CN102565398B - Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit - Google Patents
Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit Download PDFInfo
- Publication number
- CN102565398B CN102565398B CN201010619492.4A CN201010619492A CN102565398B CN 102565398 B CN102565398 B CN 102565398B CN 201010619492 A CN201010619492 A CN 201010619492A CN 102565398 B CN102565398 B CN 102565398B
- Authority
- CN
- China
- Prior art keywords
- clonorchiasis
- elisa
- liquid
- diagnostic kit
- reaction plate
- 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.)
- Expired - Fee Related
Links
- 206010009344 Clonorchiasis Diseases 0.000 title claims abstract description 30
- 238000002965 ELISA Methods 0.000 title claims abstract description 26
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 29
- 102000005720 Glutathione transferase Human genes 0.000 claims abstract description 27
- 108010070675 Glutathione transferase Proteins 0.000 claims abstract description 27
- 102000004190 Enzymes Human genes 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 239000013642 negative control Substances 0.000 claims abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 9
- 229920001184 polypeptide Polymers 0.000 claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 8
- 239000013641 positive control Substances 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 4
- 150000001413 amino acids Chemical group 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 28
- 241001327965 Clonorchis sinensis Species 0.000 claims description 24
- 238000009007 Diagnostic Kit Methods 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 13
- 238000007865 diluting Methods 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 9
- 239000003550 marker Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 5
- 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 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 230000002421 anti-septic effect Effects 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 108060003951 Immunoglobulin Proteins 0.000 abstract 1
- 239000003085 diluting agent Substances 0.000 abstract 1
- 102000018358 immunoglobulin Human genes 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 19
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 16
- 102100037478 Glutathione S-transferase A2 Human genes 0.000 description 15
- 101001026115 Homo sapiens Glutathione S-transferase A2 Proteins 0.000 description 15
- 239000000427 antigen Substances 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 12
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 235000015097 nutrients Nutrition 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 229960003180 glutathione Drugs 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- VYZAHLCBVHPDDF-UHFFFAOYSA-N Dinitrochlorobenzene Chemical compound [O-][N+](=O)C1=CC=C(Cl)C([N+]([O-])=O)=C1 VYZAHLCBVHPDDF-UHFFFAOYSA-N 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- 210000000013 bile duct Anatomy 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000009465 prokaryotic expression Effects 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 235000003969 glutathione Nutrition 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 238000012856 packing Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108010024636 Glutathione Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000037029 cross reaction Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 231100000614 poison Toxicity 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 3
- 229940033663 thimerosal Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000011941 Tilia x europaea Nutrition 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000003596 drug target Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000001155 isoelectric focusing Methods 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
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 239000004571 lime Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- WDCYWAQPCXBPJA-UHFFFAOYSA-N 1,3-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC([N+]([O-])=O)=C1 WDCYWAQPCXBPJA-UHFFFAOYSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010056375 Bile duct obstruction Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000242711 Fasciola hepatica Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 101710142847 Glutathione S-transferase class-mu 26 kDa isozyme Proteins 0.000 description 1
- 101710113304 Glutathione S-transferase class-mu 28 kDa isozyme Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 101710118186 Neomycin resistance protein Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000223960 Plasmodium falciparum Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 241001442514 Schistosomatidae Species 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 208000035472 Zoonoses Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 201000001883 cholelithiasis Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 210000003459 common hepatic duct Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 208000006275 fascioliasis Diseases 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 231100000734 genotoxic potential Toxicity 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 101150017084 gst-2 gene Proteins 0.000 description 1
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- -1 lipid peroxide Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000011546 protein dye Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000002676 xenobiotic agent Substances 0.000 description 1
- 230000002034 xenobiotic effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and discloses a clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit, which comprises an ELISA reaction plate, a negative control, a positive control, a substrate liquid A, a substrate liquid B, a sample diluent, a concentrated washing liquid and a stopping solution, wherein the ELISA reaction plate is coated with a clonorchiasis glutathione transferase 2; the clonorchiasis glutathione transferase 2 is a polypeptide which has an amino acid sequence shown as SEQ ID No.1 or a conserved variation polypeptide; and an enzyme label is horse radish peroxidase-labeled antihuman IgG4 (immunoglobulin G4) second antibody. The clonorchiasis ELISA diagnostic reagent kit disclosed by the invention has the characteristics of high specificity, high sensitivity, high accuracy and the like, and can be well applied to diagnosis of clonorchiasis; a major reagent of the reagent kit is in the form of a working liquid, and can be used directly; and compared with the conventional aetiology diagnosis method, the clonorchiasis ELISA diagnostic reagent kit has the advantages of operation accuracy, easiness and convenience and believable results.
Description
Technical field
The invention belongs to biological technical field, specifically, the invention describes a kind of clonorchiasis ELISA diagnostic kit.
Background technology
Clonorchiasis is a kind of parasitic zoonoses, adult colonizes in hepatic duct, and adult can destroy biliary tract epithelium and submucosal blood vessel, and produces secretion excretion antigen, cause the inflammatory reaction around bile duct and bile duct, cause bile duct limitation to be expanded and bile duct epithelial cell hyperplasia.Some antigenic components can also enter hepatic tissue by bile duct epithelial cell, and cause inflammation reaction and liver dysfunction.Long-term severe infections can cause proliferation of fibrous tissue and hepatocellular atrophy sex change, even causes canceration, ascites and cirrhosis.Liver fluke blocks bile duct, bile is flowed out not smooth, easily merges bacteriological infection and occur cholelithiasis.
The control of liver rot mainly relies on comprehensive preventive health measures.At the mass screening and mass treatment of Endemic Area strengthening to crowd, and strengthen the development of vaccine, to reduce the infection sources and protection Susceptible population.At present, the emphasis of liver rot study on prevention concentrates on diagnosis and vaccine candidate antigen, drug targets, pathogenic molecular mechanism research.
Transferase carries out the transfer of some group or the enzyme of exchange between catalytic substrate, is one of catalyzer important in liver cell association reaction in biosome (genus second-phase reaction).Glutathione transferase (glutathione S-transferase, GST) (EC2.5.1.18) be extensively be present in various biosome by multiple gene code, multi-functional isodynamic enzyme, it is the enzyme family that catalysis glutathione is connected with multiple electrophilic compound, the subunit forming enzyme has 7 at least, and most of enzyme is present in tenuigenin with homodimer or the existence of heterodimer form.
One of GSTs critical function in biosome is the detoxication of internal, exogenous poisonous substance.The biosome comprising people is all exposed in the environment of a large amount of xenobiontics existence, some compounds have electrophilic reactivity after metabolism activation, cause and endogenous molecule covalent bond, protein is important biomacromolecule, through often changing its function after exogenous molecules and its combination.Glutathione transferase impels glutathione to be combined with xenobiotic mainly through enzyme catalysis, or in non-enzymatic binding mode, chemical substance, dyestuff, the carcinogenic substance with genotoxic potential various in body is discharged in body, thus reach removing toxic substances object, discovered in recent years glutathione transferase for research liver diseases generation, develop, lapse to and prevent significant.Its catalytic reaction characteristic determine its preventing lipid peroxidation injury from expanding, resist and DNA plerosis damage to reduce the vital role in tumour generation and cancer cell drug resistance formation etc.
GSTs is the potential drug targets of anti-plasmodium falciparum, the GSTs of worm participates in the removing toxic substances to the lipid peroxide produced by polypide metabolism and host immune system and Cytotoxic carbonyl, is also considered to immunotherapy and a chemotherapeutic potential target.Wherein, being mainly positioned blood fluke GST-26 and GST-28 of adult essence, is one of candidate vaccine molecule of 6 most potentiality that WHO proposes.Catalytic activity is had at the restructuring GST of expression in escherichia coli plasmodium falciparum.The 26KDa GST of clonorchis sinensis and the encoding gene of 28kDa GST also have catalytic activity at prokaryotic expression, and the 26kDaGST of restructuring can be used for detecting IgG and IgE antibody.Thus, prokaryotic expression activated restructuring GST is feasible. and be easy to obtain a large amount of recombinant proteins for functional study.
The present inventor, through extensive and deep research, has been separated coding for glutathion transferase 2DNA from clonorchis sinensis, and expression and purification glutathione transferase 2, and further disclose the application based on these polynucleotide and polypeptide.
Summary of the invention
The present invention, on the basis of the clonorchis sinensis glutathione transferase 2 and preparation and application thereof that provide separation, provides a kind of clonorchiasis ELISA diagnostic kit.
The invention discloses a kind of clonorchis sinensis glutathione transferase 2 of separation, it is polypeptide or its conservative variation's polypeptide of the amino acid sequence with SEQ ID NO.1.
Method well-known to those having ordinary skill in the art can be used for building containing clonorchis sinensis glutathione transferase 2 DNA sequences encoding and the suitable expression vector of transcribing/translating control signal.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promoter in expression vector, synthesizes to instruct mRNA.The representative example of these promoters has: colibacillary lac or trp promoter; Enter bacteriophage PL promoter; Eukaryotic promoter comprise CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promoter, retroviruse LTRS and some other known can the promoter expressed in protokaryon or eukaryotic or its virus of controlling gene.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as dihyrofolate reductase, neomycin resistance and green fluorescent protein (GFP) that eukaryotic is cultivated, or for colibacillary tetracycline or amicillin resistance.Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promoter or control sequence, may be used for transforming suitable host, with can marking protein.
Host cell can be prokaryotic, as bacterial cell e. coli bl21/DE3; Or the eukaryotic such as low, as yeast cells; Or higher eucaryotic cells, as mammalian cell.Representative example has: Escherichia coli, streptomyces; The bacterial cell of salmonella typhimurium; Fungal cell is as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast etc. of CHO, COs.293 cell or Bowes melanoma cells.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotes as Escherichia coli time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eucaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.
The preparation method of the clonorchis sinensis glutathione transferase 2 of aforementioned separation, in general there are following steps: (1) uses the polynucleotide (or variant) of coding clonorchis sinensis glutathione transferase 2 of the present invention, or transform or suitable host cell of transduceing with the recombinant expression carrier containing these polynucleotide; (2) host cell cultivated in suitable nutrient culture media; (3) separation, protein purification from nutrient culture media or cell.
The transformant obtained can be cultivated by conventional method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, nutrient culture media used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promoter selected with the induction of suitable method (as temperature transition or chemical induction), cultivates a period of time again by cell.Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cell membrane.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Enumerate according to the embodiment of the present invention, an appropriate method preparing clonorchis sinensis glutathione transferase 2 is:
1) encoding gene of clonorchis sinensis glutathione transferase 2 is cloned into prokaryotic expression carrier pET-30a (+);
2) by screening positive clone after the expression vector transformation of E. coli BL21/DE3 competent cell of restructuring;
3) abduction delivering of e. coli bl21/DE3;
4) collect the nutrient solution supernatant after the abduction delivering of e. coli bl21/DE3, obtain the albumen of purifying through affinity chromatography.
Above-mentioned clonorchis sinensis glutathione transferase 2 can be used for preparation clonorchiasis diagnostic kit.
The diagnosis of clonorchiasis is except the Pathogen Biology inspection of routine, and main method uses immunological diagnostic reagent box.Mainly contain two classes: enzyme linked immunological kit (ELISA) and tachysynthesis colloidal gold kit (ICT).Namely the diagnosis of clonorchiasis judges Current Infection by the circulating antigen detected in serum or urine, also carrys out rapid screening by the specific antibody detected in serum or saliva.Because clonorchis sinensis mainly colonizes in outside tissue, the antigenic component overwhelming majority produced enters enteron aisle along with bile, and only have infectiosity high, polypide is serious to bile duct obstruction, when causing epithelial duct serious damage, its Secretory product just can enter in liver and peripheral blood.Enter circulating antigen major part in peripheral blood neutralize by antibody, only have a small amount of free circulating antigen to be detected.Therefore, although the method detecting circulating antigen can reflect Current Infection, susceptibility is lower.At present, the clinical mainly antibody assay kit for auxiliary diagnosis.The method detecting antibody is divided into two kinds, and one is combine with the antibody that the antigen be coated on reaction plate or reaction film is caught as detection reagent with mark two is anti-, and a kind of is as detection reagent with the antigen marked.Two is anti-as detecting reagent, generally can only detect a kind of antibody, and non-specific binding is higher; Labelled antigen is as detection reagent, and can detect Multiple Antibodies, specificity is higher.
The invention discloses a kind of clonorchiasis ELISA diagnostic kit, comprise: ELISA reaction plate, negative control, positive control, enzyme marker, sample diluting liquid, concentrated cleaning solution, substrate A liquid, substrate B liquid and stop buffer, wherein, ELISA reaction plate is coated with above-mentioned clonorchis sinensis glutathione transferase 2, described enzyme marker is that horseradish peroxidase-labeled anti-human igg 4 two resists.
Described clonorchis sinensis glutathione transferase 2 is the recombinant expressed clonorchis sinensis glutathione transferase 2 of genetic engineering.
Preferably, the formula of sample diluting liquid is: 0.1%BSA (0.1g/100ml)+0.05% Tween-20 (v/v)+PBS (PH7.2).Further, antiseptic is also contained in sample diluting liquid as thimerosal 0.01% (0.01g/100ml).The formula of concentrated cleaning solution is: the Tris-HCl damping fluid of pH7.8.Negative control can be: the non-Endemic Area of clonorchiasis, and without eating sashimi (raw fish) history, empirical tests does not suffer from the normal human serum of clonorchiasis; Positive control can be: clonorchiasis Endemic Area, eats sashimi (raw fish) history, and empirical tests suffers from the patients serum of clonorchiasis.
The using method of clonorchiasis diagnostic kit of the present invention, comprises the following steps:
1). dosing: concentrated cleaning solution dilution is mixed with cleansing solution;
2). application of sample: add in the respective aperture of ELISA reaction plate respectively testing sample or positive and negative contrast and dilute with sample diluting liquid;
3). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding;
4). add enzyme marker with hole every after cleansing solution washing;
5). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding;
6). with cleansing solution washing, pat dry after rear every hole adds substrate A, the mixing of B liquid, the colour developing of 37 DEG C of lucifuges;
7). measure: every hole measures each hole OD value after adding stop buffer mixing.
Clonorchiasis diagnostic kit of the present invention is using glutathione transferase 2 as diagnostic antigen, employing enzyme linked immunosorbent assay (indirect method) detects the IgG4 antibody in clonorchis sinensis patients serum, and the incubation time of one antiserum and two antienzyme labels respectively needs 30 minutes.
Accompanying drawing explanation
Product-the time curve of different volumes restructuring GST2 is added in the catalytic activity detection of figure-1 recombinant protein GST2
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that example is not for limiting the scope of the invention.
Embodiment 1: the clone of glutathione transferase 2
By open reading frame (ORF) complete in the ORF FINDER program looks Unigene of NCBI, whether be full-length gene with BLASTx programmed decision, full-length gene is wherein known or new gene, the maximum ORF being numbered c003el0a is 639bp, we by it referred to as GST2.
1) the genomic extraction of clonorchis sinensis:
From cat liver, collect 10 clonorchis sinensis adults, PBS centrifugally removes supernatant after cleaning.Add the lysis buffer (STE, containing 2%SDS, 0.2mg/ml, Proteinase K) of 10 times of volumes after homogenate, mixing, 37 DEG C of water-baths are spent the night.Next day, solution is put to room temperature, add equal-volume balance phenol, on, under put upside down about 10min gently, the centrifugal 15min of room temperature 10000rpm, get upper strata aqueous phase, repeat extracting 2 times, get upper strata aqueous phase, add the 3mol/L sodium acetate (pH5.2) of 0.1 times of volume and the absolute ethyl alcohol of 2 times of volumes, mixing, the centrifugal 10min of 12000rpm, abandon supernatant, 70% ethanol washes twice, the centrifugal 5min of 12000rpm, abandon supernatant, after ethanol volatilization to the greatest extent, add 30 μ l TE (10mmol/LTris-HCI, 1mmol/L EDTA, pH8.0) dissolve, get 5ul 1% agarose gel analysis, remaining-20 DEG C, sample saves backup.
2) preparation of competent cell:
From culture plate, the mono-bacterium colony of picking BL21/DE3 enters in 5ml LB nutrient solution, and 37 DEG C 250 revs/min (rpm) joltings are spent the night.Getting 150ul next day adds in 3ml LB nutrient solution, and 37 DEG C of 250rpm joltings are to A
600=0.6.Take out bacterium liquid, ice bath 30min, 4 DEG C, the centrifugal 5min of 4000rpm, abandons supernatant, adds the lime chloride 750 μ l of 0.1mol/L, ice bath 30min, 4 DEG C, the centrifugal 5min of 4000rpm, abandons supernatant, add the lime chloride 400 μ l of 0.1mol/L, packing 100 μ l/ manages, 4 DEG C preserve in 24h with or often pipe add-70 DEG C of preservations after 30% sterile glycerol mixing, use in 3 months.
3) amplification of GST2 gene and clone:
The amplification of 1.1 genes, the structure of prokaryotic expression plasmid and qualification
Respectively with Library plasmid cDNA and genomic DNA for template,
Upstream primer P1 is 5 ' GCGAATTCCACATGAAACACAGACACTGTG3 ', introduces two protectiveness bases and EcoR I restriction enzyme site;
Downstream primer P2 is 5 ' CGGTCGACGTTAATCGTCGCCACAGTC3 ', introduces two protectiveness bases and Sal I restriction enzyme site.
With TaKaRa Ex Taq
tMthe reaction conditions of this gene of enzymatic amplification is as follows: 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 61 DEG C of annealing 50sec, and 72 DEG C extend 1min, totally 30 circulations, and last 72 DEG C extend 8min.The agarose gel electrophoresis analysis verification of PCR primer 12g/L, obtaining molecular weight is the fragment of 639bp.PCR primer reclaims, and carries out EcoR I and Sal I double digestion, connects 18h with the prokaryotic expression carrier pET-30a (+) carrying out same double digestion by thing mass ratio 3-4:1 T4DNA ligase in 16 DEG C.Get 100 μ l BL21/DE3 competent cells, add above-mentioned connection product, gently ice bath 1h after mixing, 42 DEG C of water-bath 90sec, ice bath 5min, add LB nutrient solution 400 μ l, put into 37 DEG C of water-bath 2min after mixing, 37 DEG C of 150rpm shake 1h.All bacterium liquid paving to through preheating containing in the LB flat board of kanamycins, be inverted at 37 DEG C of incubators and cultivate 12-16h.Bacterium liquid containing positive plasmid is frozen for subsequent use with 30% sterile glycerol-70 DEG C.
Qualification: sequencing result shows, the gene order being cloned into the clonorchis sinensis glutathione transferase 2 of pET-30a (+) is SEQ ID NO.2.
AATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCG
GTTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGA
CGACAAGGCCATGGCTGATATCGGATCCGAATTCCACATGAAACACAGACACTGTGCGCTATTCTATTTCAACGTCCGT
GGCAGAGCTGAGGCGATTAGGATGGTACTGCACGCTAACGATGTTTCGTTTGAGGATGTGCGTTTCGATAAGGACCAAT
GGTCTGAACGCAAACACGAGTTTCCGGGTGGTAAATTACCGCTTCTCCAAGTAAGAGAAGAGGGATCACAAGAGAAGAA
GACTTATACAGAGAGCATGGCGATCGCCCGAGTGCTCGCAAAACACTACAGTATGATGGGCGATTCTGAAGAGGAATAT
TACAAGATTGAACGAATGATTGGGCAGTGTGCCGATTTGGATAAGGAGTTTGTCAATGTCTTTTTCGCACGAGAAGACC
AAAAGAAAGAAGTACTTGAGAAAGCAATGGGTGGAGAGGTGCCGAGACTACTGGAACTTATTTGCAAATCACTCTCTGA
ATCTGGTGGCAAGTTCGTCGCTGGTAACAAAGTAACTCTTGGAGACATATGCCTTATGGCATCCATGGAAAATGTACGG
AGAGCGGACCCTCAGCTTTTGAAAACGAAATATTCGACATTATTGGCCCTCGAGGCGGAGGTGTTCAAGGTCCTGCCGA
AACTTGCGGACTACGTCAAAACACGACCAGAAACCGTCCTGTGAAGCAGACTGTGGCGACGATTAACGTCGACAAGCTT
GCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGT
1.2CsGST2 gene is at the abduction delivering of e. coli bl21/DE3
With the BL21/DE3 containing empty plasmid pET-30a (+) for negative control.BL21/DE3 containing pET-30a (+)-CsGST2 of recombinant plasmid is inoculated in the nutrient culture media containing kanamycins, 37 DEG C, 250rpm, and shaken cultivation is transferred by 10ml/L after spending the night, and at 37 DEG C, 250rpm, shaken cultivation is to A
600=0.6, take out 1ml as pre-induction sample, all the other bacterium liquid add IPTG (final concentration is 1mmol/L) Fiber differentiation 4-5h, take out 1ml as postinduction sample.All samples 4 DEG C, 8000rpm, 15min collected after centrifugation thalline, a little precipitation adds 5 μ l DTT, 95 μ l 1 × SDS, 30 μ l resets and add 2 μ l DTT, 8 μ l 4 × SDS, resuspended mixing, the centrifugal 1min of boiling water bath 5min, precipitation 13000rpm, supernatant is crawl slightly, get the capable SDS-PAGE of upper liquid 10 μ l (separation gel 150g/L, concentrated glue 50g/L), with coomassie brilliant blue staining, after observation has expression, albumen has expression in supernatant, without expressing in precipitation, carries out great expression and purifying.
1.3CsGST2 gene is at a large amount of abduction deliverings of e. coli bl21/DE3
37 DEG C, 250rpm, the bacterium liquid that shaken cultivation is spent the night is forwarded in 1000ml LB nutrient solution in the ratio of 20ml/L, and 37 DEG C, 250rpm, shaken cultivation is to A
600=0.6, add IPTG (final concentration is 1mmol/L) Fiber differentiation 4-5h, with 4 DEG C, after 8000rpm, 15min are centrifugal, abandon most supernatant nutrient solution, collect thalline, add 1 × binding buffer liquid (5mM imidazoles+0.5M NaCl+20mM Tris-HCl, PH7.9) the resuspended mixing of 12-15ml complete, collect, be stored in-20 DEG C, next day ultrasonic degradation bacterium (power 150W, continue 1sec, stop 2sec, 15min altogether), 4 DEG C, 13000rpm, 15min, collect supernatant, 0.45 μm of filtering with microporous membrane.
1.4 affinitive layer purification albumen
Ni-NTA resin, the wash-out capable SDS-PAGE of GST2 albumen is out verified as destination protein by gradient concentration imidazoles 20mM, 30mM, 40mM, 60mM, 200mM and 400mM imidazoles, get purity high, measure large being collected together, (dislysate is 1 × PBS to load bag filter, PH7.4), 4 DEG C of dialysis 24h (changing liquid 2-3 time).Solution after dialysis 0.22 μm of filtering with microporous membrane, packing ,-80 DEG C save backup.
Qualification: protein sequencing result shows, the sequence that purifying obtains albumen (recombinant protein GST2) is SEQ ID NO.1.
MKHRHCALFYFNVRGRAEAIRMVLHANDVSFEDVRFDKDQWSERKHEFPGGKLPLLQVREEGSQEKKTYTESMAI
ARVLAKHYSMMGDSEEEYYKIERMIGQCADLDKEFVNVFFAREDQKKEVLEKAMGGEVPRLLELICKSLSESGGK
FVAGNKVTLGDICLMASMENVRRADPQLLKTKYSTLLALEAEVFKVLPKLADYVKTRPETVL。
1.5 glutathione transferase 2 determinations of activity
The research of recombinant protein catalytic activity
1). recombinant protein GST2's is quantitative:
Use Bradford method, be that standard is (see Bradford MM.A rapid and sensitivemethod for the quantitation of protein utilizing the principle of protein-dye binding.AnalytBiochem with bovine serum albumin(BSA), 1976,72:248-254).
2). the preparation of reagent:
CDNB absolute ethyl alcohol is mixed with 20mmol/L solution.GSH water is mixed with 20mmol/L solution.
3). reaction system is:
0.1mol/L sodium phosphate buffer (pH 6.5), GSH, CDNB, recombinant protein, reaction volume is 3ml, temperature of reaction is 25 DEG C (when adding the reagent of absolute ethyl alcohol preparation, should notice that the volume of absolute ethyl alcohol is no more than 1/4th of reaction cumulative volume) (see Habig WH, Pabst MJ, Jacoby WB.Glutathione S-transferase:the firstenzymatic mercapturic acid formation.J Biol Chem, 1974,249:7130-7139.).Blank 0.1mol/L sodium phosphate buffer, all incubation reaction do three pipes, average.When surveying maximum reaction velocity, make negative control with the reaction system only not adding recombinant protein, make positive control with the thick enzyme of clonorchis sinensis adult.With being first added with damping fluid, the reaction system (final concentration of GSH and CDNB is 1mmol/L) of GSH and CDNB hatches 5min in 25 DEG C of water-baths, adds different volumes recombinant protein and starts reaction, measure the A of 1-5min
340.After selected suitable recombinant protein amount, negative control is made by the reaction system only not adding CDNB, the reaction system being added with GSH (final concentration 1mmol/L), recombinant protein and damping fluid is hatched 5min in 25 DEG C of water-baths, add variable concentrations CDNB and start reaction, survey the A at 25 DEG C of reaction 5min
340, according to the mM extinction coefficient Δ e=9.6Lmmol of the product S-sweet peptide of (2,4-dinitro benzene) paddy skin (P)
-1cm
-1calculate the active unit of enzyme (see Habig WH, Pabst MJ, Jacoby WB.GlutathioneS-transferase:the first enzymatic mercapturic acid formation.J Biol Chem, 1974,249:7130-7139.).Michaelis constant Km according to Michaelis-Menten equation statistic software SPSS 13.0 using the inverse of concentration of substrate as independent variable, the inverse of speed carries out linear regression as dependent variable and calculates and obtain.
4). the catalytic activity of recombinant protein GST2
In 1-5min, add the recombinant protein GST2 of 1,5,10 μ l, generate product amount different, as shown in figure-1, react 5min when adding 5 μ l or 10 μ l GST2, the amount of product prolongation no longer in time and increasing, the active unit of GST2 is 22.76 ± 0.096 μm of olmg
-1min
-1.5 μ l GST2 react 5min A at 25 DEG C of catalysis variable concentrations CDNB
340rising value in Table-1, the average Km that the same process data obtain GST2 is 111 μm of ol.
The A of 5 μ l GST2 catalysis variable concentrations CDNB
340
Table-1
Embodiment 2: the physicochemical property of glutathione transferase 2
The glutathione transferase 2 of the affinitive layer purification that embodiment 1 is obtained, after enterokinase enzyme excision carrier sequence, through SDS-PAGE electrophoresis, and standard molecule discharge curve, record its molecular weight and be about 30kDa; Utilize Amerham isoelectric focusing electrophoresis instrument, through the isoelectric focusing electrophoresis of pH3-10 and pH7-10 gradient fixing glue, recording its isoelectric point is 6.97.The Stability Analysis of Structures of protein.
Embodiment 3: prepare diagnostic kit
The detection of clonorchis sinensis antibody before this adopts crude antigen or the antigen coated reaction plate of secretion excretion more, susceptibility, specificity are all not satisfactory, and the genetic engineering recombinant antigen GST2 that the present invention adopts embodiment 1 to obtain wraps by reaction plate, detect the IgG4 antibody in clonorchis sinensis patient body with indirect elisa method.
The assembling of kit:
1. the preparation (96 holes/plate) of reaction plate:
The clonorchis sinensis glutathione transferase-2 (GST2) of purifying embodiment 1 obtained is buffered liquid (pH9.6) with carbonate bag and is made into 5 μ g/ml, 96 hole reaction plates, every hole adds 100 μ l, wrap by rear room temperature yawing 30 minutes, 4 DEG C are spent the night, morning next day room temperature yawing after 30 minutes again, outwell coating buffer, pat dry, 0.5%BSA (w/v)+3% trehalose (w/v)+PBS (PH7.2) closes, 200 μ l/ holes, 4 DEG C close spend the night after outwell confining liquid.The liquid of falling deblocking, pats dry, and drains aluminium-foil paper and packages spare.
2. sample diluting liquid (11ml/ bottle):
0.1%BSA (w/v)+0.05% Tween-20 (v/v)+PBS (PH7.2), adds thimerosal 0.01% (w/v), 0.22 μm of membrane filtration.
3. blood sample positive and negative control treatment (0.10ml/ bottle):
Positive control:
Feces of Patients confirms there is worm's ovum through microscopy (excrement three is examined), and get venous patient whole blood room temperature static 2 hours, 4 DEG C are spent the night, 4000rpm, centrifugal 10 minutes, is separated supernatant-20 DEG C and saves backup, through the serum of ELISA experimental verification OD value 1.9-2.0;
Negative control:
Ight soil finds no worm's ovum through microscopy (excrement three is examined), static 2 hours of venous whole room temperature, and 4 DEG C are spent the night, 4000rpm, centrifugal 10 minutes, is separated supernatant-20 DEG C and saves backup, and through the serum of ELISA experimental verification OD value about 0.1.
Positive and negative contrast adds thimerosal 0.01% (w/v), and once, every bottle of 0.10ml packing is for subsequent use for 0.22 μm of membrane filtration.
4. enzyme marker thing (11ml/ bottle):
Anti-1: 1500 dilution of horseradish peroxidase-labeled anti-human igg 4 two, the packing of 11ml/ bottle.
5. concentrated cleaning solution (30ml/ bottle, 25 ×):
Tris-HCl damping fluid (PH7.8), with 0.22 μm of membrane filtration, used time adding distil water or deionized water do 25 times of dilutions.
6. stop buffer (6ml/ bottle):
2M H
2SO
4。
7. substrate solution A and substrate solution B all has the complete sale of commodity.
8. shrouding film:
3.
9. instructions:
1 part.
Embodiment 4: the use of kit
4.1 kits use step:
1). dosing: concentrated cleaning solution distilled water or deionized water are done 1: 25 dilution.
2). application of sample: add testing sample respectively or positive and negative contrast 5 μ l in respective aperture, then every hole adds 95 μ l sample diluting liquids.
3). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding.
4). washing: rinse 4 times with cleansing solution, pat dry.
5). enzyme-added: every hole adds enzyme marker 100 μ l.
6). hatch: hatch 30 minutes with rearmounted 37 DEG C of shrouding film shrouding.
7). washing: rinse 6 times with cleansing solution, pat dry.
8). colour developing: every hole adds substrate A, each 50 μ l of B liquid, and mixing of vibrating gently, 37 DEG C of lucifuges develop the color 10 minutes.
9). measure: every hole adds 50 μ l stop buffers, pats mixing.Setting microplate reader ripple is longer than 450nm place (suggestion uses double UV check), measures each hole OD value.
Serum dilution is 1: 20, two dilutabilitys resisted of enzyme labeling are 1: 1500, the incubation time of one antiserum and two antienzyme labels respectively needs 30 minutes, comparatively, the similar reaction time shortens half, improve efficiency, reaction conditions easily realizes (37 DEG C, leave standstill), this kit is operated more convenient.
4.2 kit sensitivity Detection tests:
Get 176 parts of microscopy positive serums, adopt mentioned reagent box to detect, final ELISA result is positive 161 parts, and positive rate is 91.50%.
4.3 kit specific detection tests:
Get 258 parts of negative serums, adopt mentioned reagent box to detect, ELISA result measures 233 parts of feminine genders, and negative recall rate is 90.31%.
Use kit described in the invention, method shown in by specification, carry out cross reaction experiment, result is as table-2:
Cross reaction experimental result (Results of cross-reaction)
Table-2
Conclusion: no matter kit of the present invention is that susceptibility or specificity are all better than other similar at present kits.Experimental result shows, this kit has high specificity, highly sensitive, accuracy high, the diagnosis of clonorchiasis can be applied to preferably, the main agents of this kit all adopts working fluid form, can directly use, more traditional etiological diagnosis method has very large advantage: operate accurate, easy, credible result.
Claims (7)
1. a clonorchiasis diagnostic kit, comprise: ELISA reaction plate, negative control, positive control, enzyme marker, substrate A liquid, substrate B liquid, sample diluting liquid, concentrated cleaning solution and stop buffer, it is characterized in that, described ELISA reaction plate is coated with clonorchis sinensis glutathione transferase 2, described clonorchis sinensis glutathione transferase 2 is polypeptide of the amino acid sequence with SEQ ID NO.1, and described enzyme marker is that horseradish peroxidase-labeled anti-human igg 4 two resists.
2. clonorchiasis diagnostic kit as claimed in claim 1, is characterized in that, described sample diluting liquid by percent weight in volume be 0.1%BSA, percent by volume is that the PBS mixed preparing of 0.05% Tween-20 and pH7.2 forms.
3. clonorchiasis diagnostic kit as claimed in claim 2, is characterized in that, also containing antiseptic in described sample diluting liquid.
4. clonorchiasis diagnostic kit as claimed in claim 1, it is characterized in that, described concentrated cleaning solution is the Tris-HCl damping fluid of pH7.8.
5. clonorchiasis diagnostic kit as described in as arbitrary in claim 1-4, is characterized in that, also comprise shrouding film and instructions in described clonorchiasis diagnostic kit.
6. as described in as arbitrary in claim 1-5, the using method of clonorchiasis diagnostic kit, comprises the following steps:
1). by concentrated cleaning solution dilution preparation cleansing solution;
2). add in the respective aperture of ELISA reaction plate respectively testing sample or positive and negative contrast and dilute with sample diluting liquid;
3). hatch with rearmounted 37 DEG C of shrouding film shrouding;
4). add enzyme marker with every hole after cleansing solution washing ELISA reaction plate;
5). hatch with rearmounted 37 DEG C of shrouding film shrouding;
6). add substrate A, the mixing of B liquid, 37 DEG C of lucifuge colour developings with every hole after cleansing solution washing ELISA reaction plate;
7). every hole measures each hole OD value after adding stop buffer mixing.
7. the using method of clonorchiasis diagnostic kit as claimed in claim 6, it is characterized in that, the incubation time in step 3 and 5 only needs 30 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010619492.4A CN102565398B (en) | 2010-12-31 | 2010-12-31 | Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010619492.4A CN102565398B (en) | 2010-12-31 | 2010-12-31 | Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102565398A CN102565398A (en) | 2012-07-11 |
| CN102565398B true CN102565398B (en) | 2015-06-24 |
Family
ID=46411355
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010619492.4A Expired - Fee Related CN102565398B (en) | 2010-12-31 | 2010-12-31 | Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102565398B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830231A (en) * | 2012-09-13 | 2012-12-19 | 江苏省血吸虫病防治研究所 | Liver fluke antibody IgG4 biotin-avidin enzyme linked immunodetection kit and detection method thereof |
| CN105510575A (en) * | 2015-12-04 | 2016-04-20 | 深圳市伯劳特生物制品有限公司 | Anti-phospholipase A2 acceptor antibody IgG kit and detection method |
| CN105807070B (en) * | 2016-06-08 | 2018-03-09 | 广西中医药大学附属瑞康医院 | A kind of kit for detecting hepatitis B |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384360A (en) * | 2001-05-02 | 2002-12-11 | 新丰制药株式会社 | Enzyme-linked immunosorbent assay kit for diagnosis of testicular trematodiasis, paragonimiasis, pork-measles disease and sparganosis |
| CN1670202A (en) * | 2005-03-11 | 2005-09-21 | 中山大学 | Microsome GST-1 of clonorchis sinensis, its coding acid and application |
| CN101206222A (en) * | 2006-12-18 | 2008-06-25 | 深圳市康百得生物科技有限公司 | Method and box for quick enzyme immunity detecting food-borne parasitic disease |
-
2010
- 2010-12-31 CN CN201010619492.4A patent/CN102565398B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384360A (en) * | 2001-05-02 | 2002-12-11 | 新丰制药株式会社 | Enzyme-linked immunosorbent assay kit for diagnosis of testicular trematodiasis, paragonimiasis, pork-measles disease and sparganosis |
| MXPA02004417A (en) * | 2001-05-02 | 2004-07-16 | Shin Poong Pharmaceutical Co | Elisa kit for diagnosis of clonorchiasis, paragonimiasis, cysticercosis and sparganosis. |
| CN1670202A (en) * | 2005-03-11 | 2005-09-21 | 中山大学 | Microsome GST-1 of clonorchis sinensis, its coding acid and application |
| CN101206222A (en) * | 2006-12-18 | 2008-06-25 | 深圳市康百得生物科技有限公司 | Method and box for quick enzyme immunity detecting food-borne parasitic disease |
Non-Patent Citations (4)
| Title |
|---|
| ABC-ELISA 法检测华支睾吸虫特异性IgG及其亚类;裴福全等;《中国寄生虫病防治杂志》;20040430;第17卷(第2期);第100页 * |
| Clonorchis sinensis: glutathione S-transferase as a serodiagnostic antigen for detecting IgG and IgE antibodies;Sung-Jong Hong et al;《Experimental Parasitology》;20020831;第101卷(第4期);第231－233页 * |
| Molecular cloning and characterization of cDNA encoding a novel cytosolic glutathione transferase from Clonorchis sinensis;Zhongluan Wu et al;《Parasitol Res》;20060117;第98卷(第6期);第534、535、537页 * |
| 华支睾吸虫GST的新编码基因的克隆与纯化;伍忠銮等;《中国公共卫生》;20040630;第20卷(第6期);第668–669页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102565398A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Functional expression and characterization of Echinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage | |
| Becker-Hapak et al. | TAT-mediated protein transduction into mammalian cells | |
| Goldberg et al. | Subunit Composition of the Peripherin/rds− Rom-1 Disk Rim Complex from Rod Photoreceptors: Hydrodynamic Evidence for a Tetrameric Quaternary Structure | |
| Hamamoto et al. | Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B | |
| Panner et al. | mTOR controls FLIPS translation and TRAIL sensitivity in glioblastoma multiforme cells | |
| US20200239558A1 (en) | Compositions, Devices, Kits and Methods for Detecting Hookworm | |
| Wang et al. | Intraflagellar transport (IFT) protein IFT25 is a phosphoprotein component of IFT complex B and physically interacts with IFT27 in Chlamydomonas | |
| Fujita et al. | Telmisartan, an angiotensin II type 1 receptor blocker, controls progress of nonalcoholic steatohepatitis in rats | |
| ES2315434T3 (en) | PROCEDURE FOR THE EARLY DETECTION OF THE SEROCONVERSION OF AN ANTIBODY AGAINST THE VIRUS OF HEPATITIS C. | |
| ES2369544T3 (en) | PROCEDURE FOR THE DETECTION OF ANTIBODIES IN BODY FLUIDS THROUGH AN IMMUNE REACTION WITH GLYCOPROTEIN 2 (GP2) OF GRANULES OF ZIMOGENO OF THE PANCREAS FOR THE DIFFERENTIAL DIAGNOSIS OF INFLAMMATORY INFLAMMATORY DISEASES. | |
| EP3383887B1 (en) | Mutated hev polypeptides and the use thereof for assaying anti-hev antibodies | |
| Marinelli et al. | Expression of aquaporin-4 water channels in rat cholangiocytes | |
| Rouhani et al. | Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient’s sera: a novel hypothesis to follow-up of Cystic echinococcosis after surgery | |
| CN105209616A (en) | HCV NS3 recombinant antigens and mutants thereof for improved antibody detection | |
| CN102565398B (en) | Clonorchiasis ELISA (Enzyme-Linked Immuno Sorbent Assay) diagnostic reagent kit | |
| Parvin et al. | Expression and localization of AQP5 in the stomach and duodenum of the rat | |
| KR101528169B1 (en) | Method and kit for detection of anti-avibacterium paragallinarum antibody | |
| Johnson et al. | The carboxyl terminus of the ε subunit of the chloroplast ATP synthase is exposed during illumination | |
| KR20120001741A (en) | Method for detecting substance in biological sample | |
| TW200427463A (en) | Compositions and methods for the detection and treatment of methylthioadenosine phosphorylase deficient cancers | |
| Lin et al. | Sm22. 6 antigen is an inhibitor to human thrombin | |
| CN102539781B (en) | Clonorchiasis TRFIA diagnostic kit and use thereof | |
| JP5109114B2 (en) | New detection method for fish antibody production | |
| WO1992009634A1 (en) | Non-a non-b hepatitis virus antigen protein | |
| Wu et al. | Molecular cloning, expression, and characterization of cyclophilin A from Clonorchis sinensis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: FOSHAN SHUNDE ZHIYAN BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: ZHONGSHAN UNIVERSITY Effective date: 20130123 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 510275 GUANGZHOU, GUANGDONG PROVINCE TO: 529311 FOSHAN, GUANGDONG PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20130123 Address after: 529311, Foshan Town, Shunde District, Guangdong City, Beijiao province Beijiao neighborhood committee, Penglai Road Industrial Road, Midea science and technology incubator, B building, two floor, B211 Applicant after: Foshan Shunde Zhiyan Biotechnology Co., Ltd. Address before: 510275 Xingang West Road, Guangdong, China, No. 135, No. Applicant before: Sun Yat-sen University |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150624 Termination date: 20171231 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |