CN112457397A - 一种prrsv n蛋白的纳米抗体及其制备方法和应用 - Google Patents
一种prrsv n蛋白的纳米抗体及其制备方法和应用 Download PDFInfo
- Publication number
- CN112457397A CN112457397A CN202011346127.0A CN202011346127A CN112457397A CN 112457397 A CN112457397 A CN 112457397A CN 202011346127 A CN202011346127 A CN 202011346127A CN 112457397 A CN112457397 A CN 112457397A
- Authority
- CN
- China
- Prior art keywords
- prrsv
- protein
- fusion protein
- antibody
- hrp
- 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
- 101710141454 Nucleoprotein Proteins 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 title claims abstract 10
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 33
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 33
- 210000002966 serum Anatomy 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000011161 development Methods 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 238000002965 ELISA Methods 0.000 claims description 47
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 19
- 108010001336 Horseradish Peroxidase Proteins 0.000 abstract description 10
- 230000014509 gene expression Effects 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 230000002860 competitive effect Effects 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 241000282887 Suidae Species 0.000 description 10
- 239000000427 antigen Substances 0.000 description 10
- 241000282898 Sus scrofa Species 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 241000711404 Avian avulavirus 1 Species 0.000 description 5
- 238000008157 ELISA kit Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 101100309714 Arabidopsis thaliana SD16 gene Proteins 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 241000282828 Camelus bactrianus Species 0.000 description 2
- 241000282836 Camelus dromedarius Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 2
- -1 ORF1b Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 101000621943 Acholeplasma phage L2 Probable integrase/recombinase Proteins 0.000 description 1
- 101000768957 Acholeplasma phage L2 Uncharacterized 37.2 kDa protein Proteins 0.000 description 1
- 101000823746 Acidianus ambivalens Uncharacterized 17.7 kDa protein in bps2 3'region Proteins 0.000 description 1
- 101000916369 Acidianus ambivalens Uncharacterized protein in sor 5'region Proteins 0.000 description 1
- 101000977065 Acidithiobacillus ferridurans Uncharacterized 11.6 kDa protein in mobS 3'region Proteins 0.000 description 1
- 101000769342 Acinetobacter guillouiae Uncharacterized protein in rpoN-murA intergenic region Proteins 0.000 description 1
- 101000823696 Actinobacillus pleuropneumoniae Uncharacterized glycosyltransferase in aroQ 3'region Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101000786513 Agrobacterium tumefaciens (strain 15955) Uncharacterized protein outside the virF region Proteins 0.000 description 1
- AJBVYEYZVYPFCF-CIUDSAMLSA-N Ala-Lys-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O AJBVYEYZVYPFCF-CIUDSAMLSA-N 0.000 description 1
- DYXOFPBJBAHWFY-JBDRJPRFSA-N Ala-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N DYXOFPBJBAHWFY-JBDRJPRFSA-N 0.000 description 1
- 101000618005 Alkalihalobacillus pseudofirmus (strain ATCC BAA-2126 / JCM 17055 / OF4) Uncharacterized protein BpOF4_00885 Proteins 0.000 description 1
- 101000618348 Allochromatium vinosum (strain ATCC 17899 / DSM 180 / NBRC 103801 / NCIMB 10441 / D) Uncharacterized protein Alvin_0065 Proteins 0.000 description 1
- 102100020724 Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Human genes 0.000 description 1
- YNSGXDWWPCGGQS-YUMQZZPRSA-N Arg-Gly-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O YNSGXDWWPCGGQS-YUMQZZPRSA-N 0.000 description 1
- DQTIWTULBGLJBL-DCAQKATOSA-N Asn-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)N)N DQTIWTULBGLJBL-DCAQKATOSA-N 0.000 description 1
- SPWXXPFDTMYTRI-IUKAMOBKSA-N Asp-Ile-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SPWXXPFDTMYTRI-IUKAMOBKSA-N 0.000 description 1
- RTXQQDVBACBSCW-CFMVVWHZSA-N Asp-Ile-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RTXQQDVBACBSCW-CFMVVWHZSA-N 0.000 description 1
- JSHWXQIZOCVWIA-ZKWXMUAHSA-N Asp-Ser-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JSHWXQIZOCVWIA-ZKWXMUAHSA-N 0.000 description 1
- MNQMTYSEKZHIDF-GCJQMDKQSA-N Asp-Thr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O MNQMTYSEKZHIDF-GCJQMDKQSA-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
- 101000781117 Autographa californica nuclear polyhedrosis virus Uncharacterized 12.4 kDa protein in CTL-LEF2 intergenic region Proteins 0.000 description 1
- 101000967489 Azorhizobium caulinodans (strain ATCC 43989 / DSM 5975 / JCM 20966 / LMG 6465 / NBRC 14845 / NCIMB 13405 / ORS 571) Uncharacterized protein AZC_3924 Proteins 0.000 description 1
- 101000708323 Azospirillum brasilense Uncharacterized 28.8 kDa protein in nifR3-like 5'region Proteins 0.000 description 1
- 101000770311 Azotobacter chroococcum mcd 1 Uncharacterized 19.8 kDa protein in nifW 5'region Proteins 0.000 description 1
- 101000823761 Bacillus licheniformis Uncharacterized 9.4 kDa protein in flaL 3'region Proteins 0.000 description 1
- 101000819719 Bacillus methanolicus Uncharacterized N-acetyltransferase in lysA 3'region Proteins 0.000 description 1
- 101000789586 Bacillus subtilis (strain 168) UPF0702 transmembrane protein YkjA Proteins 0.000 description 1
- 101000748761 Bacillus subtilis (strain 168) Uncharacterized MFS-type transporter YcxA Proteins 0.000 description 1
- 101000792624 Bacillus subtilis (strain 168) Uncharacterized protein YbxH Proteins 0.000 description 1
- 101000790792 Bacillus subtilis (strain 168) Uncharacterized protein YckC Proteins 0.000 description 1
- 101000765620 Bacillus subtilis (strain 168) Uncharacterized protein YlxP Proteins 0.000 description 1
- 101000819705 Bacillus subtilis (strain 168) Uncharacterized protein YlxR Proteins 0.000 description 1
- 101000916134 Bacillus subtilis (strain 168) Uncharacterized protein YqxJ Proteins 0.000 description 1
- 101000948218 Bacillus subtilis (strain 168) Uncharacterized protein YtxJ Proteins 0.000 description 1
- 101000718627 Bacillus thuringiensis subsp. kurstaki Putative RNA polymerase sigma-G factor Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101000641200 Bombyx mori densovirus Putative non-structural protein Proteins 0.000 description 1
- 101000754349 Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) UPF0065 protein BP0148 Proteins 0.000 description 1
- 101000827633 Caldicellulosiruptor sp. (strain Rt8B.4) Uncharacterized 23.9 kDa protein in xynA 3'region Proteins 0.000 description 1
- 241000712083 Canine morbillivirus Species 0.000 description 1
- 101000947628 Claviceps purpurea Uncharacterized 11.8 kDa protein Proteins 0.000 description 1
- 101000947633 Claviceps purpurea Uncharacterized 13.8 kDa protein Proteins 0.000 description 1
- 101000686796 Clostridium perfringens Replication protein Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102100031725 Cortactin-binding protein 2 Human genes 0.000 description 1
- 101000861180 Cupriavidus necator (strain ATCC 17699 / DSM 428 / KCTC 22496 / NCIMB 10442 / H16 / Stanier 337) Uncharacterized protein H16_B0147 Proteins 0.000 description 1
- CMYVIUWVYHOLRD-ZLUOBGJFSA-N Cys-Ser-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CMYVIUWVYHOLRD-ZLUOBGJFSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101000948901 Enterobacteria phage T4 Uncharacterized 16.0 kDa protein in segB-ipI intergenic region Proteins 0.000 description 1
- 101000805958 Equine herpesvirus 4 (strain 1942) Virion protein US10 homolog Proteins 0.000 description 1
- 101000790442 Escherichia coli Insertion element IS2 uncharacterized 11.1 kDa protein Proteins 0.000 description 1
- 101000788129 Escherichia coli Uncharacterized protein in sul1 3'region Proteins 0.000 description 1
- 101000788370 Escherichia phage P2 Uncharacterized 12.9 kDa protein in GpA 3'region Proteins 0.000 description 1
- 101000788354 Escherichia phage P2 Uncharacterized 8.2 kDa protein in gpA 5'region Proteins 0.000 description 1
- 101000770304 Frankia alni UPF0460 protein in nifX-nifW intergenic region Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000797344 Geobacillus stearothermophilus Putative tRNA (cytidine(34)-2'-O)-methyltransferase Proteins 0.000 description 1
- 101000748410 Geobacillus stearothermophilus Uncharacterized protein in fumA 3'region Proteins 0.000 description 1
- 101000787096 Geobacillus stearothermophilus Uncharacterized protein in gldA 3'region Proteins 0.000 description 1
- SMLDOQHTOAAFJQ-WDSKDSINSA-N Gln-Gly-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SMLDOQHTOAAFJQ-WDSKDSINSA-N 0.000 description 1
- SYZZMPFLOLSMHL-XHNCKOQMSA-N Gln-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)C(=O)O SYZZMPFLOLSMHL-XHNCKOQMSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- DIXKFOPPGWKZLY-CIUDSAMLSA-N Glu-Arg-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O DIXKFOPPGWKZLY-CIUDSAMLSA-N 0.000 description 1
- KXTAGESXNQEZKB-DZKIICNBSA-N Glu-Phe-Val Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=CC=C1 KXTAGESXNQEZKB-DZKIICNBSA-N 0.000 description 1
- RFTVTKBHDXCEEX-WDSKDSINSA-N Glu-Ser-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RFTVTKBHDXCEEX-WDSKDSINSA-N 0.000 description 1
- KRRMJKMGWWXWDW-STQMWFEESA-N Gly-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KRRMJKMGWWXWDW-STQMWFEESA-N 0.000 description 1
- QSTLUOIOYLYLLF-WDSKDSINSA-N Gly-Asp-Glu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QSTLUOIOYLYLLF-WDSKDSINSA-N 0.000 description 1
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 101000772675 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) UPF0438 protein HI_0847 Proteins 0.000 description 1
- 101000631019 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Uncharacterized protein HI_0350 Proteins 0.000 description 1
- 101000976889 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 19.2 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 101000768938 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 8.9 kDa protein in int-C1 intergenic region Proteins 0.000 description 1
- 241000724675 Hepatitis E virus Species 0.000 description 1
- 101000785414 Homo sapiens Ankyrin repeat, SAM and basic leucine zipper domain-containing protein 1 Proteins 0.000 description 1
- 101000782488 Junonia coenia densovirus (isolate pBRJ/1990) Putative non-structural protein NS2 Proteins 0.000 description 1
- 101000827627 Klebsiella pneumoniae Putative low molecular weight protein-tyrosine-phosphatase Proteins 0.000 description 1
- 101000811523 Klebsiella pneumoniae Uncharacterized 55.8 kDa protein in cps region Proteins 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- 101000818409 Lactococcus lactis subsp. lactis Uncharacterized HTH-type transcriptional regulator in lacX 3'region Proteins 0.000 description 1
- 101000878851 Leptolyngbya boryana Putative Fe(2+) transport protein A Proteins 0.000 description 1
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 1
- VZBIUJURDLFFOE-IHRRRGAJSA-N Leu-His-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VZBIUJURDLFFOE-IHRRRGAJSA-N 0.000 description 1
- PDQDCFBVYXEFSD-SRVKXCTJSA-N Leu-Leu-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O PDQDCFBVYXEFSD-SRVKXCTJSA-N 0.000 description 1
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 1
- CIDICGYKRUTYLE-FXQIFTODSA-N Met-Ser-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CIDICGYKRUTYLE-FXQIFTODSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101000758828 Methanosarcina barkeri (strain Fusaro / DSM 804) Uncharacterized protein Mbar_A1602 Proteins 0.000 description 1
- 101001122401 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF3 Proteins 0.000 description 1
- 101001130841 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF5 Proteins 0.000 description 1
- 101001055788 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) Pentapeptide repeat protein MfpA Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101150001779 ORF1a gene Proteins 0.000 description 1
- 101150063292 ORF2a gene Proteins 0.000 description 1
- 101150102680 ORF2b gene Proteins 0.000 description 1
- 101710087110 ORF6 protein Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 101000740670 Orgyia pseudotsugata multicapsid polyhedrosis virus Protein C42 Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- KUSYCSMTTHSZOA-DZKIICNBSA-N Phe-Val-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N KUSYCSMTTHSZOA-DZKIICNBSA-N 0.000 description 1
- 101000769182 Photorhabdus luminescens Uncharacterized protein in pnp 3'region Proteins 0.000 description 1
- 241000202347 Porcine circovirus Species 0.000 description 1
- 241000702619 Porcine parvovirus Species 0.000 description 1
- 101710197985 Probable protein Rev Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101000961392 Pseudescherichia vulneris Uncharacterized 29.9 kDa protein in crtE 3'region Proteins 0.000 description 1
- 101000731030 Pseudomonas oleovorans Poly(3-hydroxyalkanoate) polymerase 2 Proteins 0.000 description 1
- 101001065485 Pseudomonas putida Probable fatty acid methyltransferase Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 101000711023 Rhizobium leguminosarum bv. trifolii Uncharacterized protein in tfuA 3'region Proteins 0.000 description 1
- 101000974028 Rhizobium leguminosarum bv. viciae (strain 3841) Putative cystathionine beta-lyase Proteins 0.000 description 1
- 101000756519 Rhodobacter capsulatus (strain ATCC BAA-309 / NBRC 16581 / SB1003) Uncharacterized protein RCAP_rcc00048 Proteins 0.000 description 1
- 101000948219 Rhodococcus erythropolis Uncharacterized 11.5 kDa protein in thcD 3'region Proteins 0.000 description 1
- 101000948156 Rhodococcus erythropolis Uncharacterized 47.3 kDa protein in thcA 5'region Proteins 0.000 description 1
- 101000917565 Rhodococcus fascians Uncharacterized 33.6 kDa protein in fasciation locus Proteins 0.000 description 1
- 101000790284 Saimiriine herpesvirus 2 (strain 488) Uncharacterized 9.5 kDa protein in DHFR 3'region Proteins 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- TUYBIWUZWJUZDD-ACZMJKKPSA-N Ser-Cys-Gln Chemical compound OC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCC(N)=O TUYBIWUZWJUZDD-ACZMJKKPSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 101000936719 Streptococcus gordonii Accessory Sec system protein Asp3 Proteins 0.000 description 1
- 101000936711 Streptococcus gordonii Accessory secretory protein Asp4 Proteins 0.000 description 1
- 101000929863 Streptomyces cinnamonensis Monensin polyketide synthase putative ketoacyl reductase Proteins 0.000 description 1
- 101000788499 Streptomyces coelicolor Uncharacterized oxidoreductase in mprA 5'region Proteins 0.000 description 1
- 101000788468 Streptomyces coelicolor Uncharacterized protein in mprR 3'region Proteins 0.000 description 1
- 101001102841 Streptomyces griseus Purine nucleoside phosphorylase ORF3 Proteins 0.000 description 1
- 101000708557 Streptomyces lincolnensis Uncharacterized 17.2 kDa protein in melC2-rnhH intergenic region Proteins 0.000 description 1
- 101000845085 Streptomyces violaceoruber Granaticin polyketide synthase putative ketoacyl reductase 1 Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 101000649826 Thermotoga neapolitana Putative anti-sigma factor antagonist TM1081 homolog Proteins 0.000 description 1
- 101000711771 Thiocystis violacea Uncharacterized 76.5 kDa protein in phbC 3'region Proteins 0.000 description 1
- DKDHTRVDOUZZTP-IFFSRLJSSA-N Thr-Gln-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DKDHTRVDOUZZTP-IFFSRLJSSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- NDXSOKGYKCGYKT-VEVYYDQMSA-N Thr-Pro-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O NDXSOKGYKCGYKT-VEVYYDQMSA-N 0.000 description 1
- LXXCHJKHJYRMIY-FQPOAREZSA-N Thr-Tyr-Ala 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](C)C(O)=O LXXCHJKHJYRMIY-FQPOAREZSA-N 0.000 description 1
- MNYNCKZAEIAONY-XGEHTFHBSA-N Thr-Val-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O MNYNCKZAEIAONY-XGEHTFHBSA-N 0.000 description 1
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 1
- QJIOKZXDGFZQJP-OYDLWJJNSA-N Trp-Trp-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QJIOKZXDGFZQJP-OYDLWJJNSA-N 0.000 description 1
- FFCRCJZJARTYCG-KKUMJFAQSA-N Tyr-Cys-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N)O FFCRCJZJARTYCG-KKUMJFAQSA-N 0.000 description 1
- 101710110895 Uncharacterized 7.3 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- 101710095001 Uncharacterized protein in nifU 5'region Proteins 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- 101000711318 Vibrio alginolyticus Uncharacterized 11.6 kDa protein in scrR 3'region Proteins 0.000 description 1
- 101000827562 Vibrio alginolyticus Uncharacterized protein in proC 3'region Proteins 0.000 description 1
- 101000778915 Vibrio parahaemolyticus serotype O3:K6 (strain RIMD 2210633) Uncharacterized membrane protein VP2115 Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 208000012153 swine disease Diseases 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/56983—Viruses
-
- 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
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开一种PRRSV N蛋白的纳米抗体及其制备方法和应用,属于生物技术领域,所述纳米抗体的氨基酸序列为序列表中的SEQ ID No.1。本发明还公开了所述纳米抗体与辣根过氧化物酶(horseradish peroxidase,HRP)融合蛋白的表达制备方法,同时评价了所述纳米抗体与HRP融合蛋白在检测猪血清中PRRSV抗体中的应用,本发明将所述抗PRRSV N蛋白纳米抗体与HRP融合蛋白应用于建立检测猪血清中PRRSV抗体的竞争ELISA中,建立的方法操作简单,检测样品耗时短,不需要使用酶标二抗。为后续将所述纳米抗体应用于PRRSV抗体检测的产品的开发提供了关键的材料。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种PRRSV N蛋白的纳米抗体及其制备方法应用。
背景技术
猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)俗称猪蓝耳病,是由猪繁殖与呼吸综合征病毒(PRRS virus,PRRSV)引起的猪的一种繁殖障碍与各年龄段呼吸道疾病为特征的烈性传染病,给全球养猪业带来巨大的经济损失。PRRSV是一种有囊膜的单股正链RNA病毒,包含10多个开放阅读框(ORF)ORF1a,ORF1b,ORF2a,ORF2b,ORF3,ORF4,ORF5,ORF5a,ORF6和ORF7。PRRSV病毒粒子中ORF7(核衣壳蛋白,N蛋白)含量高于其它结构蛋白成分,占病毒蛋白的20%-40%;不同毒株之间N蛋白氨基酸序列同源性达到96-100%,高度保守。由于PRRSV具有抗原变异性、嗜巨噬细胞性、抗体依赖性增强作用和持续性感染等特征,目前该病的致病机制尚不清楚,至今无理想的预防和控制措施,因此加强对该病的监测尤为重要。猪感染PRRSV后,针对N蛋白的抗体最早出现,感染后7天即可检测出N蛋白抗体,持续时间较长,并且该蛋白具有较强的免疫原性。因此,PRRSVN蛋白作为理想抗原。
在骆驼科动物体内存在一种天然缺失轻链和重链第一个恒定区的特殊IgG,被称作重链抗体(Heavy chain-only antibodies,HcAbs),其抗原结合片段由一个单独的结构域构成,即重链抗体可变区(Variable domains of Camellidae heavy chain-onlyantibodies,VHH),又称为纳米抗体(Nanobody,Nb)。纳米抗体具有分子量小(15kDa),高亲和力,高特异性,可以识别特殊的抗原表位且生产成本低等独特优点。此外,较于传统抗体,纳米抗体更易于进行基因工程编辑操作,可以和多种标记酶(如辣根过氧化物酶、生物素等)进行偶联。基于上述优点,纳米抗体在疾病诊断与治疗中更具优势。Zhu等筛选到针对H5N1病毒的特异性纳米抗体并建立了检测H5N1病毒的纳米抗体双抗夹心ELISA,该方法可检测到的H5N1病毒最低量为14.1ng/mL,其检测灵敏度远高于未标记纳米抗体;SigalGelkop等利用FMDV的3ABC蛋白特异性纳米抗体建立检测FMDV血清抗体的竞争ELISA,和传统的间接ELISA方法相比,具有特异性更强,灵敏度更高的优势。但是目前关于纳米抗体在PRRSV诊断方法中的应用却未见相关研究报道,也未有商品化产品。
目前为止,针对猪蓝耳病的血清学检测方法目前主要是间接ELISA,该方法需要纯度较高的抗原和酶标二抗,导致其商品化试剂盒生产成本高,操作繁琐,耗费时间长,阻碍了临床应用。例如,目前市场上使用较多的IDEXX的PRRSV抗体检测试剂盒,其检测效果好,但是由于其价格较高,很难在临床基层大范围推广使用。而基于纳米抗体分子量小和易于基因工程改造的优点,可以将纳米抗体与酶或荧光蛋白等进行融合表达,从而直接用于免疫学检测中,无需抗体标记和使用二抗,从而简化了生产工艺和大大降低了生产成本。因此,筛选和制备PRRSV抗原蛋白的纳米抗体,将其作为免疫学检测的材料,具有非常广阔的应用前景。
发明内容
本发明的目的是提供一种PRRSV N蛋白的纳米抗体及其制备方法应用,以解决上述现有技术存在的问题。
为实现上述目的,本发明提供一种PRRSV N蛋白的纳米抗体,命名为PRRSV-N-Nb1,氨基酸序列如SEQ ID No.1所示。
本发明还提供一种编码所述纳米抗体氨基酸序列的核苷酸分子,所述核苷酸分子的核苷酸序列如SEQ ID No.2所示。
本发明还提供一种含有所述的纳米抗体与HRP的融合蛋白。
本发明还提供一种所述的融合蛋白的制备方法,包括以下步骤:
(1)重组真核表达载体的构建:通过扩增、双酶切,将编码纳米抗体PRRSV-N-Nb1的基因序列连接至pEGFP-N1-HRP载体中获得阳性质粒;
(2)将步骤(1)中阳性质粒转化宿主细胞,诱导表达纳米抗体与HRP融合蛋白。
进一步的,所述宿主细胞为HEK 293T细胞。
本发明还提供一种所述的融合蛋白在制备检测猪血清中PRRSV抗体的产品中的应用。
进一步的,所述的融合蛋白在制备检测猪血清中PRRSV抗体的产品中的应用,包括以下步骤:
(1)将PRRSV N蛋白包被ELISA板;
(2)将所述融合蛋白与猪血清混合后加入步骤(1)的ELISA板中,孵育;
(3)在步骤(2)的ELISA板中加入显色液避光显色,加入3M浓H2SO4终止反应;
(4)观察ELISA板的颜色变化,若猪血清中有PRRSV抗体,则ELISA板内孔为无色;若猪血清中无PRRSV抗体,则ELISA板内孔为黄色。
本发明公开了以下技术效果:本发明提供了一种PRRSV N蛋白的纳米抗体PRRSV-N-Nb1,并公开了所述纳米抗体的氨基酸序列。同时提供了所述纳米抗体PRRSV-N-Nb1与HRP融合蛋白表达的制备方法,然后利用所述融合蛋白作为竞争探针,提供了一种检测猪血清中PRRSV抗体的竞争ELISA,该方法操作简单,仅需要混匀待检血清与融合蛋白,全程只需要30min即可显示结果;此外,该竞争ELISA,不需要标记和使用HRP标记的二抗,可以简化生产工艺,降低生产成本,具有很好的市场转化前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为ELISA检测纳米抗体Nb1与PRRSV N蛋白特异性结合;
图2为PRRSV-N-Nb1-HRP融合蛋白的表达及鉴定,其中A为IFA鉴定PRRSV-N-Nb1-HRP融合蛋白在HEK 293T细胞中的表达;B为ELISA检测细胞上清中PRRSV-N-Nb1-HRP融合蛋与PRRSV N蛋白特异结合;C为ELISA检测上清中PRRSV-N-Nb1-HRP融合蛋与PRRSV N蛋白的亲和力;
图3为PRRSV-N-Nb1-HRP融合蛋白作为竞争探针的检测PRRSV抗体的抑制百分比,竞争ELISA检测PRRSV抗体阳性(20份)和阴性猪血清(20份);
图4为竞争ELISA的敏感性和特异性分析,其中A为竞争ELISA检测PRRSV NADC30毒株感染不同天数的抗体;B:IDEXX ELISA试剂盒检测PRRSV NADC30毒株感染不同天数的抗体;C为竞争ELSIA检测其它动物疫病抗体分析;
图5为竞争ELISA检测不同PRRSV毒株感染猪只不同天数的抗体的抑制百分比,其中A为竞争ELISA检测PRRSV HuN4感染猪只不同天数的抗体;B为竞争ELISA检测PRRSV JXA1毒株感染猪只后不同天数的抗体;C为竞争ELISA检测PRRSV SD16感染猪只后不同天数的抗体。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图和具体实施方式对本发明作进一步详细的说明。
实施例1抗PRRSV N蛋白的特异性纳米抗体的筛选与鉴定
(1)双峰驼免疫
2mg PRRSV N重组蛋白与弗氏佐剂等体积混合并乳化均匀,免疫一只双峰驼,每两周1次,共免疫5次,第一次使用弗氏完全佐剂,其余4次全部使用弗式不全佐剂。
(2)噬菌体文库的构建
第5次免疫后4天,采血,分离骆驼外周血淋巴细胞并提取总RNA,参照QIAGEN RNA提取试剂盒说明书操作;将RNA反转录成cDNA,以此为模板进行PCR扩增并通过PstI、NotI酶切位点连入pMECS噬菌体展示载体;将连接产物电转至TG1感受态细胞中,活化后涂于LB-AMP琼脂平板,37℃过夜培养,收集菌苔,制成甘油菌在-80℃保存。与此同时,通过菌落PCR检测所建文库的阳性率,并测定库容大小及多样性,检测结果阳性率为96%,库容大小为3×109具有较好的多样性。
(3)抗PRRSV N蛋白的特异性纳米抗体的筛选
取50μL上述的甘油菌,接种于100mL LB-AMP培养基,待细菌生长至对数期,用20MOI M13KO7辅助噬菌体感染TG1细胞,过夜培养后纯化噬菌体,得到骆驼纳米抗体噬菌展示基因库。
抗原包被:将纯化的PRRSV N重组蛋白每孔4μg包于96孔酶标板,同时选取一个孔直接加入PBS作为无抗原对照孔,4℃包被过夜;封闭:用2.5%脱脂奶粉封闭,每孔200μL,37℃孵育1h;孵育噬菌体:2.5%的脱脂奶粉稀释上述纳米抗体噬菌展示基因库至5×1010pfu/ml,每孔100μL,37℃孵育1h;洗脱:用PBS’T洗涤5次,洗去不结合的噬菌体,加入新鲜配制的0.1M三乙胺100μL每孔,洗脱与PRRSV N特异性结合的噬菌体,即P0代洗脱产物。将洗脱产物感染处于对数期生长的大肠杆菌TG1,生产并纯化噬菌体用于下一轮的筛选。经过3轮筛选,富集阳性克隆。
(4)抗PRRSV N蛋白的特异性纳米抗体的鉴定
随机挑取96个单菌落进行测序,比对纳米抗体CDR3高变区发现共筛选出3株针对PRRSV N蛋白的特异性纳米抗体,将其命名为Nb1,Nb2,Nb3。粗提物的间接ELISA检测发现与其它两株相比Nb1具有最高亲和力,因此,我们选择Nb1进行后续研究。
实施例2纳米抗体Nb1与辣根过氧化物酶融合蛋白的制备
pEGFP-N1-HRP载体(Sheng,Y.,et al.,Nanobody-horseradish peroxidasefusion protein as an ultrasensitive probe to detect antibodies againstNewcastle disease virus in the immunoassay.J Nanobiotechnology,2019.17(1):p.35.),DNA序列包括分泌信号肽,HA标签,多克隆酶切位点,辣根过氧化物酶和His标签。
将骆驼来源的VHH基因连接至pMECS载体上,以获得的pMECS-VHH为模板,使用Nb-F:AACTGCAGATGGAGACCGACACC,Nb-R:ATAAGAATGCGGCCGCTTAGTGGTGATGGTG引物扩增VHH序列,其中引入了酶切位点Pst I(CTGCAG)和Not I(GCGGCCGC)。用Pst I和Not I进行双酶切获得VHH基因,连接至pEGFP-N1-HRP载体。将连接产物转化大肠杆菌后培养,挑取单个菌落,测序鉴定后,获得阳性质粒pEGFP-N1-Nb1-HRP。
将生长状态良好的HEK 293T细胞铺于10cm平皿,37℃5%CO2恒温培养18小时,汇合度至80%。使用PEI转染试剂转染细胞。具体步骤如下:
(1)转染前2h细胞换液,弃去旧培养基加入8mL DMEM培养基;
(2)取200μL opti-MEM培养基,加入12μg pEGFP-N1-Nb1-HRP质粒,用移液枪吹打混匀,再加入72μL PEI转染试剂,缓慢吹打混匀,室温静置20min;
(3)将上述复合物逐滴均匀加入平皿中,轻轻的摇晃混匀,置于37℃CO2培养箱中培养;
(4)生长8-12h换液,将旧的细胞培养基弃去,加入8mL 10%FBS细胞培养基,置于37℃CO2培养箱中继续培养48h,收集细胞分泌表达上清液,经离心浓缩后,即获得PRRSV-N-Nb1-HRP融合蛋白。
(5)IFA和ELISA验证PRRSV-N-Nb1-HRP融合蛋白的表达分泌以及亲和力和特异性,结果如图2所示。
根据图2结果显示,PRRSV-N-Nb1-HRP融合蛋白成功在HEK293T细胞中的表达,细胞上清中的融合蛋白可特异性的与PRRSV N蛋白结合。
实施例3抗PRRSV N蛋白的特异性纳米抗体Nb1的鉴定
酶联免疫方法(ELISA)鉴定PRRSV N蛋白的特异性纳米抗体
(1)包板:将原核表达的PRRSV N蛋白、鸡新城疫病毒(NDV)NP蛋白(无关蛋白对照)包被ELISA酶标板(400ng/孔)。
(2)封闭:PBS’T洗板3次,每孔加入200μL 2.5%脱脂奶粉室温孵育1h。
(3)抗体孵育:PBS’T洗板3次,加入PRRSV-N-Nb1-HRP融合蛋白至包被的ELISA板中,室温孵育30分钟。
(4)显色:PBS’T洗板3次,加入商品化的TMB显色液(天根生化科技有限公司),避光显色10分钟后,加入3M浓H2SO4。
(5)读数:读数OD450,ELISA结果如图1所示。
根据图1的结果显示,纳米抗体Nb1特异性的与PRRSV N蛋白结合,不与NDV NP蛋白(无关蛋白对照)结合,本发明的纳米抗体Nb1具有特异性。
实施例4以纳米抗体Nb1与辣根过氧化物酶融合蛋白作为敏感探针,检测PRRSV抗体的竞争ELISA方法的建立。
4.1抗原与纳米抗体与辣根过氧化物酶融合蛋白最佳稀释比例
抗原与纳米抗体与辣根过氧化物酶融合蛋白最佳稀释比例即两者反应OD450=1.0,不同浓度抗原(PRRSV N蛋白)包板,加入不同稀释倍数的HEK293T-Nb1-HRP上清,进行ELISA试验,OD450=1.0。
4.2验证Nb1-HRP作为敏感探针,能否阻断PRRSV感染猪的阳性血清中的抗体。
选取已知PRRSV感染猪的阳性血清20份,阴性血清20份(图3),竞争ELISA试验步骤如下:
(1)包被抗原:PRRSV N蛋白包板,4℃孵育过夜;
(2)封闭:每孔加入200μL 2.5%脱脂奶粉封闭,室温孵育1h;
(3)孵育复合物:PBS’T洗板3次,加入最佳稀释比例血清和Nb1-HRP融合蛋白的复合物,室温孵育最佳反应时间;
(4)显色:PBS’T洗板3次,加入TMB显色液室温孵育10分钟,最后加入3M H2SO4终止反应;
(5)观察颜色变化。
实施例5竞争ELISA敏感性与特异性的分析
5.1竞争ELISA敏感性的分析
PRRSV NADC30毒株感染PRRSV阴性猪,分别于感染后1、3、5、7、14、21、28天采血,分离血清,分别用竞争ELISA和IDEXX ELISA试剂盒进行检测。试验结果如图4所示,竞争ELISA检测结果:三头猪中其中有一头在感染后第五天血清转阳,在感染后7天全部为阳性,但是商品化IDEXX ELISA试剂盒,在感染后7天血清首次转阳。以上结果表明竞争ELISA的灵敏度高于商品化IDEXX ELISA试剂盒。
5.2竞争ELISA特异性的分析
用建立的竞争ELISA分别检测猪细小病毒病、猪圆环病毒病、猪伪狂犬病毒病、猪传染性胃肠炎病毒病和猪流感病毒病商品化ELISA试剂盒阳性标准品以及鸡新城疫病毒病阳性血清、犬瘟热病毒病阳性血清和兔戊型肝炎病毒病阳性血清各6份,用PRRSV阳性血清作为对照。试验结果均为阴性,不仅不与其它主要猪病阳性标准品发生交叉反应,也不与其它物种病毒病阳性血清反应,表明该竞争ELISA检测方法具有较高的特异性。
实施例6竞争ELISA检测不同毒株感染猪PRRSV抗体
HuN4,JXA1,SD16毒株感染猪后不同天数的血清(由本实验室保存)分别用竞争ELISA进行检测,试验结果如图5所示HuN4,JXA1和SD16毒株感染后第7天至28天,所有猪血清PRRSV抗体均为阳性。上述结果表明,该竞争ELISA检测方法可以应用于临床PRRSV抗体检测。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 西北农林科技大学
<120> 一种PRRSV N蛋白的纳米抗体及其制备方法应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Asp Ser Val Glu Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Ser Cys Gln Tyr
20 25 30
Asp Ile Tyr Asp Ile Thr Trp Trp Arg Gln Ser Pro Gly Asp Glu His
35 40 45
Glu Phe Val Ala Ser Ile Glu Arg Asp Asp Thr Arg Thr Tyr Ala Asp
50 55 60
Phe Val Gln Gly Arg Phe Thr Ile Ser Gln Gly Ser Ala Lys Asn Thr
65 70 75 80
Leu Leu Leu Asp Met Ser Ala Leu Thr Pro Asp Asp Thr Ala Lys Tyr
85 90 95
Tyr Cys Lys Leu His Arg Asn Arg Lys Cys Ser Ala Arg Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala
115 120
<210> 2
<211> 365
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caggtgcagc tgcaggagtc tgggggagac tcggtggagt ctggagggtc tctgagactc 60
tcctgtgtag cctctggatt tacctcctgc caatatgaca tatacgacat cacctggtgg 120
cgccagtctc caggggacga gcacgagttc gtcgcaagta ttgagaggga tgacactaga 180
acatacgcag atttcgtgca gggccggttc actatctccc aaggcagcgc caagaacact 240
ctactccttg acatgtcagc tctcacgcct gacgacacgg ccaagtatta ctgtaaatta 300
caccggaacc ggaagtgcag tgctcggggc caggggaccc aggtcaccgt ctcctcagcg 360
gccgc 365
Claims (7)
1.一种PRRSV N蛋白的纳米抗体,其特征在于:命名为PRRSV-N-Nb1,氨基酸序列如SEQID No.1所示。
2.一种编码权利要求1所述纳米抗体氨基酸序列的核苷酸分子,其特征在于:所述核苷酸分子的核苷酸序列如SEQ ID No.2所示。
3.一种含有权利要求1所述的纳米抗体与HRP的融合蛋白。
4.根据权利要求3所述的融合蛋白的制备方法,其特征在于,包括以下步骤:
(1)重组真核表达载体的构建:通过扩增、双酶切,将编码纳米抗体PRRSV-N-Nb1的基因序列连接至pEGFP-N1-HRP载体中获得阳性质粒;
(2)将步骤(1)中阳性质粒转化宿主细胞,诱导表达纳米抗体与HRP融合蛋白。
5.根据权利要求4所述的纳米抗体与HRP的融合蛋白的制备方法,其特征在于:所述宿主细胞为HEK 293T细胞。
6.一种根据权利要求3所述的融合蛋白在制备检测猪血清中PRRSV抗体的产品中的应用。
7.根据权利要求6所述的融合蛋白在制备检测猪血清中PRRSV抗体的产品中的应用,其特征在于,包括以下步骤:
(1)将PRRSV N蛋白包被ELISA板;
(2)将所述融合蛋白与猪血清混合后加入步骤(1)的ELISA板中,孵育;
(3)在步骤(2)的ELISA板中加入显色液避光显色,加入3M浓H2SO4终止反应;
(4)观察ELISA板的颜色变化,若猪血清中有PRRSV抗体,则ELISA板内孔为无色;若猪血清中无PRRSV抗体,则ELISA板内孔为黄色。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011346127.0A CN112457397A (zh) | 2020-11-26 | 2020-11-26 | 一种prrsv n蛋白的纳米抗体及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011346127.0A CN112457397A (zh) | 2020-11-26 | 2020-11-26 | 一种prrsv n蛋白的纳米抗体及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112457397A true CN112457397A (zh) | 2021-03-09 |
Family
ID=74809487
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011346127.0A Pending CN112457397A (zh) | 2020-11-26 | 2020-11-26 | 一种prrsv n蛋白的纳米抗体及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112457397A (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114106158A (zh) * | 2021-12-07 | 2022-03-01 | 重庆市动物疫病预防控制中心 | 一种靶向猪伪狂犬病毒gD蛋白的纳米抗体、制备方法和应用 |
CN114181305A (zh) * | 2021-12-28 | 2022-03-15 | 山东畜牧兽医职业学院 | 一种犬瘟热病毒h蛋白纳米抗体及其制备工艺 |
CN114891095A (zh) * | 2022-04-13 | 2022-08-12 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 一种用于检测prrsv抗原的纳米抗体对、试剂盒及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20140080779A (ko) * | 2012-12-18 | 2014-07-01 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | 돼지생식기호흡기증후군 바이러스 검출용 바이오 프로브 및 이를 이용한 돼지생식기호흡기증후군 바이러스 진단 방법 |
CN104710528A (zh) * | 2015-03-13 | 2015-06-17 | 西北农林科技大学 | 一种特异性结合PRRS病毒非结构蛋白Nsp9纳米抗体及其应用 |
CA3022006A1 (en) * | 2016-05-11 | 2017-11-16 | Phibro Animal Health Corporation | A composition comprising antigens and a mucosal adjuvant and a method for using |
CN111057145A (zh) * | 2019-11-22 | 2020-04-24 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 猪繁殖与呼吸综合征病毒Nsp2蛋白纳米抗体及其应用 |
-
2020
- 2020-11-26 CN CN202011346127.0A patent/CN112457397A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20140080779A (ko) * | 2012-12-18 | 2014-07-01 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | 돼지생식기호흡기증후군 바이러스 검출용 바이오 프로브 및 이를 이용한 돼지생식기호흡기증후군 바이러스 진단 방법 |
CN104710528A (zh) * | 2015-03-13 | 2015-06-17 | 西北农林科技大学 | 一种特异性结合PRRS病毒非结构蛋白Nsp9纳米抗体及其应用 |
CA3022006A1 (en) * | 2016-05-11 | 2017-11-16 | Phibro Animal Health Corporation | A composition comprising antigens and a mucosal adjuvant and a method for using |
CN111057145A (zh) * | 2019-11-22 | 2020-04-24 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 猪繁殖与呼吸综合征病毒Nsp2蛋白纳米抗体及其应用 |
Non-Patent Citations (3)
Title |
---|
HONG DUAN等: "Development of a Nanobody-Based Competitive EnzymeLinked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
ZE-HUI LIU等: "Novel Lentivirus-Based Method for Rapid Selection of Inhibitory Nanobody against PRRSV", 《VIRUSES》 * |
宋欢等: "猪繁殖与呼吸综合征病毒Nsp2蛋白纳米抗体的筛选及其鉴定", 《中国预防兽医学报》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114106158A (zh) * | 2021-12-07 | 2022-03-01 | 重庆市动物疫病预防控制中心 | 一种靶向猪伪狂犬病毒gD蛋白的纳米抗体、制备方法和应用 |
CN114106158B (zh) * | 2021-12-07 | 2022-06-07 | 重庆市动物疫病预防控制中心 | 一种靶向猪伪狂犬病毒gD蛋白的纳米抗体、制备方法和应用 |
CN114181305A (zh) * | 2021-12-28 | 2022-03-15 | 山东畜牧兽医职业学院 | 一种犬瘟热病毒h蛋白纳米抗体及其制备工艺 |
CN114181305B (zh) * | 2021-12-28 | 2024-01-26 | 山东畜牧兽医职业学院 | 一种犬瘟热病毒h蛋白纳米抗体及其制备工艺 |
CN114891095A (zh) * | 2022-04-13 | 2022-08-12 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 一种用于检测prrsv抗原的纳米抗体对、试剂盒及其应用 |
CN114891095B (zh) * | 2022-04-13 | 2023-09-01 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 一种用于检测prrsv抗原的纳米抗体对、试剂盒及其应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112457397A (zh) | 一种prrsv n蛋白的纳米抗体及其制备方法和应用 | |
CN114957454B (zh) | 一种抗csfv e2蛋白的纳米抗体、融合蛋白及其制备方法和应用 | |
CN111269318A (zh) | 一种gapdh纳米抗体及其应用 | |
CN112920268B (zh) | ASFV-p54蛋白特异性的纳米抗体-HRP融合蛋白及制备方法、应用 | |
CN110577594A (zh) | 一种金黄色葡萄球菌肠毒素a纳米抗体a21、应用及试剂盒 | |
CN111454355B (zh) | 一种sox6纳米抗体及其应用 | |
CN110016078B (zh) | 一种基于pedv n蛋白特异性纳米抗体的阻断elisa的检测方法及其应用 | |
Chitray et al. | Diagnostic and epitope mapping potential of single-chain antibody fragments against foot-and-mouth disease virus serotypes A, SAT1, and SAT3 | |
CN110244039B (zh) | 一种检测j亚群禽白血病病毒抗体的elisa试剂盒及其应用 | |
CN109503711B (zh) | 一种用于血凝方法检测pcv2病毒的双功能纳米抗体、编码基因及其应用 | |
CN112010969A (zh) | 一种高亲和力增强绿色荧光蛋白纳米抗体及其编码基因的筛选方法 | |
CN114213532B (zh) | 高亲和力的抗鸡传染性法氏囊病毒的scFv抗体的制备及其应用 | |
CN103088033B (zh) | 抗拟除虫菊酯类农药scFv的抗体基因及应用 | |
CN113735968A (zh) | 一种猪传染性胃肠炎病毒n蛋白抗体效价测定方法 | |
CN111499745B (zh) | 热反应蛋白单价及双价纳米抗体及其制备方法、应用 | |
CN110317242B (zh) | 可特异性结合Cry1Da蛋白的多肽分子及其应用 | |
CN113740528A (zh) | 一种猪传染性胃肠炎病毒抗体检测试剂盒 | |
CN110456047B (zh) | 一种传染性法氏囊病病毒抗体竞争elisa检测方法及试剂盒 | |
CN114773462B (zh) | 用于检测牛crp蛋白的重组单链抗体及其应用 | |
CN114891095B (zh) | 一种用于检测prrsv抗原的纳米抗体对、试剂盒及其应用 | |
CN114891098B (zh) | 一种产气荚膜梭菌β毒素纳米抗体及其应用 | |
CN113278073B (zh) | 一种nkg2a纳米抗体及其应用 | |
CN110221065B (zh) | 一种禽滑液囊支原体间接elisa检测试剂盒 | |
CN112159797B (zh) | 杂交瘤细胞株3g7 1b10、抗gii.4型诺如病毒p蛋白单克隆抗体和应用 | |
CN112175912B (zh) | 杂交瘤细胞株3g4 1d6、抗gii.4型诺如病毒p蛋白单克隆抗体和应用 |
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 |