAO-ZJDVBMNYSA-N 0.000 claims description 3
- PFOYSEIHFVKHNF-FXQIFTODSA-N Asn-Ala-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PFOYSEIHFVKHNF-FXQIFTODSA-N 0.000 claims description 3
- HDHZCEDPLTVHFZ-GUBZILKMSA-N Asn-Leu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O HDHZCEDPLTVHFZ-GUBZILKMSA-N 0.000 claims description 3
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 claims description 3
- GBAWQWASNGUNQF-ZLUOBGJFSA-N Asp-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)O)N GBAWQWASNGUNQF-ZLUOBGJFSA-N 0.000 claims description 3
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 claims description 3
- NAPNAGZWHQHZLG-ZLUOBGJFSA-N Asp-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N NAPNAGZWHQHZLG-ZLUOBGJFSA-N 0.000 claims description 3
- ZCKYZTGLXIEOKS-CIUDSAMLSA-N Asp-Asp-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N ZCKYZTGLXIEOKS-CIUDSAMLSA-N 0.000 claims description 3
- VZNOVQKGJQJOCS-SRVKXCTJSA-N Asp-Asp-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VZNOVQKGJQJOCS-SRVKXCTJSA-N 0.000 claims description 3
- VAWNQIGQPUOPQW-ACZMJKKPSA-N Asp-Glu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VAWNQIGQPUOPQW-ACZMJKKPSA-N 0.000 claims description 3
- GHODABZPVZMWCE-FXQIFTODSA-N Asp-Glu-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GHODABZPVZMWCE-FXQIFTODSA-N 0.000 claims description 3
- QPDUWAUSSWGJSB-NGZCFLSTSA-N Asp-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N QPDUWAUSSWGJSB-NGZCFLSTSA-N 0.000 claims description 3
- XSQAWJCVYDEWPT-GUBZILKMSA-N Cys-Met-Arg Chemical compound SC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XSQAWJCVYDEWPT-GUBZILKMSA-N 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- BVELAHPZLYLZDJ-HGNGGELXSA-N Gln-His-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O BVELAHPZLYLZDJ-HGNGGELXSA-N 0.000 claims description 3
- IWUFOVSLWADEJC-AVGNSLFASA-N Gln-His-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O IWUFOVSLWADEJC-AVGNSLFASA-N 0.000 claims description 3
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-N 0.000 claims description 3
- WLRYGVYQFXRJDA-DCAQKATOSA-N Gln-Pro-Pro Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 WLRYGVYQFXRJDA-DCAQKATOSA-N 0.000 claims description 3
- UTKICHUQEQBDGC-ACZMJKKPSA-N Glu-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N UTKICHUQEQBDGC-ACZMJKKPSA-N 0.000 claims description 3
- RLZBLVSJDFHDBL-KBIXCLLPSA-N Glu-Ala-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RLZBLVSJDFHDBL-KBIXCLLPSA-N 0.000 claims description 3
- SRZLHYPAOXBBSB-HJGDQZAQSA-N Glu-Arg-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SRZLHYPAOXBBSB-HJGDQZAQSA-N 0.000 claims description 3
- SYWCGQOIIARSIX-SRVKXCTJSA-N Glu-Pro-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O SYWCGQOIIARSIX-SRVKXCTJSA-N 0.000 claims description 3
- IDEODOAVGCMUQV-GUBZILKMSA-N Glu-Ser-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IDEODOAVGCMUQV-GUBZILKMSA-N 0.000 claims description 3
- BDISFWMLMNBTGP-NUMRIWBASA-N Glu-Thr-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O BDISFWMLMNBTGP-NUMRIWBASA-N 0.000 claims description 3
- YQAQQKPWFOBSMU-WDCWCFNPSA-N Glu-Thr-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O YQAQQKPWFOBSMU-WDCWCFNPSA-N 0.000 claims description 3
- QXUPRMQJDWJDFR-NRPADANISA-N Glu-Val-Ser Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O QXUPRMQJDWJDFR-NRPADANISA-N 0.000 claims description 3
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 claims description 3
- QSTLUOIOYLYLLF-WDSKDSINSA-N Gly-Asp-Glu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QSTLUOIOYLYLLF-WDSKDSINSA-N 0.000 claims description 3
- QCTLGOYODITHPQ-WHFBIAKZSA-N Gly-Cys-Ser Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O QCTLGOYODITHPQ-WHFBIAKZSA-N 0.000 claims description 3
- FIQQRCFQXGLOSZ-WDSKDSINSA-N Gly-Glu-Asp Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FIQQRCFQXGLOSZ-WDSKDSINSA-N 0.000 claims description 3
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 claims description 3
- NNCSJUBVFBDDLC-YUMQZZPRSA-N Gly-Leu-Ser Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O NNCSJUBVFBDDLC-YUMQZZPRSA-N 0.000 claims description 3
- HFPVRZWORNJRRC-UWVGGRQHSA-N Gly-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN HFPVRZWORNJRRC-UWVGGRQHSA-N 0.000 claims description 3
- JNGHLWWFPGIJER-STQMWFEESA-N Gly-Pro-Tyr Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JNGHLWWFPGIJER-STQMWFEESA-N 0.000 claims description 3
- MVADCDSCFTXCBT-CIUDSAMLSA-N His-Asp-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MVADCDSCFTXCBT-CIUDSAMLSA-N 0.000 claims description 3
- 108010093488 His-His-His-His-His-His Proteins 0.000 claims description 3
- ZVKDCQVQTGYBQT-LSJOCFKGSA-N His-Pro-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O ZVKDCQVQTGYBQT-LSJOCFKGSA-N 0.000 claims description 3
- WYKXJGWSJUULSL-AVGNSLFASA-N His-Val-Arg Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)Cc1cnc[nH]1)C(=O)N[C@@H](CCCNC(=N)N)C(=O)O WYKXJGWSJUULSL-AVGNSLFASA-N 0.000 claims description 3
- HERITAGIPLEJMT-GVARAGBVSA-N Ile-Ala-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HERITAGIPLEJMT-GVARAGBVSA-N 0.000 claims description 3
- JLWLMGADIQFKRD-QSFUFRPTSA-N Ile-His-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CN=CN1 JLWLMGADIQFKRD-QSFUFRPTSA-N 0.000 claims description 3
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 claims description 3
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 claims description 3
- WCTCIIAGNMFYAO-DCAQKATOSA-N Leu-Cys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O WCTCIIAGNMFYAO-DCAQKATOSA-N 0.000 claims description 3
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 claims description 3
- ZDBMWELMUCLUPL-QEJZJMRPSA-N Leu-Phe-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 ZDBMWELMUCLUPL-QEJZJMRPSA-N 0.000 claims description 3
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 claims description 3
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 claims description 3
- ONHCDMBHPQIPAI-YTQUADARSA-N Leu-Trp-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N ONHCDMBHPQIPAI-YTQUADARSA-N 0.000 claims description 3
- OZTZJMUZVAVJGY-BZSNNMDCSA-N Leu-Tyr-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N OZTZJMUZVAVJGY-BZSNNMDCSA-N 0.000 claims description 3
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 claims description 3
- OBVHKUFUDCPZDW-JYJNAYRXSA-N Met-Arg-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OBVHKUFUDCPZDW-JYJNAYRXSA-N 0.000 claims description 3
- WXJLBSXNUHIGSS-OSUNSFLBSA-N Met-Thr-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WXJLBSXNUHIGSS-OSUNSFLBSA-N 0.000 claims description 3
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 claims description 3
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- CTNODEMQIKCZGQ-JYJNAYRXSA-N Phe-Gln-His Chemical compound C([C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=CC=C1 CTNODEMQIKCZGQ-JYJNAYRXSA-N 0.000 claims description 3
- JLLJTMHNXQTMCK-UBHSHLNASA-N Phe-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=CC=C1 JLLJTMHNXQTMCK-UBHSHLNASA-N 0.000 claims description 3
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 claims description 3
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 claims description 3
- FKKHDBFNOLCYQM-FXQIFTODSA-N Pro-Cys-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O FKKHDBFNOLCYQM-FXQIFTODSA-N 0.000 claims description 3
- LSIWVWRUTKPXDS-DCAQKATOSA-N Pro-Gln-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LSIWVWRUTKPXDS-DCAQKATOSA-N 0.000 claims description 3
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 claims description 3
- QCARZLHECSFOGG-CIUDSAMLSA-N Pro-Glu-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O QCARZLHECSFOGG-CIUDSAMLSA-N 0.000 claims description 3
- VYWNORHENYEQDW-YUMQZZPRSA-N Pro-Gly-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 VYWNORHENYEQDW-YUMQZZPRSA-N 0.000 claims description 3
- LPGSNRSLPHRNBW-AVGNSLFASA-N Pro-His-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 LPGSNRSLPHRNBW-AVGNSLFASA-N 0.000 claims description 3
- FMLRRBDLBJLJIK-DCAQKATOSA-N Pro-Leu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FMLRRBDLBJLJIK-DCAQKATOSA-N 0.000 claims description 3
- SBVPYBFMIGDIDX-SRVKXCTJSA-N Pro-Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H]2NCCC2)CCC1 SBVPYBFMIGDIDX-SRVKXCTJSA-N 0.000 claims description 3
- KBUAPZAZPWNYSW-SRVKXCTJSA-N Pro-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KBUAPZAZPWNYSW-SRVKXCTJSA-N 0.000 claims description 3
- POQFNPILEQEODH-FXQIFTODSA-N Pro-Ser-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O POQFNPILEQEODH-FXQIFTODSA-N 0.000 claims description 3
- PKHDJFHFMGQMPS-RCWTZXSCSA-N Pro-Thr-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PKHDJFHFMGQMPS-RCWTZXSCSA-N 0.000 claims description 3
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 claims description 3
- IMNVAOPEMFDAQD-NHCYSSNCSA-N Pro-Val-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IMNVAOPEMFDAQD-NHCYSSNCSA-N 0.000 claims description 3
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 claims description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 3
- LALNXSXEYFUUDD-GUBZILKMSA-N Ser-Glu-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LALNXSXEYFUUDD-GUBZILKMSA-N 0.000 claims description 3
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 claims description 3
- BEAFYHFQTOTVFS-VGDYDELISA-N Ser-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N BEAFYHFQTOTVFS-VGDYDELISA-N 0.000 claims description 3
- DINQYZRMXGWWTG-GUBZILKMSA-N Ser-Pro-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DINQYZRMXGWWTG-GUBZILKMSA-N 0.000 claims description 3
- VFWQQZMRKFOGLE-ZLUOBGJFSA-N Ser-Ser-Cys Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N)O VFWQQZMRKFOGLE-ZLUOBGJFSA-N 0.000 claims description 3
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 claims description 3
- BDMWLJLPPUCLNV-XGEHTFHBSA-N Ser-Thr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BDMWLJLPPUCLNV-XGEHTFHBSA-N 0.000 claims description 3
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 claims description 3
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 claims description 3
- PZVGOVRNGKEFCB-KKHAAJSZSA-N Thr-Asn-Val Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N)O PZVGOVRNGKEFCB-KKHAAJSZSA-N 0.000 claims description 3
- YUOCMLNTUZAGNF-KLHWPWHYSA-N Thr-His-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N)O YUOCMLNTUZAGNF-KLHWPWHYSA-N 0.000 claims description 3
- BIBYEFRASCNLAA-CDMKHQONSA-N Thr-Phe-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 BIBYEFRASCNLAA-CDMKHQONSA-N 0.000 claims description 3
- VYVBSMCZNHOZGD-RCWTZXSCSA-N Thr-Val-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(O)=O VYVBSMCZNHOZGD-RCWTZXSCSA-N 0.000 claims description 3
- BRBCKMMXKONBAA-KWBADKCTSA-N Trp-Ala-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 BRBCKMMXKONBAA-KWBADKCTSA-N 0.000 claims description 3
- NIWAGRRZHCMPOY-GMVOTWDCSA-N Trp-Ala-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N NIWAGRRZHCMPOY-GMVOTWDCSA-N 0.000 claims description 3
- OZUJUVFWMHTWCZ-HOCLYGCPSA-N Trp-Gly-His Chemical compound N[C@@H](Cc1c[nH]c2ccccc12)C(=O)NCC(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O OZUJUVFWMHTWCZ-HOCLYGCPSA-N 0.000 claims description 3
- CXPJPTFWKXNDKV-NUTKFTJISA-N Trp-Leu-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 CXPJPTFWKXNDKV-NUTKFTJISA-N 0.000 claims description 3
- ZHZLQVLQBDBQCQ-WDSOQIARSA-N Trp-Lys-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N ZHZLQVLQBDBQCQ-WDSOQIARSA-N 0.000 claims description 3
- UGFOSENEZHEQKX-PJODQICGSA-N Trp-Val-Ala Chemical compound CC(C)[C@H](NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(=O)N[C@@H](C)C(O)=O UGFOSENEZHEQKX-PJODQICGSA-N 0.000 claims description 3
- PWKMJDQXKCENMF-MEYUZBJRSA-N Tyr-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O PWKMJDQXKCENMF-MEYUZBJRSA-N 0.000 claims description 3
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 claims description 3
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 claims description 3
- HLBHFAWNMAQGNO-AVGNSLFASA-N Val-His-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCSC)C(=O)O)N HLBHFAWNMAQGNO-AVGNSLFASA-N 0.000 claims description 3
- JZWZACGUZVCQPS-RNJOBUHISA-N Val-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N JZWZACGUZVCQPS-RNJOBUHISA-N 0.000 claims description 3
- BZWUSZGQOILYEU-STECZYCISA-N Val-Ile-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BZWUSZGQOILYEU-STECZYCISA-N 0.000 claims description 3
- LYERIXUFCYVFFX-GVXVVHGQSA-N Val-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LYERIXUFCYVFFX-GVXVVHGQSA-N 0.000 claims description 3
- MIKHIIQMRFYVOR-RCWTZXSCSA-N Val-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C(C)C)N)O MIKHIIQMRFYVOR-RCWTZXSCSA-N 0.000 claims description 3
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 claims description 3
- OFTXTCGQJXTNQS-XGEHTFHBSA-N Val-Thr-Ser Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N)O OFTXTCGQJXTNQS-XGEHTFHBSA-N 0.000 claims description 3
- ZNGPROMGGGFOAA-JYJNAYRXSA-N Val-Tyr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=C(O)C=C1 ZNGPROMGGGFOAA-JYJNAYRXSA-N 0.000 claims description 3
- DFQZDQPLWBSFEJ-LSJOCFKGSA-N Val-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N DFQZDQPLWBSFEJ-LSJOCFKGSA-N 0.000 claims description 3
- WHNSHJJNWNSTSU-BZSNNMDCSA-N Val-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 WHNSHJJNWNSTSU-BZSNNMDCSA-N 0.000 claims description 3
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 claims description 3
- 108010047495 alanylglycine Proteins 0.000 claims description 3
- 108010070944 alanylhistidine Proteins 0.000 claims description 3
- 108010008355 arginyl-glutamine Proteins 0.000 claims description 3
- 108010091092 arginyl-glycyl-proline Proteins 0.000 claims description 3
- 108010038850 arginyl-isoleucyl-tyrosine Proteins 0.000 claims description 3
- 108010029539 arginyl-prolyl-proline Proteins 0.000 claims description 3
- 108010018691 arginyl-threonyl-arginine Proteins 0.000 claims description 3
- 108010060035 arginylproline Proteins 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- 108010016616 cysteinylglycine Proteins 0.000 claims description 3
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 claims description 3
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 claims description 3
- 108010089804 glycyl-threonine Proteins 0.000 claims description 3
- 108010085325 histidylproline Proteins 0.000 claims description 3
- 108010053037 kyotorphin Proteins 0.000 claims description 3
- 108010057821 leucylproline Proteins 0.000 claims description 3
- 108010056582 methionylglutamic acid Proteins 0.000 claims description 3
- 108010024654 phenylalanyl-prolyl-alanine Proteins 0.000 claims description 3
- 108010025826 prolyl-leucyl-arginine Proteins 0.000 claims description 3
- 108010031719 prolyl-serine Proteins 0.000 claims description 3
- 108010004914 prolylarginine Proteins 0.000 claims description 3
- 108010070643 prolylglutamic acid Proteins 0.000 claims description 3
- 108010029020 prolylglycine Proteins 0.000 claims description 3
- 108010048818 seryl-histidine Proteins 0.000 claims description 3
- 108010005834 tyrosyl-alanyl-glycine Proteins 0.000 claims description 3
- 229960004854 viral vaccine Drugs 0.000 claims description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 2
- 229960000723 ampicillin Drugs 0.000 claims description 2
- 102000002322 Egg Proteins Human genes 0.000 claims 2
- 108010000912 Egg Proteins Proteins 0.000 claims 2
- 235000014103 egg white Nutrition 0.000 claims 2
- 210000000969 egg white Anatomy 0.000 claims 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- FJUKMPUELVROGK-IHRRRGAJSA-N Leu-Arg-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N FJUKMPUELVROGK-IHRRRGAJSA-N 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- 238000011084 recovery Methods 0.000 claims 1
- 230000003248 secreting effect Effects 0.000 claims 1
- 230000036039 immunity Effects 0.000 abstract description 14
- 241000700605 Viruses Species 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 11
- 108020004705 Codon Proteins 0.000 abstract description 10
- 229960005486 vaccine Drugs 0.000 abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 9
- 210000004027 cell Anatomy 0.000 description 14
- 239000000047 product Substances 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 230000005847 immunogenicity Effects 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 102100033195 DNA ligase 4 Human genes 0.000 description 2
- 101710121417 Envelope glycoprotein Proteins 0.000 description 2
- 101000927810 Homo sapiens DNA ligase 4 Proteins 0.000 description 2
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- 208000032420 Latent Infection Diseases 0.000 description 2
- MHQXIBRPDKXDGZ-ZFWWWQNUSA-N Met-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@@H](N)CCSC)C(O)=O)=CNC2=C1 MHQXIBRPDKXDGZ-ZFWWWQNUSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000035415 Reinfection Diseases 0.000 description 2
- INCNPLPRPOYTJI-JBDRJPRFSA-N Ser-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N INCNPLPRPOYTJI-JBDRJPRFSA-N 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 2
- HQYVQDRYODWONX-DCAQKATOSA-N Val-His-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N HQYVQDRYODWONX-DCAQKATOSA-N 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000004279 orbit Anatomy 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000004114 suspension culture Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 241000710914 Totivirus Species 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001032 spinal nerve Anatomy 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis of the present invention and expression thereof, application relate to technique for gene engineering, vaccine and diagnostic reagent field.The present invention is to be analyzed by computer, filter out the strong antigen epi-position in HSV2 virus gE glycoprotein, 21st aminoacid is to the 414th aminoacid, totally 394 aminoacid, select the codon that eucaryon and prokaryote are all had a preference for, the brand-new gene order of chemosynthesis epitope, utilizes technique for gene engineering, expresses this genetic fragment, prepares the strong antigen epitope Fragments of HSV2 virus gE glycoprotein.The strong antigen epitope Fragments of the HSV2 virus gE glycoprotein expressed, can be used for HSV2 vaccine development, the detection of antibody and prepares anti-HSV2 virus monoclonal antibody and multi-resistance etc. for immunity.
Description
Technical field
The present invention relates to the brand-new genetic fragment of the HSV2 virus gE glucoprotein extracellular region of chemosynthesis, utilize gene
Engineering, preparation restructuring HSV2 virus gE glycoprotein.Analyzed by computer, filter out the HSV2 virus containing strong antigen epi-position
GE glucoprotein extracellular region fragment, select the codon that eucaryon and prokaryote are all had a preference for, the gene order that chemosynthesis is brand-new,
Utilizing technique for gene engineering to express, the albumen of expression can be used for vaccine and HSV2 antiviral antibody or the detection etc. of antigen, the present invention
Relate to technique for gene engineering, vaccine and diagnostic reagent field.
Background technology
Herpes simplex virus (Herpers simplex virus, HSV) belongs to herpetoviridae α subfamily, is that the mankind feel
One of common virus of dye, can be divided into two kinds of I and II type according to serotype.Wherein, I type mainly by mouth facial infection, is also deposited
Spreading through sex intercourse, II type is mainly by spreading through sex intercourse and closely related with the generation of women cervical cancer.The most increasing research
Showing, HSV I is relevant to the generation of Alzheimer disease, and infection and the HSV II of HIV are also related.HSV generally flows in the whole world
OK, HSV I type is up to more than 85% in the positives rate of HAS, and HSV II type whole world seroprevalence is about 20%, and sends out
Patient's number increases with annual 25% speed.
HSV can infected tissue or near position breeding form primary lesion, and set up eventually at nuclei quintus spinal nerves
The latent infection of body, when body by environmental stimuli or immunity degradation is, HSV virus comes downwards to epidermis and breeds in a large number, is formed
Recurrent infection.HSV recurrent exerbation is relevant to HSV immunologic escape, and HSV realizes hiding sense by hiding the attack of human immune system
Dye and recurrent infection, therefore HSV once infects, and is difficult to be eliminated, and patient is tormented by prolonged sickness.
Viral glycoprotein (glycoproteins, gp) is the antigenic determinant on virion surface, has now been found that
And the envelope glycoprotein of definite designation has 12 (gB, gC, gD, gE, gG, gH, gI, gK, gL and gM etc.).GB and gD glycoprotein
Participate in virus and the absorption of host cell and fusion, be selected as candidate vaccine, more to its research.Research in recent years finds, gE2
It it is the envelope glycoprotein that immunologic escape is relevant.HSV virus envelope protein gE and IgG Fc end is combined, thus blocks multiple complement and be situated between
The immunization route led, makes HSV virus successfully hide the immune system attack of body.Therefore he is the egg causing virus lays dormant to infect
One of white molecule.Choose gE glycoprotein for immunity body, the latent infection of blocking virus, promise to be preferable candidate's epidemic disease
Seedling.
The present invention is analyzed by computer, filters out the viral gE glucoprotein extracellular region sheet of the HSV2 containing strong antigen epi-position
Section, selects the codon that eucaryon and prokaryote are all had a preference for, the gene order that chemosynthesis is brand-new, utilizes technique for gene engineering table
Reaching, expression product has preferable antigenicity and specificity, and the albumen of expression can be used for vaccine and HSV2 antiviral antibody or antigen
Detection etc..
Summary of the invention
The present invention is the brand-new genetic fragment of the HSV2 virus gE glucoprotein extracellular region of chemosynthesis, utilizes genetic engineering skill
Art, preparation restructuring HSV2 virus gE glucoprotein extracellular region fragment.Analyzed by computer, filter out the HSV2 containing strong antigen epi-position
Virus gE glucoprotein extracellular region fragment, the 21st aminoacid the 414th aminoacid, totally 394 aminoacid, select eucaryon
The codon all having a preference for prokaryote, the gene order that chemosynthesis is brand-new, utilize technique for gene engineering to express this gene.Table
The albumen reached can be used for vaccine, HSV2 antiviral antibody or the detection of antigen and prepares anti-HSV2 virus monoclonal antibody and multi-resistance for immunity
Deng.
The present invention is analyzed by computer, filters out the viral gE glucoprotein extracellular region sheet of the HSV2 containing strong antigen epi-position
Section, selects the codon that eucaryon and prokaryote are all had a preference for, the gene order that chemosynthesis is brand-new, utilizes technique for gene engineering table
Reaching, expression product has preferable antigenicity and specificity, and the albumen of expression can be used for vaccine and HSV2 antiviral antibody or antigen
Detection etc..
The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression thereof, application are to take following steps real
Existing:
The screening of 1.HSV2 virus gE glucoprotein extracellular region epitope and the chemosynthesis of genetic fragment thereof:
Utilize the softwares such as ANTHEWIN, analyzed the aminoacid sequence of HSV2 virus gE glycoprotein by computer, find gE
The N end (the 21st aminoacid the 414th aminoacid) of glycoprotein is containing stronger antigenic determinant.Select eucaryon and protokaryon raw
The codon that thing is all had a preference for, the gene order that chemosynthesis is brand-new, and add EcoRI restriction enzyme site (underscore at 5' end
Part), start codon (ATG), signal peptide
(GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCATTCTGC C), adds His mark at 3' end
Note (CAC CAC CAC CAC CAC CAC), termination codon (TGA) and Himd III digestion site (underscore part), make
This genetic fragment is prone in EcoRI and the Himd III digestion site being cloned in plasmid pMCE5.
Epitope aminoacid sequence (-the 414 aa of the 21st aa) in the HSV2 virus gE glycoprotein of screening:
Ala Ala Pro Arg Thr Ser Trp Lys Arg Val Thr Ser Gly Glu Asp Val Val
Leu Leu Pro Ala Pro Ala Glu Arg Thr Arg Ala His Lys Leu Leu Trp Ala Ala Glu
Pro Leu Asp Ala Cys Gly Pro Leu Arg Pro Ser Trp Val Ala Leu Trp Pro Pro Arg
Arg Val Leu Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu Ala
Ile Ala Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr Ser Glu Leu Ala
Trp Arg Asp Arg Val Ala Val Val Asn Glu Ser Leu Val Ile Tyr Gly Ala Leu Glu
Thr Asp Ser Gly Leu Tyr Thr Leu Ser Val Val Gly Leu Ser Asp Glu Ala Arg Gln
Val Ala Ser Val Val Leu Val Val Glu Pro Ala Pro Val Pro Thr Pro Thr Pro Asp
Asp Tyr Asp Glu Glu Asp Asp Ala Gly Val Thr Asn Ala Arg Arg Ser Ala Phe Pro
Pro Gln Pro Pro Pro Arg Arg Pro Pro Val Ala Pro Pro Thr His Pro Arg Val Ile
Pro Glu Val Ser His Val Arg Gly Val Thr Val His Met Glu Thr Leu Glu Ala Ile
Leu Phe Ala Pro Gly Glu Thr Phe Gly Thr Asn Val Ser Ile His Ala Ile Ala His
Asp Asp Gly Pro Tyr Ala Met Asp Val Val Trp Met Arg Phe Asp Val Pro Ser Ser
Cys Ala Asp Met Arg Ile Tyr Glu Ala Cys Leu Tyr His Pro Gln Leu Pro Glu Cys
Leu Ser Pro Ala Asp Ala Pro Cys Ala Val Ser Ser Trp Ala Tyr Arg Leu Ala Val
Arg Ser Tyr Ala Gly Cys Ser Arg Thr Thr Pro Pro Pro Arg Cys Phe Ala Glu Ala
Arg Met Glu Pro Val Pro Gly Leu Ala Trp Leu Ala Ser Thr Val Asn Leu Glu Phe
Gln His Ala Ser Pro Gln His Ala Gly Leu Tyr Leu Cys Val Val Tyr Val Asp Asp
His Ile His Ala Trp Gly His Met Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala
Val Val Glu Gln His Leu Pro Gln Arg Gln Pro Glu Pro Val Glu Pro Thr Arg Pro
His Val Arg Ala Pro His Pro Ala Pro Ser Ala Arg Gly Pro Leu Arg
The DNA sequence containing HSV2 virus gE glucoprotein extracellular region antigen epitope genes (1282 bp) of chemosynthesis:
GAATTC GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC
ACT GCC ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC
GGA GAG GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG
CTG TGG GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC
CTG TGG CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT
CCC GAA CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG
TAT AGT GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC
TAC GGC GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC
GAC GAG GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT
ACT CCA ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA
CGC TCA GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC
CAC CCA CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG
ACC CTG GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC
CAC GCT ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT
GAT GTG CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT
CAG CTG CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC
TAC CGA CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG
TGT TTC GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA
GTG AAC CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG
GTC TAT GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA
CAG TAC AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG
GAA CCA ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC
CTG CGT CAC CAC CAC CAC CAC CAT TGA TAAGCTT
2. the structure of expression HSV2 virus gE glucoprotein extracellular region fragment recombiant plasmid:
Extract plasmid pMCE5, with EcoR I and Hind III double digestion, after electrophoresis, reclaim the plasmid large fragment of enzyme action, molten
In deionized water.Equally by EcoR I and the HSV2 virus gE glycoprotein gene fragment of Hind III double digestion chemosynthesis,
After electrophoresis reclaims, it is dissolved in deionized water.
Take DNA fragmentation after the above two enzyme action of equimolar concentration, in same centrifuge tube with T4 DNA ligase even
Connect, make between EcoR I and the Hind III site that HSV2 virus gE glycoprotein gene fragment is inserted in carrier pMCE5, with load
Start codon translation framework on body is consistent, expresses a fusion protein.
3. the screening of recombiant plasmid and qualification:
By recombinant plasmid transformed bacillus coli DH 5 alpha, the coating LB flat board containing 100 μ g/ml ampicillin, put 37 DEG C of mistakes
Night.Next day, random picking converted bacterium colony, and the pMCE5 transformed bacteria containing plasmid makees negative control, made containing plasmid pUC57-gC21 transformed bacteria
Positive control, bacterium solution PCR amplification HSV2 virus gE glucoprotein extracellular region gene fragment, containing HSV2 virus gE glucoprotein extracellular region base
Because of the positive recombiant plasmid of fragment, the genetic fragment being about 1282bp should be amplified.Extracting positive single bacterium colony plasmid, carrying out
DNA sequence analysis is identified and carried out by the plasmid containing exogenous gene to double digestion, and sequence analysis confirms that recombiant plasmid contains synthesis
HSV2 virus gE glycoprotein gene fragment, sequence is the most correct:
GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC ACT GCC
ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC GGA GAG
GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG CTG TGG
GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC CTG TGG
CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT CCC GAA
CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG TAT AGT
GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC TAC GGC
GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC GAC GAG
GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT ACT CCA
ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA CGC TCA
GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC CAC CCA
CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG ACC CTG
GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC CAC GCT
ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT GAT GTG
CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT CAG CTG
CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC TAC CGA
CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG TGT TTC
GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA GTG AAC
CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG GTC TAT
GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA CAG TAC
AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG GAA CCA
ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC CTG CGT
CAC CAC CAC CAC CAC CAT TGA T
The viral gE glucoprotein extracellular region fragment (394 aminoacid) of the expression of recombinant plasmid HSV2 built, melts at its N end
Close 19 aminoacid on carrier, merge 6 aminoacid on carrier, 419 aminoacid of total length, its amino at its C end
Acid sequence is as follows:
N end: Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
Val His Ser
C end: His His His His His His
4. recombiant plasmid expression identification in Chinese hamster ovary celI:
Chinese hamster ovary celI is expressed culture medium at the cell without serum and is shaken cultivation in conical flask, 37 DEG C, 5% CO2.Transfection
The previous day, with suitable concentration, Chinese hamster ovary celI is inoculated in the cell without serum and expresses in culture medium, concussion suspension culture, 37
DEG C, 5% CO2.Recombiant plasmid is transfected with DNA and PEI mixture.Within after transfection the 5th day, collect cell conditioned medium, detect protein expression
Level, collects all supernatants for purification of recombinant proteins on the 6th day.
5. the purification of recombiant protein, qualification and Concentration Testing:
Collect cell conditioned medium 100ml, after being centrifuged, supernatant is added His Trap with the speed of 1ml/minTM FF crude
In column.10 times of volumetric balance liquid (1 × PBS, 0.5mol/L NaCl, 20mmol/L imidazole, pH7.4) eluting are miscellaneous
Albumen, adds 1ml eluent (1 × PBS, 0.5mol/L NaCl, 500mmol/L imidazole, pH7.4) eluting purpose
Albumen, eluting after standing 20 minutes, collect eluent, dialysed with dialysis solution (1 × PBS, 0.5mol/L NaCl, pH7.4)
At night, being the destination protein of purification, SDS-PAGE, Western Blot detects, and measures protein concentration.
6. immune mouse and the sero-fast preparation of gE2:
10 male mouse of kunming are randomly divided into 2 groups, often group 5.Experimental group is with the restructuring gE2 glycoprotein of purification and Freund
Adjuvant equal-volume mixes, fully emulsified, respectively at the 0th, 2,4 week, and lumbar injection immune mouse, 2 ug//times, 0.1ml/,
Matched group is with PBS equal-volume mixing Freund adjuvant, fully emulsified, and 0.1ml/ is only.Eye socket blood sampling in 2 weeks after immunity every time.Blood 37
DEG C place after 1 hour 4 DEG C overnight, 4000rpm is centrifuged 10 minutes, takes supernatant and i.e. obtains recombiant protein gE2 antiserum.
7. the immunogenicity detection of recombiant protein gE2:
With 1 × CBS, the restructuring gE2 glycoprotein of purification is diluted to 1 ug/ml, is coated elisa plate, every hole 100ul, 4 DEG C of mistakes
Night.Next day 5%FBS sealase yoke plate, every hole 130ul, room temperature 2 hours.By the antiserum of preparation with Sample dilution by 1:
After 500,1:2500,1:12500 stepwise dilution, adding in the enzyme linked plate holes after closing respectively, every hole 100ul, 37 DEG C of reactions 1 are little
Time, after washing 5 times with PBST, add goat anti-mouse igg-HRP, every hole 100ul of 1:5000 dilution, 37 DEG C are reacted 30 minutes, PBST
Washing 5 times, add substrate TMB solution every hole 100 ul, 37 DEG C of lucifuges develop the color 10 minutes, add 50ul 1N hydrochloric acid and terminate reaction, use enzyme
Connection instrument measures A450Value.Result judges, is more than or equal to 2.1(P/N >=2.1 with the ratio of testing sample A value with negative control A value)
And A value >=0.15 is positive, the serum antibody titer that antibody dilution is immune mouse that display positive findings is maximum.Data are used
Mean ± standard deviation () represent, carry out statistical analysis, add up with GraphPad Prism 5.0, SPSS software
Credit is analysed, it is believed that P ＜ 0.05 is for there being significant difference.
The gene fragment order of the HSV2 virus gE glucoprotein extracellular region of described chemosynthesis, utilizes antibacterial, yeast thin
Born of the same parents, insect cell, mammalian cell and genetically modified animals and plants carry out recombinant expressed, preparation.
HSV2 virus gE glucoprotein extracellular region fragment prepared by method described above, for HSV2 subunit viral vaccine
Development and antibody test.
The present invention compared with prior art has the advantage that
The gE glycoprotein fragment of the HSV2 virus that the present invention expresses, has a more advantages:
1. the enzyme-linked immunologic detecting kit of the HSV2 antiviral antibody applied at present, its antigen used is that totivirus resists
Former, produce relatively hazardous, cost is high and other virus has the shortcomings such as cross reaction.Outside the gE glycoprotein born of the same parents of the HSV2 virus expressed
District's fragment is used as antigen can overcome disadvantages mentioned above.
2. lack HSV2 viral vaccine at present.The research of inactivated vaccine achieves greater advance, but production cost is high, danger
Danger is big.The gE glycoprotein of HSV2 virus content in the cell infected is most, is the conservative albumen of herpes virus group camber,
Each poison strain difference is less, and gE albumen has stronger immunogenicity simultaneously, body can be induced to produce neutralizing antibody and cell is exempted from
Epidemic disease is reacted, and relevant with viral escape mechanism, and therefore HSV2-gE glycoprotein is to build the preferable genes of interest of HSV vaccine.I
Have selected its strong antigen epi-position, utilize technique for gene engineering express preparation, for develop recombinant vaccine lay the foundation.Gene
Engineered vaccine safety, low cost.
3., according to the gE glucoprotein extracellular region fragment amino acid sequence of the HSV2 virus filtered out, select eucaryon and protokaryon raw
The codon that thing is all had a preference for, the gene order that chemosynthesis is brand-new, this gene suitably high expressed in eucaryon and prokaryotic cell.
4. the Chinese hamster ovary celI strain of the gE glycoprotein of the expression HSV2 virus that the present invention builds, can prepare this albumen by large-scale purification,
It is prone to purification, and there is great immunogenicity.Have no the report expressing this section of albumen at present.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is the pcr amplification product of bacterium solution PCR 8 recons of detection.1:2000 DL DNA(TaKaRa)；2:gE2 base
Because of PCR primer；3-10:1-8 picking list bacterium colony bacterium solution PCR primer；11: empty pMCE5/DH5 α bacterium solution PCR primer.
Fig. 2 is double digestion qualification result.1:5000 DL DNA(TaKaRa)；2: recombiant plasmid through EcoRI and
HindIII double digestion product；3: recombiant plasmid is through EcoRI single endonuclease digestion product；4:gE2 gene.
Fig. 3 is the SDS-PAGE analysis result of the gE glycoprotein recombinant bacterium expressing HSV2 virus.M:Protein
Marker；1: electrophoresis under reducing condition；2: non reducing conditions electrophoresis
Fig. 4 is the Western-Blots analysis result before and after the gE glycoprotein purification expressing HSV2 virus.M:Protein
Marker；1: electrophoresis under reducing condition；2: non reducing conditions electrophoresis；P:Multiple-tag joins as sun.
Fig. 5 is that ELISA detects recombiant protein gE2 immunogenicity.
Detailed description of the invention
The detailed description of embodiment of the present invention:
Analysis, gene chemical synthesis and the expression of the gE glycoprotein antigen epi-position of HSV2 virus
Analyzed whole aminoacid sequences of the gE glycoprotein of HSV2 virus by computer, filter out the gE sugar of HSV2 virus
Strong antigen epi-position in albumen, selects the codon of eucaryon preference, the gE glycoprotein strong antigen epi-position of chemosynthesis HSV2 virus
Brand-new genetic fragment.By the EcoRI/HindIII site in gene fragment clone to plasmid pMCE5, with initiateing on carrier
The translation framework of codon is consistent, can express a fusion protein.By build eukaryon expression plasmid pMCE5-gE2 and transfection extremely
Chinese hamster ovary celI carries out eukaryotic expression, and screening obtains the cell strain of high expressed gE glycoprotein.
Materials and methods
1. strain and plasmid: Host Strains DH5 α and expression vector pMCE5 is that this laboratory preserves.
2. molecular biology reagents: restricted enzyme EcoRI, HindIII and T4 DNA ligase is that TaKaRa is public
Department's product.Plasmid purification kit and the test kit reclaiming DNA fragmentation in agarose gel are TaKaRa Products. other
Reagent is import or domestic analytical reagent.
3. the synthesis of genetic fragment: helped synthesis by Dalian TaKaRa company.
4. the enzyme action of gene clone method: DNA, connection, electrophoresis；The extraction of plasmid, conversion.Other test kit is illustratively
Book operates.
5. DNA sequence analysis: with TAKARA company plasmid purification kit plasmid purification, survey with the full-automatic sequenator of DNA
Sequence.
Result
1. the gE glycoprotein antigen epi-position of HSV2 virus is screened and the synthesis of genetic fragment:
Utilize the softwares such as ANTHEWIN, analyzed whole aminoacid sequences of the gE glycoprotein of HSV2 virus by computer
(UniProtKB:P89475.1), the strong antigen epi-position in the gE glycoprotein of HSV2 virus is filtered out, i.e. from the 21st aminoacid
To the 414th aminoacid, its aminoacid sequence is as follows:
Ala Ala Pro Arg Thr Ser Trp Lys Arg Val Thr Ser Gly Glu Asp Val Val
Leu Leu Pro Ala Pro Ala Glu Arg Thr Arg Ala His Lys Leu Leu Trp Ala Ala Glu
Pro Leu Asp Ala Cys Gly Pro Leu Arg Pro Ser Trp Val Ala Leu Trp Pro Pro Arg
Arg Val Leu Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu Ala
Ile Ala Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr Ser Glu Leu Ala
Trp Arg Asp Arg Val Ala Val Val Asn Glu Ser Leu Val Ile Tyr Gly Ala Leu Glu
Thr Asp Ser Gly Leu Tyr Thr Leu Ser Val Val Gly Leu Ser Asp Glu Ala Arg Gln
Val Ala Ser Val Val Leu Val Val Glu Pro Ala Pro Val Pro Thr Pro Thr Pro Asp
Asp Tyr Asp Glu Glu Asp Asp Ala Gly Val Thr Asn Ala Arg Arg Ser Ala Phe Pro
Pro Gln Pro Pro Pro Arg Arg Pro Pro Val Ala Pro Pro Thr His Pro Arg Val Ile
Pro Glu Val Ser His Val Arg Gly Val Thr Val His Met Glu Thr Leu Glu Ala Ile
Leu Phe Ala Pro Gly Glu Thr Phe Gly Thr Asn Val Ser Ile His Ala Ile Ala His
Asp Asp Gly Pro Tyr Ala Met Asp Val Val Trp Met Arg Phe Asp Val Pro Ser Ser
Cys Ala Asp Met Arg Ile Tyr Glu Ala Cys Leu Tyr His Pro Gln Leu Pro Glu Cys
Leu Ser Pro Ala Asp Ala Pro Cys Ala Val Ser Ser Trp Ala Tyr Arg Leu Ala Val
Arg Ser Tyr Ala Gly Cys Ser Arg Thr Thr Pro Pro Pro Arg Cys Phe Ala Glu Ala
Arg Met Glu Pro Val Pro Gly Leu Ala Trp Leu Ala Ser Thr Val Asn Leu Glu Phe
Gln His Ala Ser Pro Gln His Ala Gly Leu Tyr Leu Cys Val Val Tyr Val Asp Asp
His Ile His Ala Trp Gly His Met Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala
Val Val Glu Gln His Leu Pro Gln Arg Gln Pro Glu Pro Val Glu Pro Thr Arg Pro
His Val Arg Ala Pro His Pro Ala Pro Ser Ala Arg Gly Pro Leu Arg
The epitope aminoacid sequence in HSV2 virus gE glycoprotein according to screening, selects the codon of eucaryon, changes
Learn the gene order that synthesis is brand-new, and add EcoRI restriction enzyme site (underscore part), start codon at 5' end
(ATG), signal peptide (GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCATTC TGCC), at 3'
End add His labelling (CAC CAC CAC CAC CAC CAC), termination codon (TGA) and Himd III digestion site (under
Dashed part), in making EcoRI and the Hind III digestion site that this genetic fragment is prone to be cloned in plasmid pMCE5.Chemistry closes
The DNA sequence containing HSV2 virus gE glycoprotein antigen epitope gene (1282 bp) become is as follows:
GAATTC GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC
ACT GCC ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC
GGA GAG GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG
CTG TGG GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC
CTG TGG CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT
CCC GAA CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG
TAT AGT GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC
TAC GGC GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC
GAC GAG GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT
ACT CCA ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA
CGC TCA GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC
CAC CCA CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG
ACC CTG GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC
CAC GCT ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT
GAT GTG CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT
CAG CTG CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC
TAC CGA CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG
TGT TTC GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA
GTG AAC CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG
GTC TAT GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA
CAG TAC AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG
GAA CCA ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC
CTG CGT CAC CAC CAC CAC CAC CAT TGA TAAGCTT
2. the structure of expression HSV2 virus gE glucoprotein extracellular region fragment recombiant plasmid:
Extract plasmid pMCE5, with EcoRI and Hind III double digestion, after electrophoresis, reclaim the plasmid large fragment of enzyme action, molten
In deionized water.The HSV2 virus gE glucoprotein extracellular region gene of same EcoRI and Hind III double digestion chemosynthesis
Fragment, after electrophoresis reclaims, is dissolved in deionized water.
Take DNA fragmentation after the above two enzyme action of equimolar concentration, in same centrifuge tube with T4 DNA ligase even
Connect, make EcoRI and Hind III site that HSV2 virus gE glucoprotein extracellular region gene fragment is inserted in carrier pMCE5 it
Between, consistent with the start codon translation framework on carrier, express a fusion protein.
3. the screening of recombiant plasmid and qualification:
The recombinant plasmid transformed upper step connected is to bacillus coli DH 5 alpha, and converted product coating is blue or green containing 100 g/ml ammonia benzyls
On the solid LB media of mycin, put 37 DEG C of overnight incubation.Next day random choose 1-8 transformant bacterium colony, one containing plasmid
PMCE5 transformed bacteria makees negative control and a pUC57-gE2 transformed bacteria Han plasmid makees positive control, is inoculated into 3 ml respectively containing ammonia
The LB liquid medium of benzylpcnicillin 100 g/ml, 37 DEG C of shaken cultivation 5h, take bacterium solution 1ml, centrifugal receipts bacterium.Respectively with 50 μ l
Deionized water suspension thalline, boiling water boiling 5min, centrifugal (4 DEG C, 12000rpm) 5min, take supernatant 2 μ l and be used as pcr template, PCR
HSV2 virus gE glucoprotein extracellular region gene fragment in amplification insertion vector, PCR reaction density is: plasmid template 2 μ l,
Normal chain P1(CCGGAATTCGCCGCCACCATGGG of HSV2 virus gE glucoprotein extracellular region gene fragment) and negative strand primer P2
(AAATGCGGCCGCTGACCATG ATTACGCCAAGCT) each 1 μ l, 10 × pyrobest buffer 5.0 μ l, 2.5
Mmol/L dNTP 4.0 μ l, Pyrobest DNA Taq enzyme 0.5 μ l (2.5 U), deionized water 36.5 μ l, cumulative volume 50 μ l.
Amplification condition is: 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 4 minutes, 35 circulations；Last 72 DEG C extend 7 minutes.Take PCR to expand
Volume increase thing 5 μ l, Agarose gel detection with 1.0% is as a result, No. 1 transformant amplifies the genes of interest fragment of 1282bp
(see figure 1), and the comparison bacterium containing plasmid pMCE5 does not amplify this genetic fragment.Tentative confirmation, this No. 1 transformant contains
HSV2 virus gE glycoprotein gene fragment.
Extracting the plasmid of No. 3 recons, double digestion is identified (see figure 2) and measures the HSV2 virus gE glycoprotein in plasmid
Gene order, DNA sequence analysis confirms, recombiant plasmid contains the HSV2 virus gE glycoprotein gene fragment of synthesis, and sequence is complete
Correct:
GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC ACT GCC
ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC GGA GAG
GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG CTG TGG
GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC CTG TGG
CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT CCC GAA
CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG TAT AGT
GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC TAC GGC
GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC GAC GAG
GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT ACT CCA
ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA CGC TCA
GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC CAC CCA
CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG ACC CTG
GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC CAC GCT
ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT GAT GTG
CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT CAG CTG
CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC TAC CGA
CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG TGT TTC
GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA GTG AAC
CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG GTC TAT
GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA CAG TAC
AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG GAA CCA
ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC CTG CGT
CAC CAC CAC CAC CAC CAT TGA T
The viral gE glycoprotein fragment (394 aminoacid) of the expression of recombinant plasmid HSV2 built, has merged load at its N end
19 aminoacid on body, have merged 6 aminoacid on carrier, 419 aminoacid of total length, its aminoacid sequence at its C end
As follows:
N end: Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
Val His Ser
C end: His His His His His His
4. recombiant plasmid expression identification in Chinese hamster ovary celI:
Chinese hamster ovary celI is expressed culture medium at the cell without serum and is shaken cultivation in conical flask, 37 DEG C, 5% CO2.Transfection
The previous day, with suitable concentration, Chinese hamster ovary celI is inoculated in the cell without serum and expresses in culture medium, concussion suspension culture, 37
DEG C, 5% CO2.Recombiant plasmid is transfected with DNA and PEI mixture.Within after transfection the 5th day, collect cell conditioned medium, detect protein expression
Level, collects all supernatants for purification of recombinant proteins on the 6th day.
Express the purification of HSV2 virus gE glycoprotein
According to the aminoacid sequence of expression HSV2 virus gE glycoprotein, analyze its physicochemical property, determine suitable purification side
Method.The HSV2 gE glycoprotein of expressing cho cell merges the His albumen on carrier, and therefore we determine to use affinity chromatograph
Method, uses His TrapTMFF crude column is purified.Purification obtains the gE glycoprotein of expression, specifically comprises the following steps that
Material and method
1. main agents: His TrapTMFF crude column is GE Heathcare Products, and other reagent is
Domestic or Import Analysis pure reagent.
2. the purification of recombiant protein: collect cell conditioned medium 100ml, adds supernatant with the speed of 1ml/min after being centrifuged
His TrapTMIn FF crude column.10 times of volumetric balance liquid (1 × PBS, 0.5mol/L NaCl, 20mmol/L
Imidazole, pH7.4) eluting foreign protein, add 1ml eluent (1 × PBS, 0.5mol/L NaCl, 500mmol/L
Imidazole, pH7.4) eluting destination protein, eluting after standing 20 minutes, collect eluent, with dialysis solution (1 × PBS,
0.5mol/L NaCl, pH7.4) dialysed overnight, it is the destination protein of purification, SDS-PAGE, Western Blot detects, and
Measure protein concentration.
Result
Will be from His TrapTMOn FF crude column, the albumen of eluting carries out SDS-PAGE, Western Blot and divides
Analysis, result shows (see respectively Fig. 3,4), and expression product about 55kDa, protein concentration is 27 μ g/ml.Obtain the gE sugar egg of expression
In vain.
The Analysis of Immunogenicity of the gE glycoprotein of purification HSV2 virus
With purification obtain gE2 recombiant protein as antigen, mix Freund adjuvant immune mouse, prepare antiserum, indirectly
ELISA method detection titer, the immunogenicity of the gE2 recombiant protein that evaluation and test obtains.
Material and method
1. male mouse of kunming, 6-8 week old, purchased from Chinese military medicine academy of science Experimental Animal Center.
2. complete Freund's adjuvant and incomplete Freund's adjuvant are Sigma Products.Little mouse-anti His monoclonal antibody, sheep anti-Mouse
IgG-HRP is Jin Site Products, and other reagent is domestic or Import Analysis pure reagent.
3. prepared by animal immune method and antiserum: 10 male mouse of kunming are randomly divided into 2 groups, often group 5.Experimental group
Mixing with Freund adjuvant equal-volume with the restructuring gE2 glycoprotein of purification, fully emulsified, respectively at the 0th, 2,4 week, lumbar injection was exempted from
Epidemic disease mice, 2 ug//times, only, matched group is with PBS equal-volume mixing Freund adjuvant, fully emulsified for 0.1ml/, and 0.1ml/ is only.Often
Eye socket blood sampling in 2 weeks after secondary immunity.Blood 37 DEG C place after 1 hour 4 DEG C overnight, 4000rpm is centrifuged 10 minutes, takes supernatant and i.e. obtains weight
Histone gE2 antiserum.Indirect ELISA detection antiserum titre.
4.ELISA tests: with 1 × CBS, the restructuring gE2 glycoprotein of purification is diluted to 1 ug/ml, is coated elisa plate, often
Hole 100ul, 4 DEG C overnight.Next day 5%FBS sealase yoke plate, every hole 130ul, room temperature 2 hours.By the antiserum sample of preparation
Diluent presses 1:500, after 1:2500,1:12500 stepwise dilution, adds in the enzyme linked plate holes after closing respectively, every hole 100ul,
37 DEG C are reacted 1 hour, after washing 5 times with PBST, add goat anti-mouse igg-HRP, every hole 100ul of 1:5000 dilution, 37 DEG C of reactions
30 minutes, PBST washed 5 times, added substrate TMB solution every hole 100 ul, and 37 DEG C of lucifuges develop the color 10 minutes, add 50ul 1N hydrochloric acid eventually
Only reaction, measures A with enzyme connection instrument450Value.
Result
After immunity, 2 weeks mice eyeground vein clumps take blood every time, prepare antiserum, in indirect elisa method detection serum serum
Antibody titer.Result shows, little mouse-anti gE2 serum average titer is before immunity, after immunity for the first time, after immunity for the second time,
Antiserum titre after three immunity is respectively 0.00 ± 0.00,4.10 ± 0.28,5.49 ± 0.28,6.89 ± 0.28(such as figure
, and PBS control group anti-gE2 serum titer is always 0.00 5).Mice i.e. produces the antibody of higher level after first time immunity,
After second time, immunity for the third time, in Mice Body, antibody horizontal the most slowly raises, and produces high titre levels.Use SPSS
13.0 softwares carry out one factor analysis of variance and Q inspection, P ＜ 0.05, and immunity is front and three immunized mice antiserum titres
Improve and be respectively provided with notable statistical significance.The recombiant protein gE2 expressed can produce preferable humoral immunization effect by stimulating immune system
Really.
The HSV virus gE protein extracellular gene fragment order table of chemosynthesis sees appendix document: nucleotide or aminoacid
Sequence table computer readable carrier.
Claims (3)
1. an extracellular region gene fragment for the HSV2 virus gE glycoprotein of chemosynthesis, this genetic fragment coding table Han strong antigen
Position HSV2 virus gE glucoprotein extracellular region fragment, the i.e. the 21st amino acids to the 414th amino acids, totally 394 amino
Acid, gene order total length 1282 bp of chemosynthesis, sequence is as follows:
GAATTC GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC ACT
GCC ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC GGA
GAG GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG CTG
TGG GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC CTG
TGG CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT CCC
GAA CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG TAT
AGT GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC TAC
GGC GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC GAC
GAG GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT ACT
CCA ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA CGC
TCA GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC CAC
CCA CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG ACC
CTG GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC CAC
GCT ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT GAT
GTG CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT CAG
CTG CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC TAC
CGA CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG TGT
TTC GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA GTG
AAC CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG GTC
TAT GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA CAG
TAC AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG GAA
CCA ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC CTG
CGT CAC CAC CAC CAC CAC CAT TGA TAAGCTT。
2. a method, including HSV2 virus gE using technique for gene engineering to express chemosynthesis as claimed in claim 1
The extracellular region gene fragment of glycoprotein, the protein fragments that purification is expressed, specifically comprise the following steps that
The structure of the gE glucoprotein extracellular region gene fragment recombiant plasmid of expression HSV2 virus:
With genetic fragment and plasmid pMCE5, the electrophoresis of the chemosynthesis described in EcoR I and Hind III double digestion claim 1
After recovery, connect with T4 DNA ligase, make EcoR I and Hind III position that this genetic fragment is inserted in carrier pMCE5
Between point, consistent with the start codon translation framework on carrier, express a fusion protein, this fusion protein N end contains 19
Individual amino acid whose signal peptide, C end comprises the 21st amino acids in the gE glycoprotein of HSV2 virus to the 414th amino acids and 6
Individual His amino acid label；
When the fusion protein expressed is secreted into extracellular, 19 amino acid signal peptides of its N end are cut, final secreting, expressing egg
White aminoacid sequence is as follows:
Ala Ala Pro Arg Thr Ser Trp Lys Arg Val Thr Ser Gly Glu Asp Val Val Leu
Leu Pro Ala Pro Ala Glu Arg Thr Arg Ala His Lys Leu Leu Trp Ala Ala Glu Pro
Leu Asp Ala Cys Gly Pro Leu Arg Pro Ser Trp Val Ala Leu Trp Pro Pro Arg Arg
Val Leu Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu Ala Ile
Ala Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr Ser Glu Leu Ala Trp
Arg Asp Arg Val Ala Val Val Asn Glu Ser Leu Val Ile Tyr Gly Ala Leu Glu Thr
Asp Ser Gly Leu Tyr Thr Leu Ser Val Val Gly Leu Ser Asp Glu Ala Arg Gln Val
Ala Ser Val Val Leu Val Val Glu Pro Ala Pro Val Pro Thr Pro Thr Pro Asp Asp
Tyr Asp Glu Glu Asp Asp Ala Gly Val Thr Asn Ala Arg Arg Ser Ala Phe Pro Pro
Gln Pro Pro Pro Arg Arg Pro Pro Val Ala Pro Pro Thr His Pro Arg Val Ile Pro
Glu Val Ser His Val Arg Gly Val Thr Val His Met Glu Thr Leu Glu Ala Ile Leu
Phe Ala Pro Gly Glu Thr Phe Gly Thr Asn Val Ser Ile His Ala Ile Ala His Asp
Asp Gly Pro Tyr Ala Met Asp Val Val Trp Met Arg Phe Asp Val Pro Ser Ser Cys
Ala Asp Met Arg Ile Tyr Glu Ala Cys Leu Tyr His Pro Gln Leu Pro Glu Cys Leu
Ser Pro Ala Asp Ala Pro Cys Ala Val Ser Ser Trp Ala Tyr Arg Leu Ala Val Arg
Ser Tyr Ala Gly Cys Ser Arg Thr Thr Pro Pro Pro Arg Cys Phe Ala Glu Ala Arg
Met Glu Pro Val Pro Gly Leu Ala Trp Leu Ala Ser Thr Val Asn Leu Glu Phe Gln
His Ala Ser Pro Gln His Ala Gly Leu Tyr Leu Cys Val Val Tyr Val Asp Asp His
Ile His Ala Trp Gly His Met Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala Val
Val Glu Gln His Leu Pro Gln Arg Gln Pro Glu Pro Val Glu Pro Thr Arg Pro His
Val Arg Ala Pro His Pro Ala Pro Ser Ala Arg Gly Pro Leu Arg His His His His
His His
The screening of recombiant plasmid and qualification:
By recombinant plasmid transformed bacillus coli DH 5 alpha, the coating LB flat board containing 100 μ g/ml ampicillin, put 37 DEG C overnight；
Next day, random picking converted bacterium colony, 1 comparison bacterium transformed bacteria containing plasmid pMCE5, and 1 positive containing plasmid pUC57-gE2
Comparison bacterium, bacterium solution PCR amplification HSV2 virus gE glucoprotein extracellular region gene fragment, containing HSV2 virus gE glucoprotein extracellular region gene
The positive recombiant plasmid of fragment, should amplify the genetic fragment of long 1282bp；Extract positive single bacterium colony plasmid, carry out double enzyme
Cut qualification and order-checking；
Recombiant plasmid expression identification in Chinese hamster ovary celI:
Chinese hamster ovary celI is expressed culture medium at the cell without serum and is shaken cultivation in conical flask, 37 DEG C, 5% CO2；
Day before transfection, is inoculated in the cell without serum with suitable concentration by Chinese hamster ovary celI and expresses in culture medium, and concussion suspends
Cultivate, 37 DEG C, 5% CO2；Recombiant plasmid is transfected with DNA and PEI mixture；Within after transfection the 5th day, collect cell conditioned medium, detect egg
White expression, collects all supernatants for purification of recombinant proteins on the 6th day；
The purification of recombiant protein, qualification and Concentration Testing:
Collect cell conditioned medium 100ml, after being centrifuged, supernatant is added His Trap with the speed of 1ml/minTM FF crude
In column；10 times of volumetric balance liquid eluting foreign proteins, balance liquid is 1 × PBS, 0.5mol/L NaCl, 20mmol/L
Imidazole, pH7.4；Adding 1ml elution destination protein, eluent is 1 × PBS, 0.5mol/L NaCl,
500mmol/L imidazole, pH7.4；Eluting after standing 20 minutes, collects eluent, uses dialysis solution dialysed overnight, dialysis solution
Being 1 × PBS, 0.5mol/L NaCl, pH7.4, the albumen of eluting is the destination protein of purification, SDS-PAGE, Western
Blot detects, and measures protein concentration；
The gE2 molecular weight of albumen that purification obtains is 55kD.
3. the extracellular region gene fragment sequence of the HSV2 virus gE glycoprotein of the chemosynthesis described in claim 1, for HSV2
The purposes of the development of subunit viral vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310751325.9A CN103740736B (en) | 2013-12-31 | 2013-12-31 | The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression, application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310751325.9A CN103740736B (en) | 2013-12-31 | 2013-12-31 | The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression, application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103740736A CN103740736A (en) | 2014-04-23 |
| CN103740736B true CN103740736B (en) | 2016-08-17 |
Family
ID=50497801
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310751325.9A Active CN103740736B (en) | 2013-12-31 | 2013-12-31 | The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression, application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103740736B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551187B (en) * | 2019-09-23 | 2022-09-16 | 新乡学院 | Chemically synthesized H7N9 avian influenza virus NA protein extracellular region antigen segment, preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1889976A (en) * | 2003-10-30 | 2007-01-03 | 卢斯根有限责任公司 | Pharmaceutical anti-herpetic composition, method for producing a dosage form based thereon and method for the use thereof |
| CN101583381A (en) * | 2006-09-08 | 2009-11-18 | 宾夕法尼亚大学董事会 | HSV-1 and HSV-2 vaccines and methods of use thereof |
| CN101616688B (en) * | 2006-12-28 | 2013-12-11 | 宾夕法尼亚大学理事会 | Herpes simplex virus combined subunit vaccines and methods of use thereof |
-
2013
- 2013-12-31 CN CN201310751325.9A patent/CN103740736B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1889976A (en) * | 2003-10-30 | 2007-01-03 | 卢斯根有限责任公司 | Pharmaceutical anti-herpetic composition, method for producing a dosage form based thereon and method for the use thereof |
| CN101583381A (en) * | 2006-09-08 | 2009-11-18 | 宾夕法尼亚大学董事会 | HSV-1 and HSV-2 vaccines and methods of use thereof |
| CN101616688B (en) * | 2006-12-28 | 2013-12-11 | 宾夕法尼亚大学理事会 | Herpes simplex virus combined subunit vaccines and methods of use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103740736A (en) | 2014-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113861278B (en) | Novel recombinant coronavirus RBD trimer protein vaccine capable of generating broad-spectrum cross-neutralization activity, and preparation method and application thereof | |
| CN111217918B (en) | Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase | |
| CN111234036B (en) | African swine fever virus p72 fusion protein and preparation method and application thereof | |
| CN103384531A (en) | Modified influenza hemagglutinin proteins and uses thereof | |
| CN114315989A (en) | Recombinant novel coronavirus protein vaccine, preparation method and application thereof | |
| CN114478718B (en) | Recombinant novel coronavirus protein vaccine, preparation method and application thereof | |
| CN113461787A (en) | Recombinant novel coronavirus S-RBD trimer protein, and preparation method and application thereof | |
| CN111808176B (en) | Bovine herpes virus antigen compositions and uses thereof | |
| CN112679584A (en) | Swine fever epitope peptide and application thereof | |
| CN112142851B (en) | Subunit fusion protein tG on rabies virus surface and preparation method and application thereof | |
| CN102210860B (en) | Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof | |
| CN106279378A (en) | Varicella zoster virus gE antigen and the purposes in detection varicella zoster virus antibody thereof | |
| CN109200281A (en) | The subunit vaccine of single stranded circle DNA virus GyV3 and its preparation | |
| CN111454989B (en) | Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof | |
| CN110669142B (en) | RGD-fused porcine circovirus type 2 virus-like particle, mutant infectious clone, preparation method and application thereof | |
| WO2023138333A1 (en) | Recombinant sars-cov-2 protein vaccine, and preparation method therefor and use thereof | |
| CN103740736B (en) | The HSV2 virus gE glucoprotein extracellular region gene fragment of chemosynthesis and expression, application | |
| CN105949320A (en) | Preparation and application of echovirus type 1 VP1 protein specific antigen epitope and fusion protein thereof | |
| Temoltzin-Palacios et al. | Modulation of immunodominant sites in influenza hemagglutinin compromise antigenic variation and select receptor-binding variant viruses. | |
| CN116041448A (en) | Novel coronavirus immunogenic substance, preparation method and application thereof | |
| CN102643335A (en) | Recombinant rotavirus VP6 carrier protein and preparation thereof | |
| CN105906716A (en) | Echovirus type-9 VP1 protein specific antigen epitope and preparation method and application of fusion protein thereof | |
| CN112316130A (en) | SARS-CoV2 mucosal immune vaccine and its application | |
| CN114306589B (en) | Recombinant African swine fever virus antigen cocktail vaccine and application | |
| CN113372454B (en) | Nipah virus receptor binding glycoprotein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231007 Address after: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing Patentee after: EASTERN THEATER DISEASE PREVENTION AND CONTROL CENTER OF PLA Address before: 210002 Military Medical Research Institute, Nanjing military area command, 293 East Zhongshan Road, Nanjing, Jiangsu Patentee before: Li Yuexi |