Antibodies for detecting feline calicivirus based on VP1 protein and their application

Abstract

The present invention relates to the field of biotechnology detection, and in particular to antibodies for detecting feline calicivirus based on VP1 protein and their applications. The present invention uses phage display technology to screen and obtain recombinant antibodies based on VP1 protein as an important immunogenic protein of feline calicivirus. Experimental results show that the antibodies of the present invention have strong specificity and high affinity, and Ab-FCV VP1-4 in the prepared recombinant antibodies has higher specificity and affinity, which can be used not only for indirect immunofluorescence of feline calicivirus disease, but also for indirect ELISA and other detections, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology detection, and in particular to an antibody for detecting feline calicivirus based on VP1 protein and an application thereof. Background Art

[0002] Feline calicivirus (FCV) is the causative agent of feline calicivirus disease (FCVD), also known as feline infectious rhinoconjunctivitis. It belongs to the genus Calicivirus in the family Caliciviridae and is a single-stranded, positive-sense RNA virus. FCV infection in cats can cause symptoms such as oral ulcers, fever, sneezing, rhinitis, conjunctivitis, skin edema, loss of appetite, and jaundice. FCV not only infects cats, but can also infect other felines such as lions and tigers. A highly potent variant of FCV, FCV-VSD, causes systemic infection and increased mortality in animals, resulting in significant economic losses to the wildlife and pet industries.

[0003] The FCV genome is composed of ORF1, ORF2, and ORF3. The VP1 protein encoded by ORF2 is the primary structural and capsid protein of FCV. It possesses excellent immunogenicity and can induce the production of neutralizing antibodies, making it the preferred antigen for FCV diagnostic reagents. However, traditional indirect immunofluorescence and indirect ELISA assays for detecting antibodies in serum infected with FCV have low sensitivity and specificity. Therefore, the development of a highly sensitive and specific detection antibody is of great practical significance and application value. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide antibodies for detecting feline calicivirus based on VP1 protein and applications thereof.

[0005] The present invention provides a recombinant antibody against feline calicivirus.

[0006] The amino acid sequence of CDR1 of its heavy chain variable region is GX H1 X H2 LSX H3 YX H4 , where X H1 Selected from I or F; X H2 Select from D or S, X H3 Select from T or S, X H4 Selected from A or V or Y or T;

[0007] The amino acid sequence of CDR2 of the heavy chain variable region is IX H5 X H6 X H7 X H8 X H9 X H10, where X H5 Selected from N or Y; X H6 Selected from T or G; X H7 Selected from D or S or T or G; X H8 Select from G or A; X H9 Selected from S or N or Y or I; X H10 Selected from A or T or R;

[0008] The amino acid sequence of CDR3 of the heavy chain variable region is X H11 X H12 X H13 X H14 X H15 X H16 X H17 X H18 X H19 GX H20 X H21 X H2 2X H23 X H24 X H25 , where X H11 Select A or None; X H12 Select R or None; X H13 Select A or None; X H14 Select from G or None; X H15 Select from A or P or None; X H16 Selected from G or R or none; X H17 Selected from R or S or E or G or none; X H18 Selected from G or Y; X H19 Selected from R or V or I or none; X H20 Selected from F or L or V or Y or I; X H21 Select from P or T or None; X H22 Selected from D or A or N; X H23 Selected from L or Y or F; X H24 Select D or None; X H25 Select from P or S or None;

[0009] The amino acid sequence of CDR1 of the light chain variable region is X L1 SX L2 X L3 X L4 X L5 X L6 X L7 , where X L1 Select from Q or E; X L2 Selected from V or I; X L3 Select Y or None; X L4 Selected from S or D or G or N; X L5 Selected from D or S or N; X L6 Selected from W or N; XL7 Select from Y or E or None;

[0010] The amino acid sequence of CDR2 of the light chain variable region is X L8 X L9 S, where X L8 Selected from D or Y or A; X L9 Selected from V or A;

[0011] The amino acid sequence of CDR3 of the light chain variable region is X L10 X L11 X L12 X L13 X L14 YX L15 X L16 X L17 X L18 X L19 X L20 X L 21 , where X L10 Select from C or None; X L11 Select from C or None; X L12 Selected from A or S or L; X L13 Selected from G or N; X L14 Selected from A or S or G; X L15 Select from S or D; X L16 Select from G or D or none; X L17 Select from N or D; X L18 Selected from I or V or A; X L19 Select from Y or D; X L20 Selected from T or G or A or S or N; X L16 Select from L or A or None.

[0012] Furthermore, the recombinant antibody of the present invention,

[0013] The amino acid sequence of CDR1 of the heavy chain variable region is at least one of SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47 and / or SEQ ID NO: 53;

[0014] The amino acid sequence of CDR2 of the heavy chain variable region is at least one of SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48 and / or SEQ ID NO: 54;

[0015] The amino acid sequence of CDR3 of the heavy chain variable region is at least one of SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 49 and / or SEQ ID NO: 55;

[0016] The amino acid sequence of CDR1 of the light chain variable region is at least one of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50 and / or SEQ ID NO: 56;

[0017] The amino acid sequence of CDR2 of the light chain variable region is at least one of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 51 and / or SEQ ID NO: 57;

[0018] The amino acid sequence of CDR3 of the light chain variable region is at least one of SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 52 and / or SEQ ID NO: 58.

[0019] Furthermore, the recombinant antibody of the present invention,

[0020] The amino acid sequence of the heavy chain variable region is at least one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 9;

[0021] The amino acid sequence of the light chain variable region is at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and / or SEQ ID NO: 10.

[0022] The present invention constructs a large-capacity phage single-chain antibody library through phage display technology. After two rounds of enrichment and sequencing, 5 single-chain antibodies (scFv) FCV VP1-scFv that can specifically bind to FCV VP1 protein are finally screened. VH and VL are grafted onto vectors pTT5-V5 and pTT5-V6 containing rabbit antibody skeletons using molecular biology techniques, and expressed and purified in eukaryotic cells HEK293f. Indirect ELISA experiments confirmed that the recombinant antibodies Ab-FCV VP1-1, Ab-FCV VP1-2, Ab-FCV VP1-3, Ab-FCV VP1-4, and Ab-FCV VP1-5 obtained after purification can specifically bind to the antigen with high affinity. Indirect immunofluorescence confirmed that Ab-FCV VP1-4, which has the highest affinity, can specifically bind to the FCV virus and can be used for the specific detection of feline calicivirus FCV. The detection specificity and detection limit are both better than those of existing commercial antibodies.

[0023] The recombinant antibody Ab-FCV VP1-1, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 2; or

[0024] The recombinant antibody Ab-FCV VP1-2, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 3, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 4; or

[0025] The recombinant antibody Ab-FCV VP1-3, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 5, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 6; or

[0026] The recombinant antibody b-FCV VP1-4, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 7, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 8; or

[0027] The amino acid sequence of the heavy chain of the recombinant antibody Ab-FCV VP1-5 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0028] The present invention provides a nucleic acid encoding the recombinant antibody.

[0029] Furthermore, the nucleic acid encoding the recombinant antibody of the present invention may be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. Nucleic acid may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acid may be linear or circular in topology. Nucleic acid may be part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0030] Furthermore, the nucleic acids of the present invention can be optimized or unoptimized, and the optimization includes but is not limited to: codon usage bias, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changing GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0031] The present invention also provides a transcription unit for the recombinant antibody, which refers to a DNA sequence starting from a promoter and ending at a terminator. The promoter and terminator may also be flanked or separated by regulatory segments, which may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction site, and homologous recombination site operably linked to the nucleic acid sequence, such as a promoter enhancer and a poly(A) signal.

[0032] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid of the present invention.

[0033] Furthermore, the vector backbones described in the present invention may be derived from vectors for plants, animals, bacteria, fungi, phages, or viruses, but are not limited thereto. Examples of animal vectors include, but are not limited to, pcDNA 3.1, pIRES, pTT5, pCEP4, pATX1, or pCHO1.0; bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, and pColdII; and fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, and pAUR23.

[0034] The recombinant vector described in the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that contains the desired coding sequence and appropriate nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for expression in model animals or mammalian cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably, as plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0035] In some specific embodiments of the present invention, scFv homologous recombination is used to form a single-chain antibody FCV VP1-scFv. First, the scFv sequence with higher affinity obtained by phage display is used. Secondly, VH and VL are grafted onto vectors pTT5-V5 and pTT5-V6 containing rabbit antibody backbones using molecular biology techniques. Linker (sequence is SEQ ID NO.11) is inserted between VH and VL. Ab-FCV VP1 recombinant antibodies are expressed using HEK293f, and the binding of antigen and antibody is detected by indirect ELISA. The results showed that 12 strains of FCV VP1-scFv were strongly positive in the ELISA test, and 5 strains of high-affinity antibodies Ab-FCV VP1 for the FCV VP1 target antigen were obtained by sequencing. After affinity was verified by EC50, Ab-FCV VP1-4 with the highest affinity was used for indirect immunofluorescence experiment, and it was confirmed that the recombinant antibody Ab-FCV VP1-4 can specifically bind to FCV virus and can be applied to the specific detection of feline calicivirus FCV.

[0036] The present invention provides a host, the genome of which is integrated with the nucleic acid of the present invention, or is transfected or transformed with the recombinant vector of the present invention.

[0037] Furthermore, the transformation methods include chemical transformation and electroporation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection methods include adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0038] The host cells provided by the present invention may be derived from plants, animals, bacteria, fungi, phages, or viruses, but are not limited thereto. The present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells are capable of replicating protein-encoding vectors or expressing desired proteins.

[0039] The host cell of the present invention is used for the expression of the antibody protein or the replication and storage of the nucleic acid. In some specific embodiments of the present invention, the host cell is derived from an animal, specifically HEK293f.

[0040] The present invention provides a method for preparing the recombinant antibody, which comprises culturing the host as described in the present invention to obtain a mixture containing the recombinant antibody as described in the present invention.

[0041] The present invention provides the use of at least one of the following I) to V) in the preparation of a feline calicivirus detection product:

[0042] 1) the recombinant antibody of the present invention;

[0043] II), the nucleic acid of the present invention;

[0044] III), the vector of the present invention;

[0045] IV), the host of the present invention;

[0046] V) A mixture containing the recombinant antibody prepared by the preparation method of the present invention.

[0047] The present invention uses phage display technology to screen and obtain recombinant antibodies based on VP1 protein as an important immunogenic protein of feline calicivirus. Experimental results show that compared with existing products, the antibodies of the present invention have strong specificity and high affinity, and the Ab-FCV VP1-4 in the prepared recombinant antibodies has higher specificity and affinity. It can be used not only for indirect immunofluorescence of feline calicivirus disease, but also for indirect ELISA and other detections, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The figure shows the construction of the FCV VP1-scFv phage library and the diversity analysis of the immune library provided in the embodiment of the present invention, wherein A is the antibody titer of New Zealand rabbits immunized four times; B is the amplified bands for the VL gene library and the VH gene library, M: Marker; 1: VH, 2: VL, 3: VL-linker-VH target band;

[0049] Figure 21 shows the screening of high-affinity FCV VP1-scFv provided by an embodiment of the present invention, wherein A is the PCR identification result after electroporation; B is the diversity of the antibody library after the first round of phage display screening; C is the diversity of the antibody library after the second round of phage display screening; D is the scFv antibody with high specific binding to the antigen FCV VP1 determined by the second round of phage ELISA;

[0050] Figure 3 The expression and affinity verification of Ab-FCV VP1 provided in the embodiment of the present invention are shown, wherein A is the SDS-PAGE result of Ab-FCVVP1, M: Marker, 1: Ab-FCV VP1-1, 2: Ab-FCV VP1-2, 3: Ab-FCV VP1-3, 4: Ab-FCV VP1-4, 5: Ab-FCV VP1-5; B is the EC of Ab-FCV VP1-1 antibody binding to antigen FCV VP1 50 ; C is the EC of Ab-FCVVP1-2 antibody binding to antigen FCV VP1 50 ; D is the EC of Ab-FCV VP1-3 antibody binding to antigen FCV VP1 50 ; E is the EC of Ab-FCV VP1-4 antibody binding to antigen FCV VP1 50 ; F is the EC of Ab-FCV VP1-5 antibody binding to antigen FCV VP1 50 ;

[0051] Figure 4 1 and 2 show the results of an indirect immunofluorescence experiment between the Ab-FCV VP1-4 recombinant antibody provided in an embodiment of the present invention and the FCV virus.

[0052] Figure 5 The specificity verification of the binding of Ab-FCV VP1-4 recombinant antibodies to FCV, FPV, and FHV viruses provided in the examples of the present invention is shown.

[0053] Figure 6 Figure 2 shows the plasmids pTT5-V5 and pTT5-V6 containing the rabbit antibody backbone mentioned in the examples of the present invention. DETAILED DESCRIPTION

[0054] The present invention provides antibodies for detecting feline calicivirus based on VP1 protein and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0055] The amino acid sequences of the heavy chain variable regions of the single-chain antibody FCV VP1-scFv-1 and the recombinant antibody Ab-FCV VP1-1 are: QSVKESGGRLVTPGTPLTITCTVSGIDLSTYAMSWVRQDPGKGLEYIGFINTDGSAYYAS WAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCGRGFDLWGQGTLVTVSS (SEQ ID NO: 1);

[0056] H1-CDR1: GIDLSTYA (SEQ ID NO: 29);

[0057] H1-CDR2:INTDGSA(SEQ ID NO:30);

[0058] H1-CDR3:GRGFDL (SEQ ID NO:31);

[0059] The amino acid sequences of the light chain variable regions of the single-chain antibody FCV VP1-scFv-1 and the recombinant antibody Ab-FCV VP1-1 are: DGMMTQTPSPVSAAVGGTVTIKCQSSQSVYSDWLSWFQQKPGQPPKRLIYDASTLASG VPSRFKGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYTFGGGTEVVVK (SEQ ID NO: 2);

[0060] L1-CDR1:QSVYSDW(SEQ ID NO:32);

[0061] L1-CDR2: DAS (SEQ ID NO: 33);

[0062] L1-CDR3:AGAYSGNIYT(SEQ ID NO:34);

[0063] The amino acid sequences of the heavy chain variable regions of the single-chain antibody FCV VP1-scFv-2 and the recombinant antibody Ab-FCV VP1-2 are: QSLEESGGRMVTPGTPLTLTCTVSGFSLSSYVVSWVRQAPGKGLEWIGIIYTSGNTYYAN WAKGRFTISKTSTTVDLKITSPTTEDTATYFCGRGGLPALWGQGTLVTVSS (SEQ ID NO: 3);

[0064] H2-CDR1:GFSLSSYV(SEQ ID NO:35);

[0065] H2-CDR2:IYTSGNT (SEQ ID NO:36);

[0066] H2-CDR3:GRGGLPAL (SEQ ID NO:37);

[0067] The amino acid sequences of the light chain variable regions of the single-chain antibody FCV VP1-scFv-2 and the recombinant antibody Ab-FCV VP1-2 are: DGVLTQTPSSTSAAVGGTVTISCQSSESVYDNNYLSWYQQKPGQPPKVLIYYVSTLASGV PSRFKGSGSGTQFTLTISDLECDDAATYYCAGAYSGNIYGFGGGTEVVVK (SEQ ID NO: 4);

[0068] L2-CDR1:ESVYDNNY(SEQ ID NO:38);

[0069] L2-CDR2: YVS (SEQ ID NO: 39);

[0070] L2-CDR3: AGAYSGNIYG (SEQ ID NO: 40);

[0071] The amino acid sequences of the heavy chain variable regions of the single-chain antibody FCV VP1-scFv-3 and the recombinant antibody Ab-FCV VP1-3 are: QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMIWVRQAPGKGLEWIGIIYGTAYRYYASW AKGRFTISKTSTTVDLKITSPTTEDTATYFCARSYVGVTAYDPVGQGTLVTVSS (SEQ ID NO: 5);

[0072] H3-CDR1:GFSLSTYA(SEQ ID NO:41);

[0073] H3-CDR2:IYGTAYR (SEQ ID NO:42);

[0074] H3-CDR3:ARSYVGVTAYDP (SEQ ID NO:43);

[0075] The amino acid sequences of the light chain variable regions of the single-chain antibody FCV VP1-scFv-3 and the recombinant antibody Ab-FCV VP1-3 are: DGVPTQTPSSVSAAVGGTVTISCQSSQSVYSNWLSWFQQKPGQPPKRLIYDASTLTSGVS SRFKGSGSGTQFILTISDLECDDAATYYCAGAYSGNIYAFGGGTEVVVK (SEQ ID NO: 6); L3-CDR1: QSVYSNW (SEQ ID NO: 44);

[0076] L3-CDR2:DAS (SEQ ID NO:45);

[0077] L3-CDR3: AGAYSGNIYA (SEQ ID NO: 46);

[0078] The amino acid sequences of the heavy chain variable regions of the single-chain antibody FCV VP1-scFv-4 and the recombinant antibody Ab-FCV VP1-4 are: QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYTMSWVRQAPGKGLEWIGIIYTGGITYYASW AKGRFTISRTSTTVDLKITSPTTEDTATYFCAREGVGYAFDSWGPGTLVTVSS (SEQ ID NO: 7);

[0079] H4-CDR1:GFSLSSYT(SEQ ID NO:47);

[0080] H4-CDR2:IYTGGIT (SEQ ID NO:48);

[0081] H4-CDR3:AREGVGYAFDS(SEQ ID NO:49);

[0082] The amino acid sequences of the light chain variable regions of the single-chain antibody FCV VP1-scFv-4 and the recombinant antibody Ab-FCV VP1-4 are: DGMMTQTPSPVSATVGGTVTIKCQASESIGSWLAWYQQKPGQRPKLLIYAASTLASGVP SRFSGSGSGTEFTLAISGVECADAATYYCQQSNSYSDVDSLFGGGTEVVVK (SEQ ID NO: 8);

[0083] L4-CDR1:ESIGSW(SEQ ID NO:50);

[0084] L4-CDR2: AAS (SEQ ID NO: 51);

[0085] L4-CDR3:QQSNSYSDVDSL(SEQ ID NO:52);

[0086] The amino acid sequences of the heavy chain variable regions of the single-chain antibody FCV VP1-scFv-5 and the recombinant antibody Ab-FCV VP1-5 are: QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMTWVRQAPGKGLEWIGIIYGTGYTYYAS WAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARAGPGGGIGINLWGQGTLVTVSS (SEQ ID NO: 9);

[0087] H5-CDR1:GFSLSSYY(SEQ ID NO:53);

[0088] H5-CDR2:IYGTGYT (SEQ ID NO:54);

[0089] H5-CDR3:ARAGPGGGIGINL (SEQ ID NO:55);

[0090] The amino acid sequences of the light chain variable regions of the single-chain antibody FCV VP1-scFv-5 and the recombinant antibody Ab-FCV VP1-5 are: DGVMTQTESPVSAAVGSTVTINCQSSQSVYNNNELSWYQQKPGQPPKLLIYYASTLASG VSSRFKGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADNAFGGGTEVVVK (SEQ ID NO: 10);

[0091] L5-CDR1:QSVYNNNE(SEQ ID NO:56);

[0092] L5-CDR2:YAS(SEQ ID NO:57);

[0093] L5-CDR3:LGGYDDDADNA(SEQ ID NO:58);

[0094] The name of the linker is: GGGGSGGGGGGSSRSS(SEQ ID NO:11);

[0095] VH-scFv-F1:5'-ggtgggggtggttcctctagatcttcccagtcgctggaggagtcc-3'(SEQ IDNO:12);

[0096] VH-scFv-F2:5'-ggtgggggtggttcctctagatcttcccagwcagtgaaggagtcc-3'(SEQ IDNO:13);

[0097] VH-scFv-F3:5'-ggtgggggtggttcctctctctcccagtcgctggrggagtcc-3'(SEQ IDNO:14);

[0098] VH-scFv-F4:5'-ggtgggggtggttcctctctctcccagtcggtggaggagtcc-3'(SEQ IDNO:15);

[0099] VH-scFv-R1:5'-aggaggctattggccggcctggcctgargagayggtgaccagggtgcc-3'(SEQID NO:16);

[0100] VH-scFv-R2:5'-aggaggctattggccggcctggccgcagggggccagtgggaagactgac-3'(SEQ ID NO:17);

[0101] VL-scFv-F1:5'-aaaagaggcccaggcggccgagctcgtgmtgacccagactcca-3'(SEQ IDNO:18;

[0102] VL-scFv-F2:5'-aaaagaggcccaggcggccgagctcgatmtgacccagactcca-3'(SEQ IDNO:19);

[0103] VL-scFv-F3: 5'-aaaagaggcccaggcggccgcycaagtgctgacccag-3' (SEQ ID NO:20);

[0104] VL-scFv-F4: 5'-aaaagaggcccaggcggccgcccwagtgatgacccag-3' (SEQ ID NO:21);

[0105] VL-scFv-R1: 5'-ggaagatctagaggaaccacccccaccaccgcccgagccaccgccacctttgatttccacattggtgcc-3' (SEQ ID NO:22);

[0106] VL-scFv-R2: 5'-ggaagatctagaggaaccacccccaccaccgcccgagccaccgccacctaggatctccagctcggtccc-3' (SEQ ID NO:23);

[0107] Overlap PCR-FP: 5'-gaggaggaaaaagaggcccaggcggcc-3' (SEQ ID NO:24);

[0108] Overlap PCR-RP: 5'-agaggaggctattggccggcctggcc-3' (SEQ ID NO:25);

[0109] pComb3XSS-FP: 5'-gctggtttcgctaccgtggcccaggcggcc-;

[0110] pComb3XSS-RP: 5'-gtgatggtgatggtgctggccggcctggcc-3' (SEQ ID NO:27);

[0111] Amino acid sequence of FCV VP1 protein: (SEQ ID NO: 28).

[0112] Strains, plasmids, experimental animals, and reagents: Adult New Zealand rabbits were purchased from Yongkang Qingyuan Farm in Dingyuan County, Anhui Province; FCV VP1 protein was preserved in our laboratory (amino acid sequence is SEQ ID NO.28); FCV virus was preserved in our laboratory (GenBank: KT000003.1); vectors pTT5-V5 and pTT5-V6 plasmids containing rabbit antibody backbones were preserved in our laboratory ( Figure 5); HRP-labeled recombinant protein A was purchased from Sangon Biotech (Shanghai) Co., Ltd.; HRP-labeled mouse anti-M13K07 phage was purchased from Chengdu Apac Biotechnology Co., Ltd.; HRP-labeled goat anti-rabbit IgG was purchased from Beijing Meikewande Biotechnology Co., Ltd.; pComb3XSS plasmid and Escherichia coli DH5α competent cells were purchased from Shandong Hertz Biotechnology Co., Ltd.; M13K07 helper phage and Escherichia coli ER2738 competent cells were purchased from Nanjing Heming Yinggu Biotechnology Co., Ltd.; SDS-PAGE protein gel kit was purchased from Yazyme Biotechnology Co., Ltd.; penicillin-streptomycin solution was purchased from GE (for cell culture); Freund's adjuvant was purchased from Sigma; ClonExpress II One Step Cloning Kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; Premix Taq, Plasmid mini kit, Gel Extraction, and transfection reagents were all purchased from TaKaRa.

[0113] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0114] The present invention will be further described below in conjunction with the embodiments:

[0115] Example 1 Animal Immunization

[0116] Healthy adult New Zealand rabbits were selected for the experiment. Blood was collected from the vein before immunization and the serum was separated as a negative control. 5 TCID 50 / ml FCV virus was injected into the back of the neck of New Zealand rabbits. Immunization was carried out once a week for a total of 4 times. 0.5mL of venous blood was collected before each immunization on days 0, 7, 14, and 21, and serum was separated and stored. 7 days after the fourth immunization (i.e., day 28), 0.5mL of venous blood was collected and serum was separated and stored. ELISA results showed that the antibody titer of the serum increased significantly after the first immunization, and the antibody titer of the second immunization was greater than 5×10 4 , after three exemptions, it reaches 10 5 The titer of the four immune serums reached 1.5×10 5 , compared with the serum antibody titer before immunization, there was a significant increase ( Figure 1 A) On day 28, New Zealand rabbits were sacrificed and 0.5 mL of blood was collected for serum separation and preservation. The spleen was removed and used for lymphocyte isolation.

[0117] Example 2 Extraction of total lymphocyte RNA and amplification of antibody light and heavy chain genes

[0118] Total RNA from lymphocytes was extracted using Trizol reagent and reverse transcribed using it as a template using Oligo (dT) as a primer to synthesize cDNA. Primer sequence combinations were designed based on the light and heavy chain framework regions of the rabbit antibody sequence (primer sequences are SEQ ID NO.12 to SEQ ID NO.23) to amplify the coding regions of the antibody VH and VL by PCR. The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, and after 35 cycles, extension at 72°C for another 5 minutes. The PCR product was verified by 1% agarose gel electrophoresis to have a target band of around 350 bp and was purified and recovered for later use ( Figure 1 B).

[0119] Example 3 Construction of FCV VP1-scFv Antibody Library

[0120] Using purified VH and VL as templates, a linker (sequence: SEQ ID NO. 11) was inserted between VH and VL after overlapping PCR to synthesize the scFv fragment VL-linker-VH, which contained sfiI restriction enzyme cleavage sites at both ends of the sequence.

[0121] Overlap extension first round PCR: without primers, denaturation at 98°C for 1 minute, annealing at 45°C for 30 seconds, extension at 72°C for 1 minute, repeat 10 cycles, and finally at 72°C for 5 minutes. Add primers Overlap PCR-FP and Overlap PCR-RP (SEQ ID NO. 24 to SEQ ID NO. 25), denaturation at 98°C for 30 seconds, then annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, and after 30 cycles, extension at 72°C for another 5 minutes. The PCR product was recovered by gel electrophoresis on 1% agarose gel. The band was approximately 800 bp ( Figure 1 B), stored at -20°C. The overlapping PCR primer sequences are SEQ ID NO. 24 to SEQ ID NO. 25.

[0122] The PCR products and pComb3XSS vector after gel recovery were digested with restriction endonuclease sfiI at 50°C for 30 minutes. The digested products were verified by 1% agarose gel electrophoresis, then recovered and purified on a column and ligated with T4 DNA ligase to obtain the FCV VP1-scFv-pComb3XSS library. The ligated products were transformed into ER2738 competent cells, and positive clones were screened using tetracycline and ampicillin-resistant 2YT (2×Yeast extract and Tryptone) solid medium. 20 single clones from the library were randomly selected for PCR identification (primer sequences are SEQ ID NO.26 to SEQ ID NO.27), and the calculated recombination rate of the antibody library was 60% ( Figure 2A) All colonies on the solid medium were washed off with 2YT medium and stored at 4°C.

[0123] Example 4 Phage Amplification

[0124] Take 2 mL of the bacterial solution from all clones washed with 2YT medium and inoculate it into 50 mL of 2YT liquid medium. Incubate at 37°C, 200 rpm until the OD 600 When the concentration reaches 0.8-1.0, add the final concentration of 1×10 12 Infect with M13K07 helper phage at a concentration of 50 μg / mL of M13K07. After 30 minutes of simmerment, add kanamycin (final concentration 50 μg / mL) and incubate overnight at 30°C, 200 rpm. The next day, centrifuge at 10,000 rpm for 20 minutes at 4°C. Retain the supernatant and add 1 / 2 volume of PEG8000. Incubate on ice for 2 hours to precipitate the phage. Centrifuge at 10,000 rpm for 20 minutes at 4°C and discard the supernatant. Resuspend the pellet in 5 mL of PBS and add 3 mL of PEG8000 to reprecipitate. Repeat the centrifugation. Resuspend the pellet in 4 mL of PBS, filter-sterilize, and store at 4°C until needed.

[0125] Example 5 FCV VP1-scFv screening

[0126] Biotinylate the FCV VP1 protein in advance. Amplified FCV VP1-scFv library phage, Dynabeads M-280 magnetic beads labeled with FCV VP1 protein, and 2% skim milk powder blocking buffer were mixed and incubated at 25°C ± 5°C for 1 hour. The beads were washed 5-10 times with PBST. Phage were eluted from the surface of the beads with 0.1M Glycine-HCl (pH = 2.0) and neutralized with 1M Tris to pH 7.0. The eluted and neutralized phage were used to infect ER2738 competent cells in the logarithmic growth phase, cultured and enriched on 2YT solid medium, and colonies were harvested for the next round of screening.

[0127] The above experiment was repeated twice. 20 monoclones were randomly selected from the two rounds of enrichment affinity screening libraries for PCR verification. The results showed that the positive rate of the constructed FCV VP1-scFv library in the first round of panning was 70%, and the positive rate in the second round of panning was 95% ( Figure 2 B and C).

[0128] Example 6 Phage ELISA

[0129] Coat the ELISA plate with FCV VP1 antigen at a concentration of 50 μg / mL per well at 4°C overnight. Wash five times with PBST containing 0.05% Tween-20, discard the PBST, and add 2% skim milk powder blocking buffer and block at 37°C for 1 hour. Wash five times with PBST, pat dry any residual liquid, and mix the supernatant of the positive clone antibody to be screened with 2% skim milk powder blocking buffer in a 3:2 ratio. Incubate at 25°C ± 5°C for 10 minutes to remove interference. Then, add the plate and incubate at 37°C for 1 hour. Wash five times with PBST, pat dry any residual liquid, and add HRP-conjugated mouse anti-M13K07 antibody (1:3000 dilution in blocking buffer) and incubate at 37°C for 1 hour. Wash with PBST solution 5 times, pat dry the residual liquid in the wells, add TMB colorimetric solution, keep it in the dark for 15 minutes, add 2 mol / L H2SO4 to stop the color development, and read the absorbance at 450 nm (Table 1). Figure 2 D display, select OD 450 The 12 antibodies with the highest OD values ​​were sequenced (the OD values ​​corresponding to the selected 12 antibodies are marked in bold in the table), and finally 5 antibodies with different sequences were obtained for subsequent experiments.

[0130] Table 1. Affinity test of 96 phages containing Ab-FCV VP1-1 antibody and antigen FCV VP1

[0131]

[0132]

[0133] Example 7 Recombination, Expression and Purification of FCV VP1 Recombinant Antibody Ab-FCV VP1

[0134] The VH and VL sequences of five single-chain antibody FCV VP1-scFv strains were amplified and purified. The pTT5-V5 and pTT5-V6 vectors, containing the rabbit antibody backbone, were double-digested with EcoRI and HindIII restriction enzymes. Through homologous recombination, the antibody expression plasmids VH-pTT5-V5-1 to VH-pTT5-V5-5 and VL-pTT5-V6-1 to VL-pTT5-V6-5 were generated. The recombinant plasmids VH-pTT5-V5-1 to VH-pTT5-V5-5 and VL-pTT5-V6-1 to VL-pTT5-V6-5 were aligned in a one-to-one manner and transfected into HEK293f cells via PEI for eukaryotic expression. After 48 h, the culture supernatant was collected by centrifugation at 10,000 rpm and 4°C for 20 min, and the antibodies were purified using a Protein A affinity chromatography column. The fusion proteins were finally preserved in PBS solution and named Ab-FCV VP1-1, Ab-FCV VP1-2, Ab-FCV VP1-3, Ab-FCV VP1-4, and Ab-FCV VP1-5, respectively.

[0135] The purified sample was added to the reduced protein loading buffer and subjected to SDS-PAGE electrophoresis after denaturation. The results were as follows: Figure 3 As shown in A, the disulfide bonds between the heavy and light chains of the antibody were reduced, and target bands were detected at approximately 50 kDa and 25 kDa, consistent with the expected results.

[0136] Example 8 Indirect ELISA to detect the binding of recombinant antibody Ab-FCV VP1 to FCV VP1 antigen

[0137] The affinity of Ab-FCV VP1 for the antigen FCV VP1 was determined using an indirect ELISA method. Antigen was added to the microplate and incubated overnight at 4°C. The plate was then blocked with 2% skim milk blocking buffer at 37°C for 2 hours. Twelve dilutions of Ab-FCV VP1 were added to the wells using 2% skim milk and incubated at 37°C for 1 hour. HRP-conjugated goat anti-rabbit IgG secondary antibody was added, and the color reaction was developed with TMB. The absorbance at 450 nm was measured in a microplate reader (Tables 2–6).

[0138] Table 2 EC binding of Ab-FCV VP1-1 to antigen FCV VP1 50

[0139]

[0140] Table 3 EC binding of Ab-FCV VP1-2 to antigen FCV VP1 50

[0141]

[0142]

[0143] Table 4 EC binding of Ab-FCV VP1-3 to antigen FCV VP1 50

[0144]

[0145] Table 5 EC binding of Ab-FCV VP1-4 to antigen FCV VP1 50

[0146]

[0147] Table 6 EC binding of Ab-FCV VP1-5 to antigen FCV VP1 50

[0148]

[0149] Detect the binding effect of the above antibody Ab-FCV VP1 as follows Figure 3 As shown in B to 3F, the dose-effect curves were successfully fitted with the regression model. All five Ab-FCV VP1 antibodies were able to effectively bind to FCV VP1 protein. Antibody Ab-FCV VP1-4 had the highest affinity. The EC binding to recombinantly expressed FCV VP1 protein was 50 is 1.243×10 -1 μg / mL, indicating that the recombinant antibody was active and had good affinity, and Ab-FCV VP1-4 was used in subsequent experiments.

[0150] Example 9 Indirect immunofluorescence detection of recombinant antibodies

[0151] Use EMEM containing 10% FBS to revive CRFK adherent cells. When the cell confluency reaches 80-90%, inoculate FCV venom with an MOI of 0.1 and co-infect and culture in a 37°C 5% CO2 incubator. At the same time, set up a virus-negative group as a control (i.e., no virus is added). Add an equal volume of 2% FBS EMEM and culture in a cell culture incubator. After 24 hours, wash the cells three times with PBS. Add 500 μL of 4% paraformaldehyde to fix the cells, and wash the cells three times with PBS after 30 minutes. Add 500 μL of 0.1% TritonX100, and wash the cells three times with PBS after 15 minutes. Block with PBS containing 3% sodium caseinate at 37°C for 1 hour and wash three times with PBS. The recombinant antibody Ab-FCV VP1 was used as the primary antibody for indirect immunofluorescence at a concentration of 0.5 μg / mL and incubated at 37°C for 1 hour. The secondary antibody was goat anti-rabbit FITC and incubated at 37°C for 1 hour. The plate was washed three times and stained with 500 μl DAPI for 20 minutes. The plate was washed three times with PBS and observed using an inverted fluorescence microscope. Figure 4 As shown, the recombinant antibody Ab-FCV VP1-4 produced a clear specific green fluorescent signal when it was combined with the FCV virus-positive sample, while no fluorescent signal was observed in the virus-negative control group. This demonstrates that the recombinant antibody Ab-FCV VP1-4 can specifically bind to the FCV virus and can be used for the specific detection of feline calicivirus FCV.

[0152] Example 10 Antibody specificity verification

[0153] Use 1×10 4 TCID 50 / ml of FPV, FCV, and FHV cytotoxic supernatant was coated on the ELISA plate and incubated at 4°C overnight. Wash 5 times with PBST solution containing 0.05% Tween-20, discard PBST, add 2% skim milk powder blocking solution, and block at 37°C for 1h. Wash 5 times with PBST solution, pat dry the residual liquid in the wells, mix the recombinant antibody Ab-FCV VP1-4 and 2% skim milk powder blocking solution in a ratio of 3:2, let it stand at 25°C±5°C for 10min to remove interference, add to the ELISA plate, and incubate at 37°C for 1h. Wash 5 times with PBST solution, pat dry the residual liquid in the wells, add HRP-labeled goat anti-rabbit IgG secondary antibody, and incubate at 37°C for 1h. Wash 5 times with PBST solution, pat dry the residual liquid in the wells, add TMB colorimetric solution, let it stand in the dark for 15min, add 2mol / LH2SO4 to stop colorimetric development, and read the absorbance at 450nm (Table 7). Figure 6 As shown, the recombinant antibody Ab-FCV VP1-4 showed good affinity to FCV virus, and poor binding levels to FPV and FHV viruses, showing FCV virus specificity. This proves that the recombinant antibody Ab-FCV VP1-4 can specifically bind to FCV virus, can be used for the specific detection of feline calicivirus FCV, and is not confused with the specific detection of feline parvovirus and feline herpesvirus. It is an excellent antibody for feline calicivirus detection.

[0154] Table 7 Verification of specific binding of Ab-FCV VP1-4 to FCV, FPV, and FHV

[0155] 1 2 3 FCV 2.0273 2.1523 2.0741 FPV 0.0613 0.0801 0.0609 FHV 0.0536 0.0512 0.0688

[0156] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant antibody against feline calicivirus, characterized in that: The heavy chain variable region includes CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO:47 to SEQ ID NO:49; The light chain variable region includes CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO:50 to SEQ ID NO:

52.

2. The recombinant antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. A nucleic acid encoding the recombinant antibody according to claim 1 or 2.

4. A recombinant vector, characterized in that: It comprises a vector backbone and the nucleic acid according to claim 3.

5. A host cell, characterized in that Genome integration of the nucleic acid as claimed in claim 3, or transfection or transformation of the recombinant vector as claimed in claim 4.

6. The method for preparing a recombinant antibody according to claim 1 or 2, characterized in that: Cultivate the host cell according to claim 5 to obtain a mixture containing the recombinant antibody according to claim 1 or 2.

7. Use of at least one of the following I) to V) in the preparation of a feline calicivirus detection product: 1), the recombinant antibody according to claim 1 or 2; II), the nucleic acid according to claim 3; III), the recombinant vector according to claim 4; IV), the host cell according to claim 5; V), a mixture containing the recombinant antibody according to claim 1 or 2 obtained by the preparation method according to claim 6.

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