Fiber 1 protein-based assay for detecting antibodies against avian adenovirus type 4

Abstract

The present invention relates to the field of biotechnology detection, and specifically to an antibody for detecting type 4 avian adenovirus based on Fiber1 protein. The antibody described in the present invention is based on Fiber1 protein as an important immunogenic protein of avian adenovirus, which is relatively conservative among different serotype strains of avian adenovirus group I. The prepared recombinant antibody has good specificity and stability, and can be used not only for indirect immunofluorescence of avian adenovirus group I, but also for indirect ELISA detection, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology detection, and in particular to an antibody for detecting type 4 avian adenovirus based on Fiber 1 protein. Background Art

[0002] Fowl adenovirus (FAdV) belongs to the genus Aviadenovirus of the family Adenoviridae. It is a non-enveloped linear double-stranded DNA virus. It is divided into three groups based on antigenic differences. The traditional group I fowl adenovirus (FAdV-I) is divided into 12 serotypes. FAdV infection causes symptoms such as decreased egg production and respiratory diseases in chickens. Acute infection causes pericardial effusion, gizzard erosion, and inclusion body hepatitis. In severe cases, it can lead to death, causing huge losses to the breeding industry. The main prevalent strain in my country is FAdV-4.

[0003] Mature avian adenovirus consists of a capsid and a core, including hexon, penton, fiber, and minor structural proteins. Fiber protein contains important antigens of avian adenovirus and plays an important role in the viral infection process. Fiber protein can be divided into Fiber1 (long fiber protein) and Fiber2 (short fiber protein). Fiber1 is a surface protein of FAdV, which has a strong recognition ability for specific receptors on the host cell membrane. It plays a key role in the infection and adsorption of FAdV-4, and can effectively stimulate humoral immunity and even produce neutralizing antibodies.

[0004] Currently, there is no recombinant antibody with high activity and specificity for FAdV-4Fiber 1 protein for specific detection such as indirect immunofluorescence and indirect ELISA. Summary of the invention

[0005] In view of this, the present invention provides antibodies for detecting avian adenovirus type 4 based on Fiber 1 protein.

[0006] The present invention provides an antibody for detecting type 4 avian adenovirus based on Fiber 1 protein. The antibody described in the present invention is based on Fiber 1 protein as an important immunogenic protein of avian adenovirus, which is relatively conservative among different serotype strains of avian adenovirus group I. The prepared recombinant antibody has good specificity and stability, and can be used not only for indirect immunofluorescence of avian adenovirus group I, but also for indirect ELISA detection, and has broad application prospects.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a single-chain antibody comprising a heavy chain and a light chain:

[0009] The CDR region of the heavy chain includes CDR1, CDR2 and CDR3:

[0010] (I), the CDR1 of the heavy chain has the amino acid sequence shown in SEQ ID NO.2; and

[0011] (II), the CDR2 of the heavy chain has the amino acid sequence shown in SEQ ID NO.3; and

[0012] (III), the CDR3 of the heavy chain has the amino acid sequence shown in SEQ ID NO.4; or

[0013] (IV) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (I) to (III); or

[0014] (V) a sequence having an amino acid sequence homology of more than 80% with any one of (I) to (IV);

[0015] The CDR region of the light chain includes CDR1, CDR2 and CDR3:

[0016] (VI), the CDR1 of the light chain has the amino acid sequence shown in SEQ ID NO.11; and

[0017] (VII), the CDR2 of the light chain has the amino acid sequence shown in SEQ ID NO.12; and

[0018] (VIII), the CDR3 of the light chain has the amino acid sequence shown in SEQ ID NO.13; or

[0019] (IX) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence of any one of (VI) to (VIII); or

[0020] (X) A sequence having 80% or more homology with the amino acid sequence shown in any one of (VI) to (IX).

[0021] In some specific embodiments of the present invention, the heavy chain variable region of the single-chain antibody has:

[0022] (I), the amino acid sequence shown in SEQ ID NO.5; or

[0023] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0024] (III) a sequence having an amino acid sequence homology of more than 80% with the amino acid sequence shown in (I);

[0025] Its light chain variable region has:

[0026] (IV), the amino acid sequence shown in SEQ ID NO.14; or

[0027] (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (IV); or

[0028] (VI) A sequence having a homology of more than 80% with the amino acid sequence shown in (IV).

[0029] In some specific embodiments of the present invention, the heavy chain variable region and the light chain variable region are connected by a linker peptide;

[0030] The connecting peptide linker has:

[0031] (I), the amino acid sequence shown in SEQ ID NO.16; or

[0032] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0033] (III) A sequence having a homology of more than 80% with the amino acid sequence shown in (I).

[0034] In some specific embodiments of the present invention, the single-chain antibody comprises a heavy chain and a light chain:

[0035] The heavy chain includes CH1, hinge region, CH2, and CH3:

[0036] The heavy chain has:

[0037] (I), the amino acid sequence shown in SEQ ID NO.1; or

[0038] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0039] (III) a sequence having an amino acid sequence homology of more than 80% with the amino acid sequence shown in (I);

[0040] The CH1 has:

[0041] (I), the amino acid sequence shown in SEQ ID NO.6; or

[0042] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0043] (III) a sequence having an amino acid sequence homology of more than 80% with the amino acid sequence shown in (I);

[0044] The hinge region has:

[0045] (I), the amino acid sequence shown in SEQ ID NO.7; or

[0046] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0047] (III) a sequence having an amino acid sequence homology of more than 80% with the amino acid sequence shown in (I);

[0048] The CH2 has:

[0049] (I), the amino acid sequence shown in SEQ ID NO.8; or

[0050] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0051] (III) a sequence having an amino acid sequence homology of more than 80% with the amino acid sequence shown in (I);

[0052] The CH3 has:

[0053] (I), the amino acid sequence shown in SEQ ID NO.9; or

[0054] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0055] (III) a sequence having an amino acid sequence homology of more than 80% with the amino acid sequence shown in (I);

[0056] The light chain includes CL1;

[0057] The light chain has:

[0058] (IV), the amino acid sequence shown in SEQ ID NO.10; or

[0059] (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (IV); or

[0060] (VI), a sequence having a homology of more than 80% with the amino acid sequence shown in (IV);

[0061] The CL1 has:

[0062] (I), the amino acid sequence shown in SEQ ID NO.15; or

[0063] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0064] (III) A sequence having a homology of more than 80% with the amino acid sequence shown in (I).

[0065] In some specific embodiments of the present invention, the primers for amplifying the heavy chain variable region have:

[0066] (I), the nucleotide sequence shown in SEQ ID No. 17 to 22; or

[0067] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0068] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0069] (IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III);

[0070] The primers for amplifying the light chain variable region have:

[0071] (I), the nucleotide sequence shown in SEQ ID No. 23 to 28; or

[0072] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0073] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0074] (IV) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III).

[0075] In some specific embodiments of the present invention, the PCR program for amplifying the heavy chain variable region and the light chain variable region includes: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, and after 35 cycles, extension at 72°C for another 5 min.

[0076] In some specific embodiments of the present invention, the antibody library of the single-chain antibody is constructed by overlapping PCR;

[0077] The overlapping PCR comprises: overlapping extension first round PCR: without adding primers, denaturation at 98°C for 1 minute, annealing at 45°C for 30 seconds, extension at 72°C for 1 minute, repeating 10 cycles, and finally acting at 72°C for 5 minutes; adding primers overlapping PCR-FP and overlapping PCR-RP, denaturation at 98°C for 30 seconds, then annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, and extending at 72°C for another 5 minutes after 30 cycles;

[0078] The overlapping PCR-FP has:

[0079] (I), the nucleotide sequence shown in SEQ ID No.29; or

[0080] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0081] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0082] (IV) a nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III);

[0083] The overlapping PCR-RP has:

[0084] (I), the nucleotide sequence shown in SEQ ID No.30; or

[0085] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0086] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or

[0087] (IV) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (I) to (III).

[0088] The present invention also provides a nucleic acid molecule encoding the single-chain antibody.

[0089] Based on the above research, the present invention also provides an expression element, which includes a promoter, a terminator and the nucleic acid molecule.

[0090] In some specific embodiments of the present invention, the promoter includes but is not limited to CMV promoter; the terminator includes but is not limited to poly (A).

[0091] The present invention also provides an expression vector, comprising any of the following items and an acceptable vector:

[0092] (I), the nucleic acid molecule; and / or

[0093] (II) the expression element.

[0094] In some specific embodiments of the present invention, the vector comprises pComb3XSS, pTT5-V7 and / or pTT5-V8.

[0095] The present invention also provides a recombinant antibody, including an expression vector for expressing the recombinant antibody.

[0096] The present invention also provides a host, comprising any of the following:

[0097] (I), the single-chain antibody; and / or

[0098] (II), the nucleic acid molecule; and / or

[0099] (III), the expression element; and / or

[0100] (IV), the expression vector; and / or

[0101] (V) the recombinant antibody.

[0102] In some specific embodiments of the present invention, the host comprises Escherichia coli and / or HEK293f cells.

[0103] The present invention also provides the use of any of the following items in the preparation of a product for specifically detecting avian adenovirus type 4:

[0104] (I), the single-chain antibody; and / or

[0105] (II), the nucleic acid molecule; and / or

[0106] (III), the expression element; and / or

[0107] (IV), the expression vector; and / or

[0108] (V), the recombinant antibody; and / or

[0109] (VI), the host.

[0110] In some specific embodiments of the present invention, the specific detection method includes indirect immunofluorescence and indirect ELISA.

[0111] In some specific embodiments of the present invention, the specific detection of avian adenovirus type 4 comprises specific binding to FAdV-4 Fiber 1 protein based on any of the following:

[0112] (I), the single-chain antibody; and / or

[0113] (II), the nucleic acid molecule; and / or

[0114] (III), the expression element; and / or

[0115] (IV), the expression vector; and / or

[0116] (V), the recombinant antibody; and / or

[0117] (VI), the host.

[0118] The present invention also provides a product for detecting avian adenovirus type 4, comprising any of the following items:

[0119] (I), the single-chain antibody; and / or

[0120] (II), the nucleic acid molecule; and / or

[0121] (III), the expression element; and / or

[0122] (IV), the expression vector; and / or

[0123] (V), the recombinant antibody; and / or

[0124] (VI), the host.

[0125] The present invention also provides a method for preparing a recombinant antibody, comprising culturing the host to obtain the recombinant antibody.

[0126] The present invention also provides a method for detecting avian adenovirus type 4, taking a sample to be tested and using any of the following detection methods:

[0127] (I), the single-chain antibody; and / or

[0128] (II), the nucleic acid molecule; and / or

[0129] (III), the expression element; and / or

[0130] (IV), the expression vector; and / or

[0131] (V), the recombinant antibody; and / or

[0132] (VI), the host.

[0133] The advantages of the present invention are that the phage display library constructed by the present invention has extremely high application prospects in the screening of specific antibodies, and the screened anti-FAdV-4Fiber 1 recombinant antibody Ab-FAdV-4Fiber 1 has strong specificity and high affinity, can be used for the specific detection of FAdV-4, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0135] Figure 1 The construction of the FAdV-4Fiber 1-scFv phage library and the diversity analysis of the immune library in the examples are shown; wherein, A shows the antibody titer detection of New Zealand rabbits immunized 4 times; B shows that the amplified bands are the VL gene library and the VH gene library, M: Marker; 1: VL target band; 2: VH target band; 3: VL-linker-VH target band;

[0136] Figure 2 The figure shows the screening of high affinity FAdV-4Fiber 1-scFv in the embodiment; wherein A shows the PCR identification result after electroporation; B shows the diversity of the antibody library after the first round of phage display screening; C shows the diversity of the antibody library after the second round of phage display screening; D shows the second round of phage ELISA to determine the screening of scFv antibodies with high specificity binding to the antigen FAdV-4Fiber 1;

[0137] Figure 3 The expression and affinity of Ab-FAdV-4Fiber 1 in the embodiment are shown; wherein, A shows the SDS-PAGE result of Ab-FAdV-4Fiber 1, M: Marker; 1: Ab-FAdV-4Fiber 1 target band; B shows the EC of Ab-FAdV-4Fiber1 antibody and antigen FAdV-4Fiber 1 50 ; C shows the results of indirect immunofluorescence experiment of Ab-FAdV-4Fiber 1 and FAdV-4 virus;

[0138] Figure 4Shows the plasmid maps of pTT5-V7 and pTT5-V8; wherein, A shows the plasmid map of pTT5-V7; B shows the plasmid map of pTT5-V8. DETAILED DESCRIPTION

[0139] The present invention discloses antibodies for detecting type 4 avian adenovirus based on Fiber 1 protein. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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 described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0140] The present invention constructs a large-capacity phage single-chain antibody library through phage display technology, and after two rounds of enrichment screening, a single-chain antibody (single chain antibody fragment, scFv) FAdV-4Fiber 1-scFv that can specifically bind to FAdV-4Fiber 1 protein is obtained. VH and VL are grafted onto vectors pTT5-V7 and pTT5-V8 containing mouse antibody skeletons using molecular biological techniques, and expressed and purified in eukaryotic cells HEK293f. Indirect ELISA experiments confirm that the recombinant antibody Ab-FAdV-4Fiber 1 obtained after purification can specifically bind to the antigen and has high affinity. Based on this, the present invention claims protection for the following technical solutions:

[0141] A recombinant antibody against FAdV-4Fiber 1 protein, wherein the recombinant antibody is Ab-FAdV-4Fiber 1, and the recombinant antibody comprises a heavy chain VH and a light chain VL.

[0142] The amino acid sequences of the heavy chain variable regions of Ab-FAdV-4Fiber 1 and FAdV-4Fiber 1-scFv are shown in SEQ ID NO.5, and the amino acid sequences of the light chain variable regions of Ab-FAdV-4Fiber 1 and FAdV-4Fiber 1-scFv are shown in SEQ ID NO.14;

[0143] The heavy chain variable region and the light chain variable region of the single-chain antibody FAdV-4Fiber 1-scFv are connected by a connecting peptide linker, and the amino acid sequence of the connecting peptide linker is shown in SEQ ID NO.16;

[0144] The present invention also protects all nucleotide sequences encoding the above-mentioned anti-FAdV-4Fiber 1 protein recombinant antibody.

[0145] The present invention also protects a recombinant DNA expression vector comprising the above polynucleotide molecule.

[0146] The single-chain antibody FAdV-4Fiber 1-scFv was formed by homologous recombination of scFv. First, the scFv sequence with high affinity was obtained by phage display. Secondly, VH and VL were grafted onto the vectors pTT5-V7 and pTT5-V8 containing the mouse antibody backbone using molecular biology techniques, and the Ab-FAdV-4Fiber 1 recombinant antibody was expressed using HEK293f. The binding of antigen and antibody was detected by indirect ELISA experiment. The results showed that 2 strains of FAdV-4Fiber 1-scFv were strongly positive in the ELISA test. Finally, a high-affinity Ab-FAdV-4Fiber1 for the FAdV-4Fiber 1 target antigen was screened by EC50.

[0147] Strains, plasmids, experimental animals and reagents used in the present invention

[0148] Adult New Zealand rabbits were purchased from Yongkang Qingyuan Farm, Dingyuan County, Anhui Province; FAdV-4 Fiber 1 protein was prepared and stored by Shenzhen Hertz Biotechnology Co., Ltd. (amino acid sequence is SEQ ID NO.33); FAdV-4 virus was stored by Shenzhen Hertz Biotechnology Co., Ltd. (GenBank: KM096544.1); vectors pTT5-V7 and pTT5-V8 plasmids containing mouse antibody backbone were stored by Shenzhen Hertz Biotechnology Co., Ltd. ( Figure 4 ); HRP-labeled recombinant protein G was purchased from Shanghai Bioengineering Co., Ltd.; HRP-labeled mouse anti-M13K07 phage was purchased from Chengdu Apak Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse 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 Novozyme Biotechnology Co., Ltd.; Premix taq, Plasmid mini kit, Gel Extraction and transfection reagents were all purchased from TaKaRa.

[0149] Sequences used in the present invention:

[0150] SEQ ID NO.1: Heavy chain amino acid sequences of single-chain antibody FAdV-4Fiber1-scFv and recombinant antibody Ab-FAdV-4Fiber1:

[0151]

[0152] Among them, CDR1: GIDLNSYG (SEQ ID NO.2), CDR2: IDTTDIV (SEQ ID NO.3), CDR3: ARSSGSAGVWGYGMDP (SEQ ID NO.4)

[0153] The amino acid sequence of the heavy chain variable region of the single-chain antibody FAdV-4Fiber1-scFv and the recombinant antibody Ab-FAdV-4Fiber1 (SEQ ID NO.5):

[0154] QKQLVESGGRLVTPGAALTLTCTVSGIDLNSYGMSWVRQAPGKGLEWIGMIDTTDIVDYASWANGRFTISKSSTTVDLKIISPTTEDTATYFCARSSGSAGVWGYGMDPWGPGTLVTVSS

[0155] CH1 (SEQ ID NO.6):

[0156] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVR TARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0157] Hinge region (SEQ ID NO.7):

[0158] APSTCSKPTCPP

[0159] CH2 (SEQ ID NO.8):

[0160] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0161] CH3 (SEQ ID NO.9):

[0162] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0163] Amino acid sequence of the light chain of single-chain antibody FAdV-4Fiber1-scFv and recombinant antibody Ab-FAdV-4Fiber1 of SEQ ID NO.10:

[0164] DVVMTQTPSSVEAAVGGTVTIKCQASQKINSWLSWYQQKPGQPPRLLIYQASKLASGVSSRFKGSGSGTEFTLTISDLECDDAATYYCQQGFRVSDVDNPFGGGTEV VVGGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*

[0165] Among them, CDR1: QKINSW (SEQ ID NO. 11), CDR2: QAS (SEQ ID NO. 12), CDR3: QQGFRVSDVDNP (SEQ ID NO. 13);

[0166] The amino acid sequence of the light chain variable region of the single-chain antibody FAdV-4Fiber1-scFv and the recombinant antibody Ab-FAdV-4Fiber1 (SEQ ID NO.14):

[0167] DVVMTQTPSSVEAAVGGTVTIKCQASQKINSWLSWYQQKPGQPPRLLIYQASKLAS GVSSRFKGSGSGTEFTLTISDLECDDAATYYCQQGFRVSDVDNPFGGGTEVVVG

[0168] CL1 (SEQ ID NO.15):

[0169] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKT PQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*

[0170] SEQ ID NO.16 Amino acid sequence of linker peptide

[0171] GGGGSGGGGGGSSRSS

[0172] SEQ ID NO. 17 VH-scFv-F1

[0173] 5'–GGTGGGGGTGGGTTCCTCTAGATCTTCCCAGTCGKTGGAGGAGTCC-3'

[0174] SEQ ID NO. 18 VH-scFv-F2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0175] <h2 style=";text-align:left;direction:ltr"> 5'–GGTGGGGGTGGTTCCTCTAGATCTTCCCAGWCAGTGAAGGAGTCC-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0176] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.19VH-scFv-F3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0177] <h2 style=";text-align:left;direction:ltr"> 5'–GGTGGGGGTGGTTCCTCTAGATCTTCCCAGTCCGCTGGRGGAGTCC-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0178] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.20VH-scFv-F4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0179] <h2 style=";text-align:left;direction:ltr"> 5'–GGTGGGGGTGGTTCCTCTAGATCTTCCCAGTCGGTGGAGGGAGTCC-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0180] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.21VH-scFv-R1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0181] <h2 style=";text-align:left;direction:ltr"> 5'–AGGAGGCTATTGGCCGGCCTGGCCTGARGAGAYGGTGACCAGGTGCC-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0182] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.22VH-scFv-R2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0183] <h2 style=";text-align:left;direction:ltr"> 5'–AGGAGGCTATTGGCCGGCCTGGCCGCAGCAGGGGGCCAGTGGGAAGACTGA C-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0184] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.23VL-scFv-F1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0185] <h2 style=";text-align:left;direction:ltr"> 5'–AAAAGAGGCCCAGGCGGCCGAGCTCGTGMTGACCCAGACTCCA-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0186] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.24VL-scFv-F2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0187] <h2 style=";text-align:left;direction:ltr"> 5'–AAAAGAGGCCCAGGCGGCCGAGCTCGATMTGACCCAGACTCCA-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0188] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.25VL-scFv-F3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0189] <h2 style=";text-align:left;direction:ltr"> 5'–AAAAGAGGCCCAGGCGGCCGCYCAAGTGCTGACCCAG-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0190] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.26VL-scFv-F4

[0191] 5’–AAAAGAGGCCCAGGCGGCCGCCCWAGTGATGACCCAG-3’

[0192] SEQ ID NO.27 VL-scFv-R1

[0193] 5’–GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCTTTGATTTCCACATTGGTGCC-3’

[0194] SEQ ID NO.28 VL-scFv-R2

[0195] 5’–GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCTAGGATCTCCAGCTCGGTCCC-3’

[0196] SEQ ID NO.29 Overlap PCR-FP

[0197] 5’-GAGGAGGAAAAAGAGGCCCAGGCGGCC-3’

[0198] SEQ ID NO.30 Overlap PCR-RP

[0199] 5’-AGAGGAGGCTATTGGCCGGCCTGGCC-3’

[0200] SEQ ID NO.31 pComb3XSS-FP

[0201] 5’–GCTGGTTTCGCTACCGTGGCCCAGGCGGCC-3’

[0202] SEQ ID NO.32 pComb3XSS-RP

[0203] 5’–GTGATGGTGATGGTGCTGGCCGGCCTGGCC-3’

[0204] SEQ ID NO.33 Amino acid sequence of FAdV-4 Fiber 1 protein

[0205] MSALIASAADTVSASGKKRPRRALSEPSRYLSEGDERRKPKRARPATRANGPLLDLVYPFDFNAGGGGGSGGGGGGGGGQQIAVDPDGPLELTGDLLTLNTKTPIYV SDRAVSLLIDDNTLATKQANGALMVKTAAPLNSGTGGGVTLGFDPRTMALDSVTGVLKVLVDSQGPLQADTGGITLQFDTQDFVVNNGVLALASSVGPTYLSPFATYE VTPVLGISQRNGNVKSKGLQNWSIGYYIYMVSSAGLVNGLITLELAHDLTGASGENSLTSGLNFTFVLSPMYPIETEVNLSLIVPPTVSPTNQNHVFVPNSNQSDVGY LGLPPHTRDNWYVPIDSPGLRLVSFMPTATGNEKFGQGTLGYCAATIQNTSSGTTPSDAIAFTVSLPQTSGSNWFDQNAPDTVVTTGPIPFSYQGYVYSPNGNNAPGP

[0206] In view of the technical demand for FAdV-4 detection antibodies in the art, the present invention aims to provide a recombinant antibody of FAdV-4 Fiber 1 protein with high activity and high specificity for specific detection such as indirect immunofluorescence and indirect ELISA.

[0207] The raw materials and reagents used in the antibody for detecting type 4 avian adenovirus based on Fiber 1 protein provided by the present invention can all be purchased from the market.

[0208] The present invention will be further described below in conjunction with embodiments:

[0209] Example 1 Animal Immunization

[0210] 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 FAdV-4 virus was injected into the back of the neck of New Zealand rabbits, and immunized 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 to separate serum for storage. 7 days after the fourth immunization (i.e., day 28), 0.5mL of venous blood was collected to separate serum for storage. ELISA results showed that the serum antibody titer increased significantly after the first immunization, and the antibody titer was greater than 10 after the second immunization. 4 , after the third immunization, it exceeded 6.4×10 4 , after the fourth immunization, it reached 1.28×10 5, compared with the serum antibody titer before immunization, there was a significant increase ( Figure 1 A) is shown, and the original data are shown in Table 1. On the 28th day, the New Zealand rabbits were killed, 0.5 mL of blood was collected and the serum was separated and stored, and the spleen was removed by dissection for lymphocyte separation.

[0211] Table 1 Antibody titers of rabbits immunized with avian adenovirus

[0212] Negative group One Free Second exemption Three exemptions Four free Rabbit 1 200 16000 64000 128000 256000 Rabbit 2 200 16000 16000 64000 128000 Rabbit 3 200 16000 16000 64000 128000 Rabbit 4 200 32000 64000 64000 128000

[0213] Example 2 Extraction of total RNA from lymphocytes and amplification of antibody light and heavy chain genes

[0214] Total RNA of lymphocytes was extracted with Trizol reagent, and it was used as a template for reverse transcription and synthesis of cDNA using Oligo (dT) as primer. Primer sequence combinations were designed according to the framework regions of the light and heavy chains of the rabbit antibody sequence (primer sequences were SEQ ID NO.17 to SEQ ID NO.28), and PCR was performed to amplify the coding regions of antibody VH and VL. 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 about 350 bp, and was purified and recovered for later use ( Figure 1 B) shown.

[0215] Example 3 Construction of FAdV-4Fiber 1-scFv Antibody Library

[0216] Using purified VH and VL as templates, a linker (sequence: SEQ ID NO. 16) was inserted between VH and VL after overlapping PCR to synthesize the scFv fragment VH-linker-VL, which contained sfiI restriction enzyme sites at both ends of the sequence.

[0217] Overlap extension first round PCR: without primers, denaturation at 98℃ for 1min, annealing at 45℃ for 30s, extension at 72℃ for 1min, repeat 10 cycles, and finally at 72℃ for 5min. Add primers overlap PCR-FP and overlap PCR-RP, denaturation at 98℃ for 30s, then annealing at 58℃ for 30s, extension at 72℃ for 1min, and after 30 cycles, extend at 72℃ for another 5min. PCR products were recovered by gel recovery using 1% agarose gel electrophoresis, and the band was about 800bp ( Figure 1 B) shown, stored at -20° C. The overlapping PCR primer sequences are SEQ ID NO. 29 to SEQ ID NO. 30.

[0218] The PCR product and pComb3XSS vector after gel recovery were digested with restriction endonuclease sfiI at 50°C for 30 minutes. The digestion products were verified by 1% agarose gel electrophoresis and then purified by column recovery and ligated with T4 DNA ligase to obtain the FAdV-4Fiber 1-scFv-pComb3XSS library. The ligation products were transformed into ER2738 competent cells, and positive clones were screened with tetracycline and ampicillin resistant 2YT (2×Yeast extract and Tryptone) solid culture medium. 20 single clones in the library were randomly selected for PCR identification (primer sequences were SEQ ID NO.31 to SEQ ID NO.32), and the antibody library recombination rate was calculated to be 65% ( Figure 2 All clones on the solid medium were washed off with 2YT medium and stored at 4°C.

[0219] Example 4 Amplification of bacteriophage

[0220] Take 2 mL of the bacterial culture solution of all clones washed with 2YT medium and inoculate it into 50 mL of 2YT liquid medium. Activate at 37°C and 200 rpm until OD 600 When the concentration reaches 0.8-1.0, add the final concentration of 1×10 12 pfu / mL of M13K07 helper phage for infection, let stand for 30 minutes, add kanamycin (final concentration of 50μg / mL), and culture overnight at 30℃200rpm. The next day, centrifuge at 10000rpm at 4℃ for 20 minutes, retain the supernatant and add 1 / 2 volume of PEG8000 to it, let stand on ice for 2 hours to precipitate the phage. Centrifuge at 10000rpm at 4℃ for 20 minutes, discard the supernatant. Resuspend the precipitate with 5mL PBS, add 3mL PEG8000 to re-precipitate, repeat the centrifugation operation, resuspend the precipitate with 4mL PBS, filter and sterilize, and store at 4℃ for use.

[0221] Example 5 FAdV-4 Fiber 1-scFv Screening

[0222] Biotinylate the FAdV-4Fiber 1 protein in advance. After mixing the amplified scFv library phage, Dynabeads M-280 magnetic beads labeled with FAdV-4Fiber1 protein and 2% skim milk powder blocking solution, incubate at 25℃±5℃ for 1h, and wash the magnetic beads with PBST 5-10 times. Elute the phage on the surface of the magnetic beads with 0.1mol / L Glycine-HCl (pH=2.0), and neutralize with 1mol / L Tris to pH 7.0 after elution. Infect ER2738 competent cells in the logarithmic growth phase with the neutralized phage after elution, culture and enrich on 2YT solid medium, and harvest the colonies for the next round of screening.

[0223] 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 FAdV-4Fiber 1-scFv library in the first round of panning was 90%, and the positive rate in the second round of panning was 100% ( Figure 2 B shown and C shown).

[0224] Example 6 Phage ELISA

[0225] The purified FAdV-4Fiber 1 antigen was coated on the ELISA plate at a concentration of 50μg / mL per well and incubated at 4℃ 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℃ for 1h. Wash 5 times with PBST solution, pat dry the residual liquid in the well, mix the supernatant of the positive clone antibody to be screened and 2% skim milk powder blocking solution in a ratio of 3:2, let stand at 25℃±5℃ for 10min to remove interference, add to the ELISA plate, and incubate at 37℃ for 1h. Wash 5 times with PBST solution, pat dry the residual liquid in the well, add HRP-labeled mouse anti-M13K07 antibody (diluted with blocking solution 1:3000), and incubate at 37℃ for 1h. Wash 5 times with PBST solution, pat dry the residual liquid in the well, add TMB colorimetric solution, let stand for 15min away from light, and add 2mol / LH 2 SO 4 Stop color development and read the absorbance at 450 nm (Table 2, bold numbers indicate increasing numbers from left to right and from top to bottom). Figure 2 As shown in D, two high-affinity antibodies were screened, and No. 10 with higher affinity was selected for subsequent experiments.

[0226] Table 2 Affinity test of Ab-FAdV-4Fiber 1 and antigen FAdV-4Fiber 1

[0227] 1 2 3 4 5 6 7 8 9 10 11 12 1 0.0456 0.0473 0.046 0.0467 0.0549 0.047 0.045 0.0483 0.2534 0.7321 0.0443 0.0421 13 0.051 0.0517 0.0432 0.0445 0.3142 0.2488 0.0532 0.1443 0.0675 0.0534 0.1548 0.0451 25 0.0547 0.0419 0.2768 0.0491 0.0603 0.0517 0.0489 0.0499 0.054 0.3606 0.0493 0.0509 37 0.0336 0.0236 0.0252 0.0211 0.0228 0.1069 0.0239 0.0459 0.2168 0.0167 0.0184 0.0152 49 0.0621 0.0547 0.0568 0.0561 0.0604 0.0596 0.0521 0.052 0.6147 0.0574 0.0607 0.0622 61 0.0568 0.0531 0.0539 0.0527 0.0618 0.0559 0.0533 0.0532 0.0566 0.0536 0.0538 0.064 73 0.0653 0.0609 0.0571 0.0667 0.2615 0.0572 0.0602 0.0605 0.2405 0.0576 0.0586 0.0593 85 0.064 0.0554 0.0584 0.0622 0.0537 0.289 0.0923 0.0552 0.0536 0.2543 0.054 0.0503

[0228] Example 7 Recombination, expression and purification of FAdV-4Fiber 1 recombinant antibody Ab-FAdV-4Fiber 1

[0229] The VH and VL sequences of the single-chain antibody scFv were amplified and purified, and the pTT5-V7 and pTT5-V8 vectors containing the mouse antibody backbone were double-digested with EcoRI and HindIII restriction endonucleases, and the antibody expression plasmids VH-pTT5-V7 and VL-pTT5-V8 were obtained by homologous recombination. The recombinant plasmids VH-pTT5-V7 and VL-pTT5-V8 were co-transfected into HEK293f cells by PEI for eukaryotic expression. After 48 hours, the culture supernatant was collected by centrifugation at 10000rpm and 4℃ for 20 minutes, and the antibody was purified using a Protein A affinity chromatography column, and the fusion protein was finally stored in PBS solution.

[0230] The purified samples were added to the reduced protein loading buffer and subjected to SDS-PAGE electrophoresis after denaturation. Figure 3 As shown in A, the disulfide bonds between the heavy chain and the light chain of the antibody were reduced, and target bands were detected at around 50 kDa and 25 kDa, which was consistent with the expected results.

[0231] Example 8 Indirect ELISA method to detect the binding of recombinant antibody Ab-FAdV-4Fiber 1 and FAdV-4Fiber 1 antigen

[0232] The affinity of Ab-FAdV-4Fiber 1 to antigen FAdV-4Fiber 1 was determined by indirect ELISA. Antigen was added to the ELISA plate and incubated overnight at 4°C, and then blocked with 2% skim milk powder blocking solution at 37°C for 2h. The antibody was diluted 12 times with 2% skim milk powder and added to the wells, and incubated at 37°C for 1h. HRP-labeled goat anti-mouse IgG secondary antibody was added, and TMB was used for color development, and the absorbance at a wavelength of 450nm was detected in an ELISA instrument (Table 3).

[0233] The binding effect of the above antibody Ab-FAdV-4Fiber 1 was tested as follows Figure 3 As shown in Figure B, the dose-effect curve was successfully fitted with the regression model, and Ab-FAdV-4Fiber 1 was able to effectively bind to the FAdV-4Fiber 1 protein. The EC of the binding between the antibody Ab-FAdV-4Fiber1 and the recombinantly expressed FAdV-4Fiber 1 protein was 50 1.95×10 -1 μg / mL, indicating that the recombinant antibody was active and had good affinity.

[0234] Table 3 EC of the binding of Ab-FAdV-4Fiber 1 and antigen FAdV-4Fiber 1 50

[0235]

[0236] Example 9 Indirect immunofluorescence detection of recombinant antibodies

[0237] Use DMEM / F12 containing 10% FBS to resuscitate LMH adherent cells. When the cell confluency reaches 80-90%, inoculate FAdV-4 venom with an MOI of 0.1. After infection in a 37°C 5% CO2 incubator for 1 hour, discard the culture medium, add 2% FBSDMEM / F12, and culture in a cell culture incubator. At the same time, set up LMH cells that are not infected with FAdV-4 virus as negative controls. After 5 days, wash the cells 3 times with PBS, add 300μL 4% paraformaldehyde to fix the cells, and wash the cells 3 times with PBS after 30 minutes. Add 500μL 0.1% TritonX100, and wash the cells 3 times with PBS after 15 minutes. Block with PBS containing 3% sodium caseinate at 37°C for 1 hour and wash 3 times with PBS. The recombinant antibody Ab-FAdV-4Fiber 1 was used as the primary antibody for indirect immunofluorescence at a concentration of 1 μg / mL and incubated at 37°C for 1 h. The secondary antibody was goat anti-mouse FITC and incubated at 37°C for 1 h. The inverted fluorescence microscope was used for observation ( Figure 3 C). Figure 3 As shown in C, the recombinant antibody Ab-FAdV-4Fiber 1 can specifically detect the FAdV-4 virus, and the reaction result is positive, which proves that the recombinant antibody has high specificity and can be used for the specific detection of avian adenovirus FAdV-4.

[0238] The above is only a preferred embodiment 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A single-chain antibody, characterized in that It consists of heavy and light chains: The CDR region of the heavy chain includes CDR1, CDR2 and CDR3: (I), the amino acid sequence of CDR1 of the heavy chain is shown in SEQ ID NO.2; and (II), the amino acid sequence of CDR2 of the heavy chain is shown in SEQ ID NO.3; and (III), the amino acid sequence of CDR3 of the heavy chain is shown in SEQ ID NO.4; The CDR region of the light chain includes CDR1, CDR2 and CDR3: (IV), the amino acid sequence of CDR1 of the light chain is shown in SEQ ID NO.11; and (V), the amino acid sequence of CDR2 of the light chain is shown in SEQ ID NO.12; and (VI) The amino acid sequence of CDR3 of the light chain is shown in SEQ ID NO.

13.

2. The single-chain antibody according to claim 1, characterized in that The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.5; The amino acid sequence of its light chain variable region is shown in SEQ ID NO.

14.

3. The single-chain antibody according to claim 2, characterized in that The heavy chain variable region and the light chain variable region are connected by a connecting peptide linker; The amino acid sequence of the linker peptide is shown in SEQ ID NO.

16.

4. A nucleic acid molecule, characterized in that Encodes the single-chain antibody according to any one of claims 1 to 3.

5. An expression element, characterized in that It comprises a promoter, a terminator and the nucleic acid molecule according to claim 4.

6. An expression vector, characterized in that Includes any of the following and acceptable carriers: (I), the nucleic acid molecule according to claim 4; and / or (II) The expression element as described in claim 5.

7. A recombinant antibody, characterized in that Comprising expression of the expression vector as described in claim 6.

8. A host, characterized in that Include any of the following: (I), the single-chain antibody according to any one of claims 1 to 3; and / or (II), the nucleic acid molecule according to claim 4; and / or (III), the expression element according to claim 5; and / or (IV), the expression vector according to claim 6; and / or (V) The recombinant antibody according to claim 7.

9. Use of any of the following in the preparation of a product for specific detection of avian adenovirus type 4: (I), the single-chain antibody according to any one of claims 1 to 3; and / or (II), the nucleic acid molecule according to claim 4; and / or (III), the expression element according to claim 5; and / or (IV), the expression vector according to claim 6; and / or (V) the recombinant antibody according to claim 7; and / or (VI) The host according to claim 8; The specific detection methods include indirect immunofluorescence and indirect ELISA.

10. A product for detecting avian adenovirus type 4, characterized in that: Include any of the following: (I), a single-chain antibody as described in any one of claims 1 to 3; and / or (II), a nucleic acid molecule as described in claim 4; and / or (III), an expression element as described in claim 5; and / or (IV), an expression vector as described in claim 6; and / or (V), a recombinant antibody as described in claim 7; and / or (VI), a host as described in claim 8.

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