CN109030825A - Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis - Google Patents
Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis Download PDFInfo
- Publication number
- CN109030825A CN109030825A CN201710438304.XA CN201710438304A CN109030825A CN 109030825 A CN109030825 A CN 109030825A CN 201710438304 A CN201710438304 A CN 201710438304A CN 109030825 A CN109030825 A CN 109030825A
- Authority
- CN
- China
- Prior art keywords
- rv2031c
- albumen
- mycobacterium tuberculosis
- sequence
- detection
- 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.)
- Pending
Links
- 101100054729 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) hspX gene Proteins 0.000 title claims abstract description 52
- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 37
- 208000015181 infectious disease Diseases 0.000 title abstract description 11
- 238000011895 specific detection Methods 0.000 title abstract description 5
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 238000006467 substitution reaction Methods 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000013642 negative control Substances 0.000 claims description 35
- 238000003745 diagnosis Methods 0.000 claims description 10
- 239000003550 marker Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 5
- 238000003908 quality control method Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 108010047620 Phytohemagglutinins Proteins 0.000 claims description 3
- 239000012930 cell culture fluid Substances 0.000 claims description 3
- 230000001885 phytohemagglutinin Effects 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000012217 deletion Methods 0.000 abstract description 2
- 230000037430 deletion Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 33
- 201000008827 tuberculosis Diseases 0.000 description 30
- 239000000427 antigen Substances 0.000 description 15
- 102000036639 antigens Human genes 0.000 description 15
- 108091007433 antigens Proteins 0.000 description 15
- 239000000243 solution Substances 0.000 description 12
- 208000036981 active tuberculosis Diseases 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 101000588258 Taenia solium Paramyosin Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000012224 working solution Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 206010036790 Productive cough Diseases 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 239000012139 lysis buffer Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000002331 protein detection Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000003802 sputum Anatomy 0.000 description 3
- 208000024794 sputum Diseases 0.000 description 3
- 101710166488 6 kDa early secretory antigenic target Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- UIEZQYNXCYHMQS-BJDJZHNGSA-N Ile-Lys-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)O)N UIEZQYNXCYHMQS-BJDJZHNGSA-N 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- 108010092854 aspartyllysine Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 1
- OSRZOHXQCUFIQG-FPMFFAJLSA-N Ala-Phe-Pro Chemical compound C([C@H](NC(=O)[C@@H]([NH3+])C)C(=O)N1[C@H](CCC1)C([O-])=O)C1=CC=CC=C1 OSRZOHXQCUFIQG-FPMFFAJLSA-N 0.000 description 1
- KMSHNDWHPWXPEC-BQBZGAKWSA-N Arg-Asp-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KMSHNDWHPWXPEC-BQBZGAKWSA-N 0.000 description 1
- ISJWBVIYRBAXEB-CIUDSAMLSA-N Arg-Ser-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISJWBVIYRBAXEB-CIUDSAMLSA-N 0.000 description 1
- LRPZJPMQGKGHSG-XGEHTFHBSA-N Arg-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N)O LRPZJPMQGKGHSG-XGEHTFHBSA-N 0.000 description 1
- AUZAXCPWMDBWEE-HJGDQZAQSA-N Arg-Thr-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O AUZAXCPWMDBWEE-HJGDQZAQSA-N 0.000 description 1
- XRNXPIGJPQHCPC-RCWTZXSCSA-N Arg-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)O)C(O)=O XRNXPIGJPQHCPC-RCWTZXSCSA-N 0.000 description 1
- XAJRHVUUVUPFQL-ACZMJKKPSA-N Asp-Glu-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O XAJRHVUUVUPFQL-ACZMJKKPSA-N 0.000 description 1
- KESWRFKUZRUTAH-FXQIFTODSA-N Asp-Pro-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O KESWRFKUZRUTAH-FXQIFTODSA-N 0.000 description 1
- MJJIHRWNWSQTOI-VEVYYDQMSA-N Asp-Thr-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O MJJIHRWNWSQTOI-VEVYYDQMSA-N 0.000 description 1
- WAEDSQFVZJUHLI-BYULHYEWSA-N Asp-Val-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O WAEDSQFVZJUHLI-BYULHYEWSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- MLSKFHLRFVGNLL-WDCWCFNPSA-N Gln-Leu-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MLSKFHLRFVGNLL-WDCWCFNPSA-N 0.000 description 1
- TWIAMTNJOMRDAK-GUBZILKMSA-N Gln-Lys-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O TWIAMTNJOMRDAK-GUBZILKMSA-N 0.000 description 1
- PDLGMYVCPJOYAR-DKIMLUQUSA-N Glu-Leu-Phe-Ala Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 PDLGMYVCPJOYAR-DKIMLUQUSA-N 0.000 description 1
- YGLCLCMAYUYZSG-AVGNSLFASA-N Glu-Lys-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 YGLCLCMAYUYZSG-AVGNSLFASA-N 0.000 description 1
- SOEPMWQCTJITPZ-SRVKXCTJSA-N Glu-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N SOEPMWQCTJITPZ-SRVKXCTJSA-N 0.000 description 1
- FGSGPLRPQCZBSQ-AVGNSLFASA-N Glu-Phe-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O FGSGPLRPQCZBSQ-AVGNSLFASA-N 0.000 description 1
- MLILEEIVMRUYBX-NHCYSSNCSA-N Glu-Val-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O MLILEEIVMRUYBX-NHCYSSNCSA-N 0.000 description 1
- XUORRGAFUQIMLC-STQMWFEESA-N Gly-Arg-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CN)O XUORRGAFUQIMLC-STQMWFEESA-N 0.000 description 1
- NSTUFLGQJCOCDL-UWVGGRQHSA-N Gly-Leu-Arg Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N NSTUFLGQJCOCDL-UWVGGRQHSA-N 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- SOYCWSKCUVDLMC-AVGNSLFASA-N His-Pro-Arg Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CCCNC(=N)N)C(=O)O SOYCWSKCUVDLMC-AVGNSLFASA-N 0.000 description 1
- DMZOUKXXHJQPTL-GRLWGSQLSA-N Ile-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N DMZOUKXXHJQPTL-GRLWGSQLSA-N 0.000 description 1
- SNHYFFQZRFIRHO-CYDGBPFRSA-N Ile-Met-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)O)N SNHYFFQZRFIRHO-CYDGBPFRSA-N 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 1
- PKKMDPNFGULLNQ-AVGNSLFASA-N Leu-Met-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O PKKMDPNFGULLNQ-AVGNSLFASA-N 0.000 description 1
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 1
- DTUZCYRNEJDKSR-NHCYSSNCSA-N Lys-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN DTUZCYRNEJDKSR-NHCYSSNCSA-N 0.000 description 1
- LOGFVTREOLYCPF-RHYQMDGZSA-N Lys-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-RHYQMDGZSA-N 0.000 description 1
- HUKLXYYPZWPXCC-KZVJFYERSA-N Met-Ala-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HUKLXYYPZWPXCC-KZVJFYERSA-N 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 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 description 1
- NOFBJKKOPKJDCO-KKXDTOCCSA-N Phe-Ala-Tyr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O NOFBJKKOPKJDCO-KKXDTOCCSA-N 0.000 description 1
- CSYVXYQDIVCQNU-QWRGUYRKSA-N Phe-Asp-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O CSYVXYQDIVCQNU-QWRGUYRKSA-N 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- JDJMFMVVJHLWDP-UNQGMJICSA-N Pro-Thr-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JDJMFMVVJHLWDP-UNQGMJICSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 1
- VZQRNAYURWAEFE-KKUMJFAQSA-N Ser-Leu-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VZQRNAYURWAEFE-KKUMJFAQSA-N 0.000 description 1
- KCGIREHVWRXNDH-GARJFASQSA-N Ser-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N KCGIREHVWRXNDH-GARJFASQSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- NCXVJIQMWSGRHY-KXNHARMFSA-N Thr-Leu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O NCXVJIQMWSGRHY-KXNHARMFSA-N 0.000 description 1
- BZTSQFWJNJYZSX-JRQIVUDYSA-N Thr-Tyr-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O BZTSQFWJNJYZSX-JRQIVUDYSA-N 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- XTAUQCGQFJQGEJ-NHCYSSNCSA-N Val-Gln-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N XTAUQCGQFJQGEJ-NHCYSSNCSA-N 0.000 description 1
- CELJCNRXKZPTCX-XPUUQOCRSA-N Val-Gly-Ala Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O CELJCNRXKZPTCX-XPUUQOCRSA-N 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000012152 bradford reagent Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000015355 drug-resistant tuberculosis Diseases 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 201000009671 multidrug-resistant tuberculosis Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the albumen Rv2031c infected for specific detection mycobacterium tuberculosis.The present invention provides application of the Rv2031c albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；The Rv2031c is following albumen a) or b): a) protein of the composition of amino acid sequence shown in the sequence 1 in sequence table；B) by the amino acid residue sequence of the sequence 1 in sequence table by one or several amino acid residues substitution and/or deletion and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 1.The experiment proves that infection due to Mycobacterium tuberculosis recall rate can be improved more effectively.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to the albumen for the infection of specific detection mycobacterium tuberculosis
Rv2031c。
Background technique
Since multiple centuries, tuberculosis continues to cannot be neglected public health problem as one in the whole world.The whole world at present
The population of existing one third carries mycobacterium tuberculosis, only 1 year 2010 just newly-increased cases of tuberculosis 8,800,000, and dead 145
Ten thousand, average to have a people to die of pulmonary tuberculosis less than 22 seconds, tuberculosis is in first of Death of Infectious Diseases number.And China is the whole world
One of 22 tuberculosis high burden countries, tuberculosis patient number height ranks the second in the world, and infected number is more than 500,000,000, only
Just there are new cases 90~1,200,000 within 1 year 2010, occupies about the 12% of global total new cases, such as take not in time effectively
Measure may have 30,000,000 human hairs sick within 10 years futures, it will lead to serious public health problem and social concern, so
It must realize as early as possible from national strategy level to phthisical effective control.
Science proves to show easily propagated due to active tuberculosis, diagnosis to active tuberculosis and disappears as early as possible
Except the infection sources, controls TB endemic and have very great significance.However phthisical radical cure can't be accomplished completely at present, according to
Research shows that active tuberculosis patient has the patient of 1%-9% that will recur after different short-term chemotherapies is cured, and one
In a little special populations, up to 20% (Hong Kong Chest Service/British Medical Research
Council.Am Rev Respir Dis, 1991,143:700-706), and drug-resistant tuberculosis is propagated further and recurs
The tubercular diagnosed not in time has very big relationship.So curing the active tuberculosis diagnosis of patient for anti-about tuberculosis
The overall strategy for controlling tuberculosis has a very big significance.Activity judgement at present answers complex clinical, x-ray performance and sputum bacteria to determine, and
Main foundation is sputum bacteria and x-ray.Sputum smear examination is simple and easy to do, and accuracy is higher, is the goldstandard that active tuberculosis is made a definite diagnosis,
But its recall rate is even lower than 10% in certain researchs.Diagnostic method based on nucleic acid amplification, such as real-time quantitative PCR and DNA
The advantages of chip is that sensitivity is costly and time consuming short, and problem is that specificity is relatively difficult to guarantee, and is easy to cause false positive results.And base
It is clinical to be had become due to its simplicity, rapidity for important active tuberculosis in the serodiagnosis of antigen-antibody reaction
Complementary diagnosis means, and it is able to satisfy the requirement for distinguishing tuberculosis rehabilitation crowd and active tuberculosis.Therefore, in the long run
In general, should be dedicated to finding the preferable tuberculosis marker of sensibility and specificity.
Ideal Diagnosis of Tuberculosis marker should meet the following conditions: (1) sensibility is high；(2) specificity is high；(3) it is present in
In body fluid, especially blood, it is easy to detect.But the antigen such as antigen that Mycobacterium tuberculosis serodiagnosis at present is targeted
5,38KD antigen, 30/31KD antigen and antigen 60 etc., have that positive rate is low, exempt from intersecting for other mycobacteriums
The problems such as epidemic disease, although there is certain value in curative effect monitoring, prompt recurrence, judging prognosis and the generaI investigation of people at highest risk,
But it still cannot be used for making a definite diagnosis for active tuberculosis at present.In order to realize the high sensitivity to active tuberculosis, high specific
Diagnosis, urgent need search out more sensitive, more special active tuberculosis biomarker on a molecular scale.
Summary of the invention
It is an object of the present invention to provide the purposes of Rv2031c albumen.
The present invention provides application of the Rv2031c albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；
The Rv2031c is following albumen a) or b):
A) protein of the composition of amino acid sequence shown in the sequence 2 in sequence table；
B) by the amino acid residue sequence of the sequence 2 in sequence table by one or several amino acid residues substitution and/
Or deletion and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 2.
Rv2031c is known albumen, and according to individual difference, which may have different sequences, they there may be
The difference of one or several amino acid, but be within, albumen used in one embodiment of the present of invention
Sequence be sequence 2.
Above-mentioned Rv2031c albumen detects whether patient to be measured infects in mycobacterium tuberculosis product in preparation detection or auxiliary
Application be also the scope of protection of the invention.
It is a further object to provide a kind of detections or auxiliary detection sample to be tested whether to infect tuberculosis branch bar
The product of bacterium.
Product provided by the invention, including Rv2031c albumen.
The said goods further include positive quality control and negative control hole；
The positive quality control is phytohemagglutinin HA；
The negative control hole is nonantigenic cell culture fluid.
In the said goods, the product further includes the readable carrier (can be specification) for being described below content:
If testing result meets following condition: negative control hole spot number is 0-5, and Rv2031c detection hole spot number-yin
Property control wells spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv2031c detection hole spot number-feminine gender is right
According to hole spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If testing result meets condition as described below, sample to be tested candidate is uninfected by mycobacterium tuberculosis；
The negative control hole is hole where negative control,
The Rv2031c detection hole is hole where the Rv2031c albumen.
The said goods are kit, and mentioned reagent box is specially T-SPOT kit, or ELISA kit.
Above-mentioned T-SPOT kit further includes well plates in T-SPOT kit, concentration labelled antibody, colour developing bottom
Object solution.Mentioned reagent box is the product of Oxford immunotec (Britain).
Third purpose of the present invention be to provide it is a kind of for identifying, diagnosing, auxiliary diagnosis, screening and/or auxiliary screening knot
The marker of core mycobacteria.
Marker provided by the invention is Rv2031c albumen.
The experiment proves that the present invention has screened the protein fragments in mycobacterium tuberculosis source, a kind of inspection is provided
Mycobacterium tuberculosis marker antigen RV2031C lungy is surveyed, it is anti-come vitro detection Specific T cell immunity by the antigen
It answers, can be used as the reference of diagnosis of tuberculosis patient, for diagnosing whether patient is infected by mycobacterium tuberculosis.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Portion of reagent is as follows:
Lysis buffer:20mM Tris-HCl, 500mM sodium chloride, 10% glycerol, pH 8.0
Wash buffer:20mM Tris-HCl, 20mM imidazoles, 500mM sodium chloride, 10% glycerol, pH 8.0
Elution buffer:20mM Tris-HCl, 250mM imidazoles, 500mM sodium chloride, 10% glycerol, pH 8.0
Chromatography buffer: potassium dihydrogen phosphate 13.609g (preparing 1000ml) is taken, adds 0.1mol/l sodium hydroxide solution, adjusts
PH to 7.5 is saved, the PBS buffer solution that pH value is 7.5 is obtained.
Solidify Ni+ chromatographic resin Novagen, Ni-NTA His, (Cat.NO 70691-5, Beijing are magnificent；2-8 degree is protected
It deposits)；Protein binding capacity is greater than 5mg/ml resin chromatographic column (glass or polypropylene).Superdex 75:(GE).
It is the T-SPOT of Oxford immunotec (Britain) that following embodiments, which use tuberculosis infection T cell detection kit,
Kit is 10 pairs of antigenic reagent box of ESAT-6 and CFP.
Embodiment 1, prokaryotic expression destination protein Rv2031c
The preparation of destination protein Rv2031c: the recombinant vector pET28a- of Rv2031c protein coding gene will be expressed
Rv2031c is imported in e. coli bl21, obtains recombinant bacterium BL21/pET28a-Rv2031c；IPTG induces recombinant bacterium BL21/ again
PET28a-Rv2031c collects supernatant, as destination protein Rv2031c.
It is specific as follows:
1, the building of express express target protein recombinant bacterium
The amino acid sequence of Rv2031c albumen is sequence 2, and the nucleotides sequence of encoding gene is classified as sequence 1.
By the site NdeI and EcoRI of the insertion pET28a carrier of Rv2031c protein coding gene shown in sequence 1, obtain
Recombinant vector pET28a-Rv2031c；
Recombinant vector is imported in e. coli bl21, recombinant bacterium BL21/pET28a-Rv2031c is obtained.
2, protein expression
1) recombinant bacterium BL21/pET28a-Rv2031c stands overnight (16h or so) at 37 DEG C.
2) 37 DEG C of 200rpm of 5ml LB liquid medium (5ul kan is added) are inoculated in, it is (big that culture to OD is greater than 0.6-0.8
About 3h).
3) it is inoculated in 300ml LB liquid medium by inoculum concentration 5ml, 37 DEG C, 200rpm is cultivated to OD600 ≈ 0.6-
0.8 (about 2h).16 DEG C are cooled to, final concentration 0.4mM IPTG is added；
4) 16 DEG C, 200rpm culture culture 16h, 4 DEG C, 4000rpm is centrifuged 10min and collects thallus.
3, protein purification
1) thallus is resuspended: thallus collected by every 1L culture medium is resuspended with 40ml or so lysis buffer and (1% egg is added
White enzyme inhibitor PMSF, then final concentration of 0.5-1mM)
2) bacterial cell disruption: 6s/ times ultrasonic, power 38%, ultrasonic 20min.(bright suspension is standard, is adjusted as one sees fit
Ultrasound works total time).
3) remove bacterial debris: 4 DEG C, 12000g is centrifuged 30 minutes.
4) Ni column handle: 3mlNi column first uses water process 8cv (8 times of column volumes), then use 8cv lysis buffer as put down
Weigh liquid balance, flow velocity 30cm/h.
5) loading: broken bacterium centrifugation supernatant carries out loading with the flow velocity of 30cm/ml, and collection flows through peak.
6) it balances: balancing 5cv with equilibrium liquid after sample loading；
7) it washs: washing 5cv with wash buffer；
8) it elutes: being eluted with elution buffer, be in charge of collection.Eluent is detected with bradford reagent, understands egg
It is white whether complete elution.Such as contain albumen, then continue to elute, until completely, obtaining elution albumen.
9) dialyse: it is slow that elution albumen is fitted into the ion exchange for being put into the bag filter of 7KD and eluting albumen volume equipped with 100 times
Dialysed overnight is carried out in the beaker of fliud flushing A (formula 10mM PB, 50mM NaCl, pH 7.5).Centrifuging and taking supernatant.
10) sieve chromatography
A) chromatographic column: superdex 75 (determines the volume of chromatography media according to the amount of sample)
B) flow velocity；30cm/h
C) chromatography process: water process 5cv is first used, then handles half an hour with 0.6M NaOH aqueous solution, then use chromatography buffer
(PBS, pH 7.5) balance chromatography, until chromatographic column conductance and pH efflux are consistent with chromatography buffer.It 9) is obtained above-mentioned
Supernatant loading elutes (flow velocity 30cm/h) with chromatography buffer, collects corresponding eluting peak, is purpose albumen Rv2031c (size
For 19.516KDa).
The application of embodiment 2, mycobacterium tuberculosis marker antigen Rv2031c in detection mycobacterium tuberculosis
1, T-SPOT kit detects
The specific method is as follows, and some solution of the inside are all from the T-SPOT reagent of Oxford immunotec (Britain)
Box:
1) using heparin vacuum blood collection tube, to different patients to be measured, (35 are tuberculosis patient example clinical detection shown in table 1
4 clinical detection non-tuberculosis people shown in patient and Biao 2 lungy, and know) peripheric venous blood is directly adopted, it obtains anticoagulant
Blood sample；
2) anticoagulation sample is mixed by volume 1:1 and RPMI1640 culture solution；Carefully blood sample is added in 3:1 ratio
Ficoll lymphocyte separation medium upper layer (GE Healthcare 17-1440-02) (utilizes Ficoll-PaqueTMplusAccording to
Operating instruction separates PBMCs)；25 DEG C of room temperature, 1000g is centrifuged 22 minutes；Horizontal rotor delays and rises slow drop；
3) by mononuclear cell layer (being located at centrifuge tube middle layer, be the tunica albuginea shape of layer) from Ficoll separating pipe transfer
The sterile 15ml centrifuge tube for having added 10ml AIM-V culture solution (Invitrogen 12055091) is moved on to, is mixed gently, room temperature
600g is centrifuged 7min；
4) supernatant is carefully removed, 1ml AIM-V culture solution is added, AIM-V culture solution is added extremely in light and slow resuspension cell
10ml, 350g are centrifuged 7min；
5) supernatant is carefully abandoned, 1ml AIM-V culture solution is added, cell is resuspended, obtain cell suspension (PBMCs)；
6) it takes 10 μ l cell suspensions that the 1%trypan blue of 40 μ l is added, is diluted with AIM-V culture solution, it is dilute to obtain cell
Release working solution；
7) take out 24 hole pvdf membrane plates from aluminium envelope, be sequentially added into: 50 μ l phytohemagglutinin HA (sigma) are extremely
The Rv2031c protein solution that Positive control wells, 50 μ l are prepared (initial concentration 60ug/ml, solvent are AIM V culture solution)
It is added to detection hole, 50 μ l AIM V culture solutions to negative control hole；It is separately added into above-mentioned 3 hole and has prepared cell dilution work
Make liquid 100 μ l (every hole cell quantity 2.5 × 105), and in sample well Rv2031c protein solution final concentration of 20ug/ml；
8) culture plate is put into 37 DEG C, 5%CO2Incubator is incubated for 16 hours；
9) fresh enzyme labelled antibody working solution is prepared by 1:200 with sterile PBS；Culture plate is taken out from incubator, is discarded in hole
Liquid.It is washed 4 times with the sterile PBS of 200 μ l/well；
10) 50 μ l/well enzyme labelled antibody working solutions are added, culture plate is incubated for 1 hour at 2-8 DEG C.It is washed 4 times with PBS
Remove unbonded enzyme labelled antibody；
11) the 50 μ l of substrate working solution that equilibrium at room temperature is crossed is added in every hole, reacts at room temperature 7 minutes.It is terminated with distilled water flushing
Reaction, in 37 DEG C of 2-3hrs or ambient temperature overnight desiccation culture plate, obtains the culture plate containing reaction product.
2, spot counts
1) culture plate containing reaction product is taken out from incubator discard cell culture fluid；
2) 200ul PBS buffer solution (pH 7.5) is added in each reacting hole；
3) PBS buffer solution is discarded；It is washed repeatedly at least 3 times with fresh PBS buffer solution；
4) with the concentration labelled antibody reagent (mouse of alkali phosphatase enzyme mark in 200 times of PBS buffer solution dilution kits
Anti-human gamma interferon monoclonal antibody)；
5) 50ul labelled antibody working solution is added in each reacting hole, and 2-8 DEG C is incubated for 1 hour；
6) labelled antibody working solution is discarded, is washed by above-mentioned step 2 and 3；
7) each reacting hole is added 50ul substrate chromophoric solution and is incubated at room temperature 7 minutes；
8) culture plate is thoroughly washed with distilled water or deionized water terminate reaction；
9) in ventilation or 37 DEG C of incubator desiccation culture plates；
10) navy blue that counts in each reacting hole clearly spot.
As a result judge:
If negative control hole spot number is 0-5, and Rv2031c detection hole spot number-negative control hole spot number >=6；
If or negative control hole spot number is more than or equal to 6, and (Rv2031c detection hole spot number-negative control hole spot
Number) >=2 × (negative control hole spot number)；
Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and Rv2031c do not generate immune response, and sample to be tested candidate is uninfected by tuberculosis
Mycobacteria.
35 tuberculosis patient samples to be tested in table 1 are detected in aforementioned manners, and the results are shown in Table 1.
The sample to be tested of 4 Healthy Peoples in table 2 is detected in aforementioned manners, and the results are shown in Table 2.
Comparative example:
35 tuberculosis patients shown in T-SPOT kit detection table 1 using Oxford immunotec (Britain) are to be measured
The sample to be tested of 4 Healthy Peoples in sample and table 2, method is same as Example 1, uniquely the difference is that two detection holes are arranged,
And A detection hole is antigen A hole, addition is antigen ESAT-6, and B detection hole is the hole antigen B, and addition is antigens c FP 10.
According to the reaction and judgement result of antigen A and the hole antigen B:
If negative control hole spot number is 0-5, and (antigen A spot number)-(negative control hole spot number) >=6 or (antigen B
Spot number)-(negative control hole spot number) >=6；
Or work as negative control hole spot number >=6, and (antigen A spot number) >=2 × (negative control hole spot number) or (antigen
B spot number) >=2 × (negative control hole spot number).
Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；
If being unsatisfactory for above-mentioned, sample to be tested and antigen A or antigen B do not generate immune response, and sample to be tested candidate does not feel
Contaminate mycobacterium tuberculosis.
Table 1 is sample and its testing result
In table ,-it is negative control ,+it is Positive control wells (culture medium for having added PHA), X is Rv2031c Protein Detection hole,
A is antigen A detection hole, and B is antigen B detection hole.
Table 2 is Healthy People sample and its testing result
| Serial number | - | A | B | + | X |
| 1 | 1 | 3 | 4 | Greater than 1000 | 1 |
| 2 | 0 | 0 | 0 | 690 | 2 |
| 3 | 0 | 23 | 6 | 562 | 3 |
| 4 | 0 | 4 | 10 | 759 | 2 |
In table ,-it is negative control (culture medium for being added without any antigen) ,+it is that Positive control wells (have added the culture of PHA
Base), X is Rv2031c Protein Detection hole.
As can be seen that
Rv2031c is detected as antigen, 29 in the identified sample to be tested for infecting mycobacterium tuberculosis of 35 clinics
The sample of mycobacterium tuberculosis is infected for Rv2031c Protein Detection, this is consistent with clinical identification result.But remaining 6 sample
It is not detected and, therefore, the present invention uses Rv2031c to detect whether that the recall rate of infection mycobacterium tuberculosis reaches as antigen
83%.
Mycobacterium tuberculosis is not detected in 4 Healthy People samples, this is consistent with clinical identification result.
Comparative example detects the identified sample to be tested for infecting mycobacterium tuberculosis of 35 clinics, wherein 27 are comparative example
The sample of detection infection mycobacterium tuberculosis, this is consistent with clinical identification result；But remaining 8 sample is not detected and.
Therefore, comparative example detects whether that the recall rate of infection mycobacterium tuberculosis is only 77%, examines well below Rv2031c as antigen
It surveys.
In comparative example in 4 Healthy People samples 2 mycobacterium tuberculosis is not detected, this is different from clinical identification result.
Can be seen that Rv2031c albumen from above-mentioned experiment can detecte or assist whether detection patient to be measured infects tuberculosis
Mycobacteria, it is specific as follows: using Rv2031c albumen as antigen, to be detected by tuberculosis infection T cell detection kit, with thin
Born of the same parents' culture medium is as negative control hole, if negative control hole spot number is 0-5, and Rv2031c detection hole spot number-negative control
Hole spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv2031c detection hole spot number-negative control hole spot
Points) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；If discontented
Foot is above-mentioned, then sample to be tested and Rv2031c do not generate immune response, and sample to be tested candidate is uninfected by mycobacterium tuberculosis.
Sequence table
<110>Guangdong Ti Bikang Biotechnology Co., Ltd
<120>the albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 432
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
atggccacca cccttcccgt tcagcgccac ccgcggtccc tcttccccga gttttctgag 60
ctgttcgcgg ccttcccgtc attcgccgga ctccggccca ccttcgacac ccggttgatg 120
cggctggaag acgagatgaa agaggggcgc tacgaggtac gcgcggagct tcccggggtc 180
gaccccgaca aggacgtcga cattatggtc cgcgatggtc agctgaccat caaggccgag 240
cgcaccgagc agaaggactt cgacggtcgc tcggaattcg cgtacggttc cttcgttcgc 300
acggtgtcgc tgccggtagg tgctgacgag gacgacatta aggccaccta cgacaagggc 360
attcttactg tgtcggtggc ggtttcggaa gggaagccaa ccgaaaagca cattcagatc 420
cggtccacca ac 432
<210> 2
<211> 144
<212> PRT
<213> 1
<400> 2
Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro
1 5 10 15
Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg
20 25 30
Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu
35 40 45
Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys
50 55 60
Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu
65 70 75 80
Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly
85 90 95
Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp
100 105 110
Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val
115 120 125
Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn
130 135 140
Claims (7)
- Application of the 1.Rv2031c albumen in preparation detection or auxiliary detection mycobacterium tuberculosis product；The Rv2031c is following albumen a) or b):A) protein of the composition of amino acid sequence shown in the sequence 2 in sequence table；B) amino acid residue sequence of the sequence 2 in sequence table by the substitution of one or several amino acid residues and/or is lacked Lose and/or addition and with the protein with the same function as derived from a) of albumen shown in sequence 2.
- 2.Rv2031c albumen detects whether patient to be measured infects answering in mycobacterium tuberculosis product in preparation detection or auxiliary With.
- 3. whether a kind of detection or auxiliary detection sample to be tested infect the product of mycobacterium tuberculosis, including Rv2031c albumen.
- 4. product according to claim 3, it is characterised in that: the product further includes positive quality control and negative control hole；The positive quality control is phytohemagglutinin HA；The negative control hole is nonantigenic cell culture fluid.
- 5. product according to claim 3 or 4, it is characterised in that: the product further includes be described below content readable Carrier:If testing result meets following condition: negative control hole spot number is 0-5, and Rv2031c detection hole spot number-feminine gender is right According to hole spot number >=6；If or negative control hole spot number is more than or equal to 6, and (Rv2031c detection hole spot number-negative control hole Spot number) >=2 × (negative control hole spot number)；Then sample to be tested candidate infects mycobacterium tuberculosis, is positive sample；If inspection It surveys result and meets condition as described below, then sample to be tested candidate is uninfected by mycobacterium tuberculosis；The negative control hole is hole where negative control,The Rv2031c detection hole is hole where the Rv2031c albumen.
- 6. according to the product any in claim 3-5, it is characterised in that: the product is kit.
- 7. it is a kind of for identifying, diagnosing, auxiliary diagnosis, screening and/or assist screening mycobacterium tuberculosis marker, be Rv2031c albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710438304.XA CN109030825A (en) | 2017-06-12 | 2017-06-12 | Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710438304.XA CN109030825A (en) | 2017-06-12 | 2017-06-12 | Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109030825A true CN109030825A (en) | 2018-12-18 |
Family
ID=64629089
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710438304.XA Pending CN109030825A (en) | 2017-06-12 | 2017-06-12 | Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109030825A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116559433A (en) * | 2022-01-28 | 2023-08-08 | 安博智联(北京)生物科技有限公司 | Pharmaceutical composition containing tuberculosis allergen EH and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6403100B1 (en) * | 1997-07-10 | 2002-06-11 | The United States Of America As Represented By The Department Of Health And Human Services | Method of attenuating pathogenic mycobacteria and strains of mycobacteria so attenuated |
| US20040146933A1 (en) * | 2001-01-08 | 2004-07-29 | Quinn Frederick D | Latent human tuberculosis model, diagnostic antigens, and methods of use |
| CN101203239A (en) * | 2005-03-31 | 2008-06-18 | 莱顿大学医药中心 | Methods and devices for diagnosing, preventing and treating mycobacterial infection and tuberculosis |
| CN101812464A (en) * | 2009-02-23 | 2010-08-25 | 华中农业大学 | New functions and applications of mycobacterium tuberculosis WhiB3 gene |
| CN105218648A (en) * | 2015-07-10 | 2016-01-06 | 邓林红 | Mycobacterium tuberculosis antigen, nucleic acid sequence and its application |
| CN106349350A (en) * | 2015-07-16 | 2017-01-25 | 广东体必康生物科技有限公司 | Protein capable of specifically detecting mycobacterium tuberculosis infection |
| CN106645733A (en) * | 2015-07-16 | 2017-05-10 | 广东体必康生物科技有限公司 | Protein for specific detection of mycobacterium tuberculosis infection |
-
2017
- 2017-06-12 CN CN201710438304.XA patent/CN109030825A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6403100B1 (en) * | 1997-07-10 | 2002-06-11 | The United States Of America As Represented By The Department Of Health And Human Services | Method of attenuating pathogenic mycobacteria and strains of mycobacteria so attenuated |
| US20040146933A1 (en) * | 2001-01-08 | 2004-07-29 | Quinn Frederick D | Latent human tuberculosis model, diagnostic antigens, and methods of use |
| CN101203239A (en) * | 2005-03-31 | 2008-06-18 | 莱顿大学医药中心 | Methods and devices for diagnosing, preventing and treating mycobacterial infection and tuberculosis |
| CN101812464A (en) * | 2009-02-23 | 2010-08-25 | 华中农业大学 | New functions and applications of mycobacterium tuberculosis WhiB3 gene |
| CN105218648A (en) * | 2015-07-10 | 2016-01-06 | 邓林红 | Mycobacterium tuberculosis antigen, nucleic acid sequence and its application |
| CN106349350A (en) * | 2015-07-16 | 2017-01-25 | 广东体必康生物科技有限公司 | Protein capable of specifically detecting mycobacterium tuberculosis infection |
| CN106645733A (en) * | 2015-07-16 | 2017-05-10 | 广东体必康生物科技有限公司 | Protein for specific detection of mycobacterium tuberculosis infection |
Non-Patent Citations (1)
| Title |
|---|
| 余琴 等: "RV2031c、38kDa以及融合蛋白CFP10-ESAT6用于结核病血清学诊断的评价", 《中国病原生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116559433A (en) * | 2022-01-28 | 2023-08-08 | 安博智联(北京)生物科技有限公司 | Pharmaceutical composition containing tuberculosis allergen EH and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Geluk et al. | New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy | |
| JP5805368B2 (en) | Immunological monitoring based on IP-10 | |
| WO2018076404A1 (en) | Method for in vitro detection of active tuberculosis | |
| CN111443208B (en) | Composition to differentiate active tuberculosis from latent tuberculosis | |
| US20150204885A1 (en) | Method for the direct detection of mycobacterium tuberculosis | |
| CN103760345B (en) | A kind of kit and application thereof utilizing peripheral blood detection m tuberculosis infection | |
| CN106939035A (en) | A kind of mycobacterium tuberculosis T cell antigen epitope polypeptide and its application | |
| CN103063836A (en) | Reagent, method and kit for detecting mycobacterial infection | |
| CN109030825A (en) | Albumen Rv2031c for the infection of specific detection mycobacterium tuberculosis | |
| CN106349350A (en) | Protein capable of specifically detecting mycobacterium tuberculosis infection | |
| CN107831316A (en) | A flow cytometry detection kit for diagnosing bovine tuberculosis | |
| CN109061191B (en) | Application of S100P protein as a marker in the diagnosis of active tuberculosis | |
| CN105388291B (en) | Gamma delta T cell surface activation molecule and kit for quickly diagnosing active tuberculosis | |
| CN109406776A (en) | Albumen Rv2818c for the infection of specific detection mycobacterium tuberculosis | |
| CN109187987B (en) | Application of MS4A3 protein as marker in diagnosis of active tuberculosis | |
| CN106645733A (en) | Protein for specific detection of mycobacterium tuberculosis infection | |
| Song et al. | Neutrophil Surface CD64 Stimulation Index Detection Assay in Diagnosing Mycobacterium tuberculosis Infection. | |
| CN109406777A (en) | Albumen Rv2824c for the infection of specific detection mycobacterium tuberculosis | |
| CN109030826A (en) | Albumen Rv3539 for the infection of specific detection mycobacterium tuberculosis | |
| CN108267589B (en) | Use of Mycobacterium tuberculosis protein in the preparation of products for diagnosing latent tuberculosis infection and/or active pulmonary tuberculosis | |
| CN105384805A (en) | Tuberculosis detection polypeptide and application thereof | |
| CN108254553A (en) | For the albumen Rv1791 of specific detection mycobacterium tuberculosis infection | |
| CN109813903A (en) | A method for the diagnosis of Mycobacterium tuberculosis infection based on the detection of tuberculosis-specific T cell immunophenotype by flow cytometry | |
| CN108267588B (en) | Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis | |
| Yong et al. | Eri R |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 528315 room 401-421, floor 1-3, building a, Foshan China Europe service center, No. 2, South Lingnan Avenue, xingle community, Lecong Town, Shunde District, Foshan City, Guangdong Province Applicant after: Tibikon Biotechnology (Guangdong) Co.,Ltd. Address before: 528000 6 / F, block a, China Europe center, No. 2, Lingnan Avenue South, Lecong Town, Shunde District, Foshan City, Guangdong Province Applicant before: TB Healthcare Co.,Ltd. |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181218 |