CN111704665B

CN111704665B - Recombinant caninized antibody scFv-Fc for H3N2 canine influenza virus

Info

Publication number: CN111704665B
Application number: CN202010105415.0A
Authority: CN
Inventors: 刘永杰; 赵丹; 韩德敏; 董雨豪; 赵艳兵
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-02-20
Filing date: 2020-02-20
Publication date: 2022-04-01
Anticipated expiration: 2040-02-20
Also published as: CN111704665A

Abstract

The invention discloses a recombinant caninized antibody scFv-Fc aiming at H3N2 canine influenza virus, which comprises the steps of extracting total RNA of infected cells and obtaining cDNA through reverse transcription; amplifying VH, VL and Fc genes; then carrying out PCR fusion, and extracting recombinant plasmid T-scFv and/or T-CE-Fc; amplifying scFv and/or Fc by the T-scFv and/or T-CE-Fc, connecting the scFv and/or the Fc target fragment into a cloning vector, and selecting a positive strain; and (2) amplifying by using the scFv as a template and using the CE-scFv-F and/or CE-scFv-R as a primer, performing homologous recombination and connection on a product and a carrier, transforming Rosetta competent cells after completion, and selecting a positive strain to be named as ET-32a-scFv-Fc after screening a resistant plate. The invention can be used for preparing similar biological agents. The invention aims at the recombinant caninized antibody of H3N2 subtype CIV, and lays a material foundation for preventing and controlling the infection of the canine groups caused by the virus and coping with the potential threats to human health and public health.

Description

Recombinant caninized antibody scFv-Fc for H3N2 canine influenza virus

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant caninized antibody scFv-Fc for H3N2 canine influenza virus.

Background

Canine Influenza Virus (CIV) is an RNA virus that causes respiratory symptoms such as cough, watery nasal discharge, and fever in dogs, and is a highly contagious respiratory infectious disease virus that can cause epidemics in dogs. In 2004, equine derived influenza virus subtype H3N8 was first reported in the united states to spread horizontally among canine populations, changing the view that canines are resistant to influenza viruses. Subsequently, canine respiratory diseases caused by influenza viruses of H3N2 subtype appeared in korea in 2007. In 2015, chicago in the united states developed subtype CIV H3N2, which then passed to other continents of the united states and even canada. Currently, the main canine influenza virus subtype in china is H3N 2.

CIV threatens the survival and health of dogs and at the same time carries a potential risk to human health. Firstly, after the canine influenza virus is adapted to sialic acid receptors in mammals such as dogs, the canine influenza virus may obtain the ability to infect humans, and the canine influenza virus serving as a companion animal of humans is likely to be used as an intermediate host to transmit the influenza virus to humans; secondly, researches show that the influenza viruses with various subtypes and sources can infect dogs, and when more than two influenza virus subtypes exist in dogs, reassortment is likely to be carried out in the dogs, so that new influenza viruses are recombined, and great pressure is brought to influenza prevention and control. In 2013-2015 years, researchers find a novel influenza virus formed by reassortment of 3 swine influenza viruses and CIV genes. Scholars even believe that dogs may become the next new influenza virus "blender" that causes pandemics. Therefore, the control of the spread and prevalence of CIV is necessary and has great significance in public health.

The passive immunity of the antibody is an ideal treatment strategy for treating the acute infection of the virus, can effectively block the invasion of the pathogen in time, can effectively capture the pathogen, and can quickly neutralize and eliminate the pathogen. Currently, most of the therapeutic antibodies studied are mainly monoclonal antibodies (abbreviated as monoclonal antibodies), while most of the monoclonal antibodies are of murine origin, and the heterogeneity thereof limits the application in dogs. Qiu winter et al prepared CIV single chain antibody (scFv), which was confirmed to have better neutralizing activity. However, the biggest drawback of scFv is that it binds monovalently to the target antigen and does not stay in vivo for a long time. An effective way to extend the half-life of an scFv is to fuse to the Fc region of an IgG molecule. To date, there has been no report of treatment against canine influenza virus to prepare recombinant caninized antibodies.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above-mentioned technical drawbacks. The invention aims to provide a recombinant caninized antibody scFv-Fc aiming at H3N2 subtype CIV, the recombinant caninized antibody can be specifically combined with H3N2 subtype CIV, has certain neutralization activity, and lays a foundation for applying the antibody to the prevention and treatment of canine influenza.

Therefore, in one aspect of the invention, the defects in the prior art are overcome, and the recombinant caninized antibody scFv-Fc aiming at the H3N2 canine influenza virus is provided.

In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing recombinant caninized antibody scFv-Fc aiming at H3N2 subtype CIV comprises the steps of extracting total RNA of infected cells and carrying out reverse transcription to obtain cDNA; amplifying VH, VL and Fc genes by taking the cDNA as a template and one or more of VH-F and/or VH-R, VL-F and/or VL-R, Fc-F and/or Fc-R as primers; taking the amplified VH, VL and Fc groups as templates, taking one or more of CIV-VH-F and/or CIV-VH-R, CIV-VL-F and/or CIV-VL-R, CIV-Fc-F and/or CIV-Fc-R as primers, amplifying VH, VL and Fc genes again, then carrying out PCR fusion, carrying out gel recovery and connecting into a cloning vector, transforming DH5 alpha competent cells, selecting positive bacteria, and extracting recombinant plasmid T-scFv and/or T-CE-Fc; amplifying scFv and/or Fc by the T-scFv and/or T-CE-Fc, connecting the scFv and/or the Fc target fragment into a cloning vector, transforming Rosetta competent cells after completion, screening, and selecting a positive strain to be named as pET-32a-scFv and/or pET-32 a-Fc; and (2) amplifying by using the scFv as a template and using the CE-scFv-F and/or CE-scFv-R as a primer, performing homologous recombination and connection on a product and a carrier, transforming Rosetta competent cells after completion, and selecting a positive strain to be named as ET-32a-scFv-Fc after screening a resistant plate.

As a preferable embodiment of the method for producing the recombinant caninized antibody scFv-Fc against H3N2 canine influenza virus according to the present invention, wherein: and the scFv and/or the Fc target fragment are subjected to homologous recombination connection with a carrier, and the connection sequence comprises one or more of VL- (Gly4Ser)4-VH and VH- (Gly4Ser) 4-VL.

As a preferred embodiment of the method for preparing the recombinant caninized antibody scFv-Fc against H3N2 subtype CIV according to the invention, wherein: and (3) carrying out homologous recombination connection on the scFv and/or the Fc target fragment and a carrier, wherein the connection sequence is VH- (Gly4Ser) 4-VL-Fc.

As a preferred embodiment of the method for preparing the recombinant caninized antibody scFv-Fc against H3N2 subtype CIV according to the invention, wherein: the plasmid is connected into a cloning vector, and is connected into any one of the vectors pClone007 and pMD-19T after gel recovery.

As a preferable embodiment of the method for producing the recombinant caninized antibody scFv-Fc against H3N2 canine influenza virus according to the present invention, wherein: the annealing temperature of the amplification is 55-60 ℃.

As a preferable embodiment of the method for producing the recombinant caninized antibody scFv-Fc against H3N2 canine influenza virus according to the present invention, wherein: the final PCR amplification system is 25 μ L, wherein the template cDNA is 2.0 μ L, Prime STAR HS is 12.5 μ L, the upstream primer and the downstream primer are respectively 1.0 μ L (10 μ M/. mu.L), ddH₂O 8.5μL。

As another aspect of the invention, the invention provides a primer for preparing a recombinant caninized antibody scFv-Fc against H3N2 canine influenza virus in the method of any one of claims 1 to 6, which comprises the VH-F sequence shown in SEQ ID NO. 1; the sequence of the VH-R is shown as SEQ ID NO. 2; the sequence of the VL-F is shown as SEQ ID NO. 3; the sequence of the VL-R is shown as SEQ ID NO. 4; the sequence of the Fc-F is shown as SEQ ID NO. 5; the sequence of the Fc-R is shown as SEQ ID NO. 6; the sequence of the CIV-VH-F is shown as SEQ ID NO. 7; the sequence of the CIV-VH-R is shown as SEQ ID NO. 8; the sequence of the CIV-VL-F is shown as SEQ ID NO. 9; the sequence of the CIV-VL-R is shown as SEQ ID NO. 10; the sequence of the CIV-Fc-F is shown as SEQ ID NO. 11; the sequence of the CIV-Fc-R is shown in SEQ ID NO. 12; the sequence of the CE-scFv-F is shown as SEQ ID NO. 13; the sequence of the CE-scFv-R is shown as SEQ ID NO. 14; the sequence of the VH is shown as SEQ ID NO. 15; the sequence of the VL is shown as SEQ ID NO. 16; the sequence of the Fc is shown as SEQ ID NO. 17; the sequence of the scFv-Fc is shown as SEQ ID NO. 18; the scFv has a sequence shown in SEQ ID NO. 19-SEQ ID NO. 29.

As another aspect of the invention, the invention provides an application of the recombinant caninized antibody scFv-Fc aiming at H3N2 subtype CIV, which is an application in preparing a medicine or an immune preparation for preventing or controlling influenza virus.

The invention has the beneficial effects that:

the preparation method of the recombinant caninized antibody of the invention, which aims at the H3N2 subtype CIV, can be used for preparing similar biological agents. The invention aims at the recombinant caninized antibody of H3N2 subtype CIV, and lays a material foundation for preventing and controlling the infection of the canine groups caused by the virus and coping with the potential threats to human health and public health. In the invention, the applied production technology is mature, the operation is simple, and the preparation period is short. The recombinant canine antibody produced by the technology has the advantages of high specificity and purity, stable repeatability, low production cost and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 shows the PCR identification of VH (A), VL (B), scFv and scFv-Fc (C), in which, panel A is DNA Marker, 1: VH; b, picture M is a DNA Marker; 1: VL; c, picture M is DNA Marker; 1: scFv-Fc; 2: fc; 3: scFv.

FIG. 2 shows scFv expression, purification and Western Blot identification, in which A is a diagram M: protein Marker, 1: whole bacteria, 2: supernatant after sonication, 3: precipitation after sonication (inclusion bodies); b, picture M: 1-4 of the protein Marker are purified proteins; FIG. 1-3 show original bacteria, supernatant and inclusion body, respectively.

FIG. 3 shows the expression of Fc and Western Blot identification, panel A, M: protein Marker, 1: original bacteria, 2: supernatant after sonication, 3: pellet after sonication (inclusion bodies), 4: comparison with idle load bacteria; b, picture M: a protein Marker; 1: the result of Western Blot of the Fc protein and an anti-canine secondary antibody marked by HRP is obtained.

FIG. 4 shows scFv-Fc expression, purification and Western Blot identification, panel A, M: a protein Marker; 1: original bacteria, 2: pellet after sonication (inclusion bodies), 3: comparison with idle load bacteria; b, picture M: a protein Marker; 1. 2 and 3 are both purified elution effluent liquid; c, the Western Blot results of the actions of the original bacteria, the inclusion bodies, the known positive protein and His primary antibody and the HRP-labeled anti-mouse IgG secondary antibody are shown in the figures 1, 2 and 3 respectively; d, the Western Blot results of the original bacteria, the inclusion bodies, the known positive protein and the HRP-labeled anti-canine secondary antibody are shown in the figures 1, 2 and 3 respectively.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The invention separates peripheral lymphocyte of CIV infected hair disease dog, lifts RNA, obtains cDNA through reverse transcription, obtains VH, VL and Fc segment through PCR, obtains single chain antibody (scFv) gene segment which connects VH and VL through flexible peptide through overlapping extension PCR (SOE-PCR), and connects with pET-32a prokaryotic expression vector, constructs prokaryotic expression vector of recombinant antibody scFv-Fc, detects specificity, ELISA and Hemagglutination Inhibition (HI) titer of antibody through colibacillus expression and purification, and analyzes neutralization activity.

The method comprises the following steps:

1. peripheral blood of clinically symptomatic H3N2 subtype CIV infected dogs was drawn and peripheral lymphocytes were isolated. The original genome is obtained by extracting RNA and reverse transcription. Obtaining antibody heavy chain and light chain variable region genes and heavy chain constant region Fc genes through PCR amplification. The heavy chain variable region and the light chain variable region are connected into scFv genes by flexible peptides. The amino acid sequence of the heavy chain variable region is shown as SEQ ID No.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 16; the amino acid sequence of the connecting peptide is GGGGSGGGGSGGGGS GGGGS; the amino acid sequence of the heavy chain constant region Fc fragment is shown as SEQ ID No. 17.

The Fc fragment amplified by the Fc specific primer is not only one, 2 Fc sequence characteristic-conforming fragments are obtained by sequencing screening, are connected with an expression vector and are expressed, 2 Fc proteins can be expressed, but after the anti-canine secondary antibody is detected, only 1 Fc fragment can be combined with the anti-canine secondary antibody, so that the other Fc fragment can not be subjected to subsequent experiments.

The sequences of scFv and Fc are selected in a large number, and due to the characteristics that a stop codon exists in the middle of a partial sequence or the sequences do not accord with the related sequence characteristics found in NCBI or the sequences accord with the related characteristics but only comprise limited structural domains, the preferred sequences shown in the patent SEQ ID NO. 19-SEQ ID NO.29 are selected for subsequent experiments.

2. Carrying out double enzyme digestion on a prokaryotic expression vector pET-32a through EcoR I and Hand III, Hand III and Xho I to obtain a linear vector, and then respectively connecting the linear vector with the scFv gene and the Fc gene by using a homologous recombination method to construct pET-32a-scFv and pET-32a-Fc expression vectors;

3. and transforming Rosetta (DE3) competent cells, sequencing correct positive bacteria, analyzing and selecting relatively optimal pET-32a-scFv, extracting a plasmid, specifically amplifying an scFv gene, carrying out double enzyme digestion on the pET-32a-Fc plasmid by EcoRI and HindIII, connecting the scFv gene to the pET-32a-Fc plasmid by using a homologous recombination method, and constructing a pET-32a-scFv-Fc expression vector. The amino acid sequence of the scFv-Fc is shown as SEQ ID No. 18;

4. inducing expression of the selected positive bacterium pET-32a-scFv and pET-32a-scFv-Fc with correct sequencing by using IPTG, and purifying by using a His-labeled Ni column to obtain scFv and scFv-Fc antibodies;

5. the scFv and scFv-Fc antibody are subjected to protein renaturation and characteristic analysis, including Western Blot detection, hemagglutination inhibition titer determination, ELISA titer and cell micro-neutralization activity determination.

Example 1: construction of pET-32a-scFv-Fc expression vector

1 strains, plasmids and cell lines and reagents

H3N2 subtype Canine influenza virus strain A/Canine/Jiangsu/06/2010(H3N2) (abbreviation 06), isolated and stored in the laboratory with GenBank accession numbers ranging from JN247615 to JN 247623. Strains and their sequence reference websites: http:// www.ncbi.nlm.nih.gov/nuccore. Rosetta (DE3) was purchased from Shanghai Tokyo Biotechnology Inc. Coli DH5 alpha competent cells, pClone007 Blunt Simple Vector Kit cloning Vector Kit was purchased from Biotech limited of the Kyoto engine, Beijing, Inc. (hereinafter called Ongjingkidake). The pET-32a (+) prokaryotic expression vector is stored in a laboratory. Canine kidney cells (MDCK) were purchased from ATCC in the USA and stored in the laboratory.

The kit for the animal peripheral blood lymphocyte separation solution and the TMB single-component color developing solution are purchased from Beijing Soilebao scientific technology GmbH; the RNA extraction kit, the plasmid extraction kit and the gel recovery kit are all purchased from Omega company; the cDNA reverse transcription kit is purchased from Nanjing Novozam biotech Inc., high fidelity enzyme Prime STAR HS, restriction enzymes EcoRI, Xho I and Hind III, and DNA ligase is purchased from Bao bioengineering (Dalian) Inc.; the His-Tag mouse monoclonal antibody is purchased from Eibomart (Abmart) biomedicine (Shanghai) Co., Ltd.), a horseradish peroxidase (HRP) labeled goat anti-dog IgG antibody, and an HRP labeled goat anti-mouse IgG antibody is purchased from Beijing Boaosen Biotech Co., Ltd; nitrocellulose membrane (NC) available from china biotechnology limited, beijing dingding; his (histidine) protein purification nickel column and a super-sensitive chemiluminescence detection kit, which are purchased from Shanghai Yazyme Biotechnology limited; DMEM complete medium, Fetal Bovine Serum (FBS), purchased from Gibco; t-25 cell flasks and 96-well cell culture plates were purchased from Corning.

2CIV infects experimental animal and obtains peripheral blood separated lymphocyte, extracts RNA, reverse transcription obtains template

7-week-old beagle dog, supplied by Yizheng Anli Mao Biotech Co., Ltd, Nanjing. Purified CIV is adopted to infect MDCK cells, the cell viruses obtained by proliferation are used to infect healthy beagle dogs, and anticoagulated blood is extracted from the forearm veins for standby when clinical symptoms appear. Separating lymphocytes by adopting a peripheral blood lymphocyte separation kit, extracting total RNA according to the instruction of the RNA extraction kit, and carrying out reverse transcription by taking the total RNA as a template according to the instruction of a manufacturer: 4 XgDNA wiper Mix 4. mu.L, template RNA 1. mu.L, RNase free ddH₂O11 mu L, mixing uniformly, and then keeping the temperature at 42 ℃ for 2 min; after the reaction is finished, adding 4 mu L of 5 XHiScript qRT Super Mix into the reaction solution, slowly and uniformly mixing, and storing the product cDNA at-80 ℃ after 15min at 50 ℃ and 5s at 85 ℃.

Cloning and identification of variable region genes

3.1 design and Synthesis of primers and amplification of related Gene fragments

Primers were designed according to accession numbers AF354264.1 and XM532962.3, respectively. Wherein, the upstream and downstream primers used for cloning the heavy chain variable region (VH) gene of the antibody are VH-F and VH-R. The upstream and downstream primers used for cloning the antibody light chain variable region (VL) gene are VL-F and VL-R. The upstream and downstream primers used to clone the antibody heavy chain Fc gene were Fc-F and Fc-R. VH, VL and Fc genes were amplified separately using cDNA synthesized by reverse transcription as a template. The final system of PCR amplification is 25. mu.L: template cDNA 2.0. mu.L, Prime STAR HS 12.5. mu.L, upstream and downstream primers 1.0. mu.L (10. mu.M/. mu.L), ddH₂O8.5. mu.L. PCR amplification conditions: denaturation at 98 ℃ for 15s, annealing at 57 ℃ for 15s, and extension at 72 ℃ for 30s for 35 cycles; final extension at 72 ℃ for 10 min. The product was electrophoresed on a 1% agarose gel to recover the desired fragment. The recovered product was ligated with cloning Vector pClone007 using Pclone007simple Blunt Vector Kit and transformed into DH 5. alpha. competent cells. After screening by a plate containing ampicillin, selecting a single colony, and identifying whether the fragment is inserted successfully by PCR. The results are shown in FIG. 1.

Primers for fusion of VH (CIV-VH-F, CIV-VH-R), VL (CIV-VL-F, CIV-VL-R) and Fc (CIV-Fc-F, CIV-Fc-R) fragments were designed and synthesized. With VH- (Gly4Ser)4-VL chain linking cisIn sequence, Linker fragments (Linker sequence in italic) were introduced downstream of the VH and upstream of the VL. According to the sequence of pET-32a (+), restriction enzymes EcoRI and HindIII are inserted as the cleavage sites of the 5 'end and the 3' end of the scFv full-length gene (the underlined parts are the cleavage sites and the homologous arms thereof), and HindIII and XhoI are used as the cleavage sites of the upstream and downstream of the Fc gene. The sequence information of the primers is shown in Table 1, and all primers were synthesized by Jinzhi Biotech, Suzhou, Inc. Using the recovered VH, VL and Fc fragments as templates, using CIV-VH-F, CIV-VH-R as primer to amplify VH, and CIV-VL-F, CIV-VL-R as primer to amplify VL. The products were electrophoresed with 1% agarose gel, and 1. mu.L each of the gel-recovered products was used as a template for fusion PCR. The final system was 25 μ L: prime STAR HS 12.5. mu.L, CIV-VH-F, CIV-VL-R each 1. mu.L (10. mu.M/. mu.L), gel recovery products each 1. mu.L, ddH₂O6.5. mu.L. PCR amplification conditions: denaturation at 98 ℃ for 15s, annealing at 57 ℃ for 15s, and extension at 72 ℃ for 30s for 35 cycles; final extension at 72 ℃ for 10 min. Meanwhile, CIV-Fc-F and CIV-Fc-R are used as upstream and downstream primers, and the reaction conditions are the same as above, and amplification is carried out. After the PCR products (fusion products and amplified Fc) were electrophoresed with 1% agarose gel, the gel was recovered and ligated into the cloning vector pClone007, DH 5. alpha. competent cells were transformed, positive bacteria were selected, recombinant plasmids were extracted, and the positive plasmids with correct sequencing were named T-scFv, T-CE-Fc. The results are shown in FIG. 1. BLAST of the sequences at NCBI showed that the sequences obtained were consistent with the sequence characteristics of canine heavy and light chains, i.e., sequence similarity of 95% and above.

4 construction of pET-32a-scFv, pET-32a-Fc and pET-32a-scFv-Fc recombinant expression vectors

scFv was amplified from sequenced T-scFv using CIV-VH-F and CIV-VL-R, and Fc was amplified from sequenced T-CE-FC using CIV-Fc-F and CIV-Fc-R. The final system of PCR amplification is 25. mu.L: template cDNA 2.0. mu.L, Prime STAR HS 12.5. mu.L, upstream and downstream primers 1.0. mu.L (10. mu.M/. mu.L), ddH₂O8.5. mu.L. PCR amplification conditions: denaturation at 98 ℃ for 15s, annealing at 57 ℃ for 15s, and extension at 72 ℃ for 30s for 35 cycles; final extension at 72 ℃ for 10 min. pET-32a (+) performs double enzyme digestion of EcoRI and HindIII, and HindIII and XhoI, and the enzyme digestion system is as follows: cut Buffer 5. mu.L, vector not more than 1. mu.g, fast-cutting enzymeEach 1 μ L, ddH₂Adding O to 50 μ L, and performing water bath for 30min at the optimal temperature. Detecting the enzyme digestion product and the PCR product by agarose gel electrophoresis, cutting and recovering the gel, and respectively carrying out homologous recombination connection on the scFv and the Fc target fragment and a carrier pET-32a (+), wherein the reaction system is as follows: 5 × CE Buffer 4 μ L, vector 0.02 × vector size (bp), fragment 0.04 × fragment size (bp), Exnase II 2 μ L, ddH₂O was added to 20. mu.L. After the transformation, Rosetta (DE3) competent cells are transformed, positive colonies are selected for PCR identification after screening by a resistance plate containing ampicillin, and positive plasmids (operated according to the operation flow of an OMEGA plasmid extraction kit) are extracted, sequenced and analyzed. The positive strains were designated pET-32a-scFv and pET-32 a-Fc. Reference [ Tengchun ] protein function prediction method based on sequence and PPI networks [ D]Harbin university of industry, 2016]The 11 scFv sequences obtained were analyzed, wherein 4 sequences did not contain a terminator and the nucleotide sequence similarity between them was 77.86%. The correlation properties of the 4 sequences are shown in Table 2. By combining the structural domain, homology and comparison results of SWISS-MODEL software, the amino acid sequence similarity of scFv-8 and 5yd5.1.A (scFv 4B08) annotated in a database reaches 53.01 percent and covers more than 90 percent, the result is reliable, the antibody sequence is an ideal candidate antibody sequence, and the rest are failure results.

Obtained 11 scFv sequence analysis Table

And (3) redesigning a primer based on the screened target scFv, amplifying the scFv fragment from the target pET-32a-scFv vector by using CE-scFv-F and CE-scFv-R, and constructing the pET-32a-scFv-Fc recombinant expression vector by using the VH- (Gly4Ser)4-VL-Fc connection sequence. And carrying out double enzyme digestion on pET-32a-Fc by using EcoRI and Hind III, carrying out agarose gel electrophoresis detection, recovering a product, carrying out homologous recombination connection on a target fragment and a vector pET-32a-Fc, transforming Rosetta (DE3) competent cells, screening by using a resistance plate, selecting positive colonies, carrying out PCR identification and sequencing. The positive strain was named pET-32 a-scFv-Fc. The results are shown in FIG. 1.

(II) expression, identification and related potency assays for scFv, Fc and scFv-Fc proteins

1 inducible expression of scFv, Fc and scFv-Fc proteins

The positive bacteria pET-32a-scFv, pET-32a-Fc and pET-32a-scFv-Fc were inoculated into LB medium containing 100. mu.g/mL ampicillin at a ratio of 1:100, respectively, and cultured at 37 ℃ and 180rpm for 2 to 3 hours (OD: 100)₆₀₀About 0.4-0.6), adding IPTG (isopropyl-beta-D-thiogalactoside) to a final concentration of 1mM, and inducing expression at 16 ℃ for 12-16 h. Meanwhile, pET-32a (+) empty carrier bacteria expression is set as a negative control group. After centrifugation of the culture, the cells were resuspended in a supernatant Bindingbuffer, sonicated, centrifuged at 12000rpm at 4 ℃ for 20min, and total cell protein, supernatant and inclusion bodies (precipitates after sonication) were taken separately and subjected to SDS-PAGE to detect the expression of the target protein, the results of which are shown in FIGS. 2A, 3A and 4A. The protein purity in lane 4 of FIG. 2, B, was as high as 92% as analyzed by Image-Pro Plus6.0 software. The protein purity of the B picture of FIG. 4,

lanes

1, 2 and 3 was up to 93%, 92% and 92% respectively as analyzed by Image-Pro Plus6.0 software.

Purification of 2scFv, Fc and scFv-Fc protein and Western Blot identification

Centrifuging the recombinant bacteria culture, resuspending with a supernatant Binding Buffer, performing ultrasonication, purifying the target protein with a His protein purification nickel column, collecting the eluate, dialyzing with PBS, and performing SDS-PAGE detection on the dialyzed purified protein, wherein the result is shown in FIGS. 2B and 4B. His-tag Western Blot identification of scFv and scFv-Fc proteins: the antibody was diluted with His-Tag murine monoclonal antibody 1:5000 as primary antibody, and diluted with HRP-labeled goat anti-murine IgG 1:5000 as secondary antibody, and the results are shown in FIGS. 2C and 4C. The purified Fc protein and scFv-Fc protein were subjected to Western Blot specific for canine antibodies, and the results are shown in FIGS. 3B and 4D, in which HRP-labeled goat anti-canine IgG diluted at 1:5000 was directly used as a primary antibody for incubation and washing, followed by direct exposure detection.

SDS-PAGE: SDS-PAGE (12.5%) fast gel preparation kit was purchased from Shanghai Yazyme Biotech Co., Ltd and prepared according to the instructions. Adding SDS protein loading buffer into the sample, boiling for 10 min. The gel was concentrated at 80V and separated at 120V. And (5) electrophoresis is carried out for 2 h. The Coomassie brilliant blue is shaken at 37 ℃ for dyeing for 1.5h, and the decolorized solution is decolorized. The molecular weight of the obtained scFv was about 50kD, the molecular weight of Fc was about 55kD and the molecular weight of recombinant antibody scFv-Fc was about 89 kD.

Identifying Western blot: after SDS-PAGE is complete, the gel of size marker containing the upper and lower 3 protein positions is excised. Two commercial transfer filters and one NC membrane (nitrocellulose membrane) were cut according to the size of the gel. The NC membrane is immersed in a membrane-transfer ionic buffer (Tris 3.03g, glycine 14.4g, methanol 200mL, deionized water constant volume of 1L) for about 5 min. The filter paper, the NC membrane, the gel and the filter paper are superposed and placed correctly in a hamburger mode from bottom to top according to the sequence of 0.8mA/cm²And electrifying and transferring for 1.5 h. NC membrane was incubated in blocking solution (TBST buffer containing 5% skimmed milk, TBST buffer: NaCl 8.8g, 1M Tris-HCl (pH 8.0)20mL, Tween-200.5 mL, deionized water to constant volume of 1L) at 37 ℃ for 2 h. His-tag Western Blot identification of scFv and scFv-Fc proteins: diluting with His-Tag mouse monoclonal antibody 1:5000 to obtain primary antibody, and diluting with HRP-labeled goat anti-mouse IgG 1:5000 to obtain secondary antibody. Western Blot identification is carried out on the purified Fc protein and the scFv-Fc protein, and then exposure detection is directly carried out after incubation washing by using HRP-labeled goat anti-canine IgG diluted at the ratio of 1:5000 as a primary antibody. The washes were all performed with TBST 3 times for 5min each. The exposure is carried out after the development for 5-30min by using an ultrasensitive ECL luminescent solution as a substrate solution.

3 detection of hemagglutination-inhibiting Activity

And (3) taking the purified scFv and scFv-Fc protein, adjusting the initial concentration of the scFv and scFv-Fc protein to be 1mg/mL, reacting with the 06 canine influenza virus with the same volume of 4 units for half an hour, and adding 1% of chicken blood erythrocytes to detect the hemagglutination inhibition titer of the scFv and scFv-Fc protein. The hemagglutination inhibition titer of the scFv protein is 1:2⁷(0.0078mg/mL), and the hemagglutination inhibition titer of the scFv-Fc protein is 1:2⁶(0.0156 mg/mL). The scFv and the scFv-Fc have hemagglutination inhibition activity, can perform specific binding reaction with CIV, and the Fc fragment has no significant influence on the HI titer of the scFv.

The method comprises the following specific operations:a hemagglutination assay was first performed to determine the hemagglutination titer of H3N2 canine influenza virus strain 06. Adding 50 mu L PBS into the first row of 1-12 holes of the disposable blood coagulation plate, adding 50 mu L virus allantoic fluid into the first hole, mixing uniformly, sucking 50 mu L virus allantoic fluid into the second hole, diluting sequentially by multiple times until reaching the 11 th hole, mixing uniformly, and discarding 50 mu L virus allantoic fluid. The last 1 well (12) is a red blood cell control. Then 50. mu.L of 1% red blood cells were added to each well and mixed by gentle shaking. Shaking by hand, standing at 37 ℃ for 30min, and observing the result. Taking the highest dilution of virus suspension with 50% of erythrocytes agglutinated as the erythrocyte agglutination titer of the virus liquid, and measuring the HA titer of the 06 strain to be 2^-6。

Hemagglutination inhibition titer determination of the antibody protein was then performed. Namely: 50 μ LPBS was added to wells 1-12 of the hemagglutinated plate. Adding 50 mu L of the diluted antibody to be picked up into the first hole, mixing uniformly, sucking 50 mu L to the 2 nd hole, diluting to the 10 th hole in a multiple ratio, mixing uniformly, and discarding 50 mu L. In 1-11 wells, 50. mu.L of diluted virus suspension with 4 hemagglutination units was added to each well, and left to stand at 37 ℃ for 30min to allow the virus to react with the antibody sufficiently. Adding 50 μ L of 1% red blood cells into each of 1-12 wells, shaking, mixing, standing at 37 deg.C for 30min, and observing. The highest dilution factor of the antibody which can inhibit the effect of the virus agglutinating erythrocytes by 100 percent is the erythrocyte agglutination inhibition titer of the antibody.

4ELISA detection

The ELISA plate was coated with 10. mu.g/mL of the purified canine influenza virus 06 strain, incubated overnight at 4 ℃ and blocked with 5% skim milk at 37 ℃ for 2 h. The initial concentration of scFv and scFv-Fc is adjusted to 1mg/mL, after 2-fold dilution of purified scFv, 100 μ l/well is added into a 96-well reaction plate, a negative control and a mouse positive serum control are set, the mixture is statically incubated at 37 ℃ for 1.5h and then washed, then His-Tag murine monoclonal antibody is added, the mixture is statically incubated at 37 ℃ for 1.5h and then washed, an HRP-labeled goat anti-mouse IgG antibody is added, and the static incubation is carried out for 1h and then washed, and the result is shown in Table 3. Finally adding TMB single-component developing solution for developing, stopping developing with 2M sulfuric acid stop solution, reading OD of each well with enzyme-labeling instrument₄₅₀The value is obtained. For scFv-Fc protein, dog positive serum control and blank control were set, and goat anti-dog IgG was directly labeled with HRP diluted at 1:5000 as secondary antibody, and the other procedures were the same. The results are shown in Table 4.

5 micro neutralization test

The starting concentration of scFv and scFv-Fc proteins was adjusted to 1mg/mL, diluted 2-fold in 1.5EP tubes, and the required amount was calculated in 100. mu.l/well for 4 replicates per dilution and added to 96-well plates, respectively. Diluting 06 Virus to 200TCID₅₀mu.L/well of the single-chain antibody was added with different dilutions (10-fold dilution of the antibody according to the 1 st well to eliminate incomplete renaturation and residual urea in the resulting antibody, and 2-fold dilution was performed in the 2 nd and subsequent wells, i.e., dilutions: 1:10, 1:20, 1: 40, 1:80 …), incubated at 37 ℃ for 1h, and then the liquid in each well was aspirated and added to a 96-well plate filled with MDCK cells, and a positive control (known positive antibody), a negative control (virus only) and a blank control (culture medium only) were set. Culturing for 48-72 h. The cell monolayer of the blank control group only added with the culture solution is rhombic and grows well; the 1:10 diluted scFv can protect 100% of MDCK cells from cytopathic effect, and the 1:20 diluted scFv and scFv-Fc can protect 50% of MDCK cells from cytopathic effect; the negative control group cells only added with virus have obvious cytopathic effects of falling off, wiredrawing, death and the like. The experiment was repeated 3 times to determine that the neutralizing titer of scFv and scFv-Fc was 1:20(0.05mg/mL)

TABLE 1 related primer sequences

TABLE 2 related features of scFv sequences

IgV _ H: the heavy (H) variable domain (V) of an immunoglobulin (Ig); IgC _ CH1_ IgAEGM: a first immunoglobulin constant domain of the alpha, epsilon, gamma and mu heavy chains; l1: hypervariable region 1 on IgV _ H; l2: hypervariable region 2 on IgV _ H; l3: hypervariable region 3 on IgV _ H. NCBI highest similarity (%): highest similarity of the corresponding gene sequences in the NCBI database.

TABLE 3 Indirect ELISA detection of scFv protein Activity

TABLE 4 Indirect ELISA detection of scFv-Fc protein Activity

In order to solve the problem of the occurrence of heterogeneous immune reaction, the invention directly separates a single-chain antibody gene (scFv) and a constant region gene (Fc) from a virus-infected dog body, and fuses the scFv and the Fc to obtain a fully caninized antibody, and no rejection reaction occurs in the application process. The design inspiration is obtained through a classical monoclonal antibody preparation way, the classical monoclonal antibody preparation way is to immunize a mouse by using an antigen, and the spleen cells of the mouse are harvested to be fused with a myeloma cell line of the mouse to obtain hybridoma cells. And screening the target antigen or the protein of the specific epitope to obtain the target monoclonal antibody. When an animal is immunized with an antigen, the spleen cells or blood will contain a large amount of antibodies against the particular antigen, which is a polyclonal antibody. We hypothesize that if the gene fragment of the antibody (including both scFv and Fc genes) is amplified directly from the blood of influenza virus infected dogs, it should be possible to screen for the desired antibody by constructing it on an expression vector and screening for a specific antigen. This procedure has the advantage of eliminating the cumbersome preparation of monoclonal antibodies and the high cost of culturing the cells. The scheme is more economical and rapid for preparing the antibody. However, this method has a disadvantage that the difficulty in developing an antibody according to the present invention is that the sequencing of the amplified scFv antibody fragment shows that the scFv antibody gene has a structure that does not conform to the characteristics of the antibody (specific problems include 1. gene contains a terminator therein; 2. gene sequence does not conform to the characteristics of scFv; 3. even if the gene sequence conforms to the characteristics of scFv antibody gene, there is a great difference in the number and homology of domains contained in the obtained gene). In the experiment, the carrier is used for screening the used resistant plates, and the transformation process has the reason of transformation rate, so that a large number of positive strains cannot be obtained. In order to solve the problem, after obtaining the scFv gene, the scFv gene is connected and transformed for several times, and after the sample size is selected and enlarged as much as possible in the bacteria selection process, the success rate is greatly improved.

Compared with the classical method, the method has the advantages of simplicity, rapidness and economy. On the other hand, the antibody obtained by the method is completely of animal origin at the gene level, so that the risk of generating heterologous immune injury on animals is avoided. To prepare recombinant caninized antibody scFv-Fc of H3N2 subtype CIV, CIV infected canine peripheral blood lymphocytes were collected and total RNA was extracted, and IgG heavy chain (VH), light chain (VL) and Fc fragments were amplified using PCR technology. Connecting VH and VL into single-chain antibody (scFv) fragments by an elastic peptide chain linker by utilizing overlap extension PCR, converting Escherichia coli, and selecting positive bacteria for sequencing. After analysis and screening, the target scFv is linked with the Fc fragment to construct the scFv-Fc recombinant canine antibody fragment. After induction expression, recombinant antibody scFv-Fc with the molecular weight of 89kD is obtained. The antibody activity measurement result shows that the Hemagglutination Inhibition (HI) titer of 1mg/mL scFv-Fc is 1:2⁶ELISA titre 1:2⁵The neutralizing antibody titer was 1: 20. The research successfully prepares the recombinant canine scFv-Fc aiming at the H3N2 subtype CIV, and provides a material basis for the treatment of the virus infection.

The invention provides a recombinant caninized antibody scFv-Fc aiming at H3N2 subtype CIV, which can be specifically combined with H3N2 subtype CIV, has certain neutralization activity, and lays a foundation for applying the antibody to the prevention and treatment of canine influenza. The prepared recombinant antibody can be applied to the preparation of a medicament or an immune preparation for preventing or controlling influenza viruses, or the preparation of a medicament or an immune preparation composition for preventing or controlling the influenza viruses.

The invention provides a preparation technology of a recombinant caninized antibody scFv-Fc aiming at H3N2 subtype CIV, and successfully obtains the recombinant caninized antibody with certain neutralization activity. Compared with the traditional mouse monoclonal antibody, the caninized antibody can not stimulate the heterogenous immune rejection of an organism and has stronger superiority. Because the antibody constant region gene is derived from the animal, the antibody constant region gene can more effectively perform a synergistic effect with the immune system of the animal, and has the advantages of high affinity, no xenogenesis and the like.

1. After the Fc fragment of the dog IgG molecule is fused, the size of the antibody protein is increased, the half-life period of the antibody in an animal body is prolonged, and the treatment effect of the antibody is enhanced; 2. the Fc fragment of the caninization is fused, and on the premise of not influencing the therapeutic effect of the antibody, compared with the traditional murine monoclonal antibody and the murine single-chain antibody, the caninized antibody does not stimulate the heterologous immune rejection of an organism and has stronger superiority; 3. the recombinant caninized antibody can effectively cooperate with the immune system of the animal to play a role due to the fact that the constant region gene of the antibody is derived from the animal, and has the advantages of high affinity, no xenogenesis and the like; 4. the obtained recombinant caninized antibody has neutralizing activity, the titer is 1:20, and the virus adsorption can be effectively prevented, so that the infection of influenza virus can be effectively prevented.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

<110> Nanjing university of agriculture

<120> a recombinant caninized antibody scFv-Fc against H3N2 canine influenza virus

<141> 2020-02-18

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 1

gggaaggggc ttcagcgggt 20

<210> 2

<211> 18

<212> DNA/RNA

<213> Artificial Sequence

<400> 2

ggagacggtg accagggt 18

<210> 3

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 3

agcagagtgg aggctgacga 20

<210> 4

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 4

gctgaggctg taggtactgt c 21

<210> 5

<211> 31

<212> DNA/RNA

<213> Artificial Sequence

<400> 5

accgtctcct cagcctccac cacggccccc t 31

<210> 6

<211> 15

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

tttacccgga gaatg 15

<210> 7

<211> 41

<212> DNA/RNA

<213> Artificial Sequence

<400> 7

gctgatatcg gatccgaatt cgggaagggg cttcagcggg t 41

<210> 8

<211> 65

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

ggagccgccg ccgccagaac caccaccacc agaaccacac ccaccggagg gcactgtcac 60

catgc 65

<210> 9

<211> 50

<212> DNA/RNA

<213> Artificial Sequence

<400> 9

ggcggcggcg gctccggtgg tggtggatcc agcagagtgg aggctgacga 50

<210> 10

<211> 42

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

ctcgagtgcg gccgcaagct tgctgaggct gtaggtactg tc 42

<210> 11

<211> 41

<212> DNA/RNA

<213> Artificial Sequence

<400> 11

tcgagctccg tcgacaagct taccgtctcc tcagcctcca c 41

<210> 12

<211> 42

<212> DNA/RNA

<213> Artificial Sequence

<400> 12

gtggtggtgg tggtgctcga gtttacccgg agaatgggag ag 42

<210> 13

<211> 41

<212> DNA/RNA

<213> Artificial Sequence

<400> 13

gctgatatcg gatccgaatt cgggaagggg cttcagcggg t 41

<210> 14

<211> 47

<212> DNA/RNA

<213> Artificial Sequence

<400> 14

gtggaggctg aggagacggt aagcttgctg aggctgtagg tactgtc 47

<210> 15

<211> 152

<212> PRT

<213> Artificial Sequence

<400> 15

Pro Asp Ser Val Ile Arg Thr Gln Gln Arg Gln Thr Thr Met Glu Ser

1 5 10 15

Val Leu Ser Trp Val Phe Leu Val Ala Ile Leu Gln Gly Val Gln Gly

20 25 30

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

35 40 45

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Phe

50 55 60

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

65 70 75 80

Ala Ala Ile Ala Tyr Asp Gly Ser Ser Thr Tyr Tyr Thr Asp Ala Val

85 90 95

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr

100 105 110

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

115 120 125

Ala Ser Pro Thr Thr Val Pro Thr Ile Asp Trp Phe Tyr Tyr Trp Gly

130 135 140

Gln Gly Thr Leu Val Thr Val Ser

145 150

<210> 16

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 16

Met Leu Trp Ile Pro Gly Ser Thr Gly Glu Ala Val Met Thr Gln Thr

1 5 10 15

Pro Leu Ser Leu Ala Val Thr Pro Gly Glu Leu Ala Thr Ile Ser Cys

20 25 30

Arg Ala Asn Gln Ser Leu Leu Arg Ser Asp Gly Lys Ser Tyr Leu Asn

35 40 45

Trp Tyr Leu Gln Lys Pro Gly Gln Thr Pro Arg Pro Leu Ile Tyr Glu

50 55 60

Ala Ser Lys Arg Phe Ser Gly Val Ser Gly Arg Phe Ser Gly Ser Gly

65 70 75 80

Ser Gly Thr Asp Phe Thr Leu Lys Ile Thr Arg Val Glu Ala Glu Asp

85 90 95

Val Gly Val Tyr Tyr Cys Gln Gln Gly Leu His Phe Pro Pro Thr Phe

100 105 110

Gly Gln Gly Thr Lys Val Glu Ile

115 120

<210> 17

<211> 339

<212> PRT

<213> Artificial Sequence

<400> 17

Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser Val Phe Pro Leu Ala

1 5 10 15

Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu

20 25 30

Val Ser Gly Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

35 40 45

Ser Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser

50 55 60

Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ser Ser Arg Trp

65 70 75 80

Pro Ser Glu Thr Phe Thr Cys Asn Val Ala His Pro Ala Ser Lys Thr

85 90 95

Lys Val Asp Lys Pro Val Pro Lys Arg Glu Asn Gly Arg Val Pro Arg

100 105 110

Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro Glu Met Leu Gly Gly Pro

115 120 125

Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Ala

130 135 140

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Leu Asp Pro Glu Asp

145 150 155 160

Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys Gln Met Gln Thr

165 170 175

Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe Asn Gly Thr Tyr Arg Val

180 185 190

Val Ser Val Leu Pro Ile Gly His Gln Asp Trp Leu Lys Gly Lys Gln

195 200 205

Phe Thr Cys Lys Val Asn Asn Lys Ala Leu Pro Ser Pro Ile Glu Arg

210 215 220

Thr Ile Ser Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val

225 230 235 240

Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys Asn Thr Val Ser Leu Thr

245 250 255

Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp Ile Asp Val Glu Trp Gln

260 265 270

Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys Tyr Arg Thr Thr Pro Pro

275 280 285

Gln Leu Asp Glu Gly Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val

290 295 300

Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr Phe Ile Cys Ala Val Met

305 310 315 320

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser His Ser

325 330 335

Pro Gly Lys

<210> 18

<211> 614

<212> PRT

<213> Artificial Sequence

<400> 18

Pro Asp Ser Val Ile Arg Thr Gln Gln Arg Gln Thr Thr Met Glu Ser

1 5 10 15

Val Leu Ser Trp Val Phe Leu Val Ala Ile Leu Gln Gly Val Gln Gly

20 25 30

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

35 40 45

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Phe

50 55 60

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val

65 70 75 80

Ala Ala Ile Ala Tyr Asp Gly Ser Ser Thr Tyr Tyr Thr Asp Ala Val

85 90 95

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr

100 105 110

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

115 120 125

Ala Ser Pro Thr Thr Val Pro Thr Ile Asp Trp Phe Tyr Tyr Trp Gly

130 135 140

Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Leu Trp Ile

165 170 175

Pro Gly Ser Gly Gly Asp Ile Val Met Ile Gln Thr Pro Pro Ser Leu

180 185 190

Ser Val Ser Pro Arg Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln

195 200 205

Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln

210 215 220

Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg

225 230 235 240

Phe Thr Gly Val Ser Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

245 250 255

Val Thr Leu Arg Ile Ser Arg Val Glu Ala Asp Asp Thr Gly Val Tyr

260 265 270

Tyr Cys Gly Gln Gly Thr Gln Leu Pro Pro Thr Phe Gly Gln Gly Thr

275 280 285

Lys Val Glu Ile Lys Leu Thr Val Ser Ser Ala Ser Thr Thr Ala Pro

290 295 300

Ser Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr

305 310 315 320

Val Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu Pro Val Thr

325 330 335

Val Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly Val His Thr Phe Pro

340 345 350

Ser Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr

355 360 365

Val Pro Ser Ser Arg Trp Pro Ser Glu Thr Phe Thr Cys Asn Val Ala

370 375 380

His Pro Ala Ser Lys Thr Lys Val Asp Lys Pro Val Pro Lys Arg Glu

385 390 395 400

Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala Pro

405 410 415

Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

420 425 430

Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val

435 440 445

Asp Leu Asp Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val Asp

450 455 460

Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln Phe

465 470 475 480

Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln Asp

485 490 495

Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala Leu

500 505 510

Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala His

515 520 525

Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser Lys

530 535 540

Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Asp

545 550 555 560

Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser Lys

565 570 575

Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Gly Gly Ser Tyr Phe Leu

580 585 590

Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp Thr

595 600 605

Phe Ile Cys Ala Val Met

610

<210> 19

<211> 840

<212> DNA/RNA

<213> Artificial Sequence

<400> 19

gggaaggggc ttcagcgggt cgcagctatt agctatgatg gaagtagcac atactacact 60

gacgctgtga agggccgatt caccatctcc agagacaacg ccaggaacac agtgtatctg 120

cagatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgaa ggatctaatc 180

gatagtagca gctggtactc cgaggattac tatggtatgg actactgggg ccatggcacc 240

tcactcttcg tgtcctcagc ctccaccacg gccccctcgg ttttcccact ggcccccagc 300

tgcgggtcca cttccggctc cacggtggcc ctggcctgcc tggtgtcagg ctacttcccc 360

gagcctgtaa ctgtgtcctg gaattccggc tccttgacca gcggtgtgca caccttcccg 420

tccgtcctgc agtcctcagg gctctactcc ctcagcagca tggtgacagt gccctccggt 480

gggtgtggtt ctggtggtgg tggttctggc ggcggcggct ccggtggtgg tggatccagc 540

agagtggagg ctgacgatac tggactttat tactgcgggc aaggtataca gcttccgttc 600

acttttggcc aagggaccaa actggagatc aaacggaatg atgcccagcc agccgtctat 660

ttgttccaac catctccaga ccagttacac acaggaagtg cctctgttgt gtgtttgctg 720

aatagcttct accccaaaga catcaatgtc aagtggaaag tggatggtgt catccaagac 780

acaggcatcc aggaaagtgt cacagagcag gacaaggaca gtacctacag cctcagcagc 840

<210> 20

<211> 816

<212> DNA/RNA

<213> Artificial Sequence

<400> 20

ggagggcact gtcaccatgc tgctgaggga gtagagccct gaggactgca ggacggacgg 60

gaaggtgtgc acaccgctgg tcaaggagcc ggaattccag gacacagtta caggctcggg 120

gaggtagcct gacaccaggc aggccagggc caccgtggag ccggaagtgg acccgcagct 180

gggggccagt gggaaaaccg agggggccgt ggtggaggct gaggagacgg tgaccagggt 240

tccctggccc cagtagtcaa attctccagc tgctagtata cgatccttcg gacagtaata 300

cacggctgtg tcctcggatc tcaggctgtt catctgcaga tacagtgtgt tcctggcatt 360

gtctctggag atggtgaacc ggcccttcac agcatctgcg tagtatgtgc tacttccacc 420

actgttaatg tatgcgaccc gctgaagccc cttcccggtg ggtgtggttc tggtggtggt 480

ggttctggcg gcggcggctc cggtggtggt ggatccagca agtggaggct gacgatattg 540

gagtctatta ctgcgggcaa ggtatacatc ttcctccgac gttcggagca ggaaccaagg 600

tggagctcaa acggaatgat gcccagccag ccgtctattt gttccaacca tctccagacc 660

agttacacac aggaagtgcc tctgttgtgt gtttgctgaa tagcttctac cccaaagaca 720

tcaatgtcaa gtggaaagtg gatggtgtca tccaagacac aggcatccag gaaagtgtca 780

cagagcagga caaggacagt acctacagcc tcagca 816

<210> 21

<211> 828

<212> DNA/RNA

<213> Artificial Sequence

<400> 21

ggagggcact gtcaccatgc tgctgaggga gtagagccct gaggactgca ggatggacgg 60

gaaggtgtgc acaccgctgg tcaaggagcc ggaattccag gacacagtta caggctcggg 120

gatgtagcct gacaccaggc aggccagggc caccgtggag ccggattggg acccacagct 180

gggggccagt gggaaaaccg agggggccgt ggtggaggct gaggagacgg tgaccagggt 240

tccctggccc cagtagtcaa taagcggatt actagaaagc caaggggaaa aatcctttgc 300

acagtaatac atagccttgt cctcggctct caggctgttc atctgcagat acagcgtgtt 360

cttggcattg tctctggaga cggtgaatcg gcccttcaca gcgtctgcgt agtatgtgct 420

acttccatca ctgctaatcc gtgcgacccg ctgaagcccc ttcccggtgg gtgtggttct 480

ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg gatccgctgc tgaggctgta 540

ggtactgtcc ttgtcctgct ctgtgacact ttcctggatg cctgtgtctt ggatgacacc 600

atccactttc cacttgacat tgatgtcttt ggggtagaag ctattcagca aacacacaac 660

agaggcactt cctgtgtgta actggtctgg agatggttgg aacaaataga cggctggctg 720

ggcatcattc cgtcttatct ccatcttggt cccctggctg aaagtataag gaacttgtgt 780

accttgcccg cagtaataaa ctccagtatc gtcagcctcc actctgct 828

<210> 22

<211> 807

<212> DNA/RNA

<213> Artificial Sequence

<400> 22

ggagggcact gtcaccatgc tgctgaggga gtagagccct gaggactgca ggacggacgg 60

gaaggtgtgc acaccgctgg tcaaggagcc ggaattccag gacacagtta caggctcggg 120

gaagtagcct gacaccaggc aggccagggc caccgtggag ccggaagtgg acccgcagct 180

gggggccagt gggaaaaccg agggggccgt ggtggaggct gaggagacgg tgaccagggt 240

tccctggccc cagtagtcaa aagagcttga taggtgcgca cagtaataca cggccgtgtc 300

ctcggctctc aggctgttca tctgaagata cagcgtgttc ttggcgttgt ctctggagat 360

ggtgaatcgg cccttcacag cgtctgcata gcctatggta cttccaccac tattaatgta 420

tgcgacccgc tgaagcccct tcccggtggg tgtggttctg gtggtggtgg ttctggcggc 480

ggcggctccg gtggtggtgg atccgctgct gaggctgtag gtactgtcct tgtcctgctc 540

tgtgacactt tcctggatgc ctgtgtcttg gatgacacca tccactttcc acttgacatt 600

gatgtctttg gggtagaagc tattcagcaa acacacaaca gaggcacttc ctgtgtgtaa 660

ctggtctgga gatggttgga acaaatagac ggctggctgg gcatcattcc gtttgagctc 720

caccttggtt cctgctccga acgtcggagg aagctgtgta ccttgcccgc agtaataaat 780

tccagtatcg tcagcctcca ctctgct 807

<210> 23

<211> 819

<212> DNA/RNA

<213> Artificial Sequence

<400> 23

ggagggcact gtcaccatgc tgctgaggga gtagagccct gaggactgca ggacggacgg 60

gaaggtgtgc acaccgctgg tcaaggagcc ggaattccag gacacagtta caggctcggg 120

gaggtagcct gacaccaggc aggccagggc caccgtggag ccggaagtgg acccgcagct 180

gggggccagt gggaaaaccg agggggccgt ggtggaggct gaggagacgg tgaccagggt 240

tccctggccc cagtagtcaa attctccagc tgctagtata cgatccttcg gacagtaata 300

cacggctgtg tcctcggatc tcaggctgtt catctgcaga tacagtgtgt tcctggcatt 360

gtctctggag atggtgaacc ggcccttcac agcatctgcg tagtatgtgc tacttccacc 420

actgttaatg tatgcgaccc gctgaagccc cttcccggtg ggtgtggttc tggtggtggt 480

ggttctggcg gcggcggctc cggtggtggt ggatccagca gagtggaggc tgacgatact 540

ggagtttatt actgtgggca agttatacaa gatcctcgta cgttcggagc aggaaccaag 600

gtggagctca aacggaatga tgcccagcca gccgtctatt tgttccaacc gtctccagac 660

cagttacaca caggaagtgc ctctgttgtg tgtttgctga atagcttcta ccccaaagac 720

atcaatgtca agtggaaagt ggatggtgtc atccaagaca caggcatcca ggaaagtgtc 780

acagagcagg acaaggacag tacctacagc ctcagcagc 819

<210> 24

<211> 719

<212> DNA/RNA

<213> Artificial Sequence

<400> 24

gggaaggggc ttcagcgggt cgcagctatt agctatgatg gaagtagcac atactacact 60

gacgctgtga agggccgatt caccatctcc agagacaacg ccaggaacac agtgtatctg 120

cagatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgcc ttactgtact 180

gatgattact gttcccctaa gatttttgac tactggggcc agggaaccct ggtcaccgtc 240

tcctcagcct ccaccacggc cccctcggtt ttcccactgg cccccagctg cgggtccact 300

tccggctcca cggtggccct ggcctgcctg gtgtcaggct acttccccga gcctgtaact 360

gtgtcctgga attccggctc cttgaccagc ggtgtgcaca cctcccgtcc gtcctgcagt 420

cctcagggct ctactccctc agcagcatgg tgacagtgcc ctccggtggg tgtggttctg 480

gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg atccgctgct gaggctgtag 540

gtactgtcct tgtcctgctc tgtgacactt tcctggatgc ctgtgtcttg gatgacacca 600

tccactttcc acttgacatt gatgtctttg gggtagaagc tattcagcaa acacacaaca 660

gaggcacttc ctgtgtacct tgcccgcagt aataaactcc agtatcgtca gcctccact 719

<210> 25

<211> 726

<212> DNA/RNA

<213> Artificial Sequence

<400> 25

ggagggcact gtcaccatgc tgctgaggga gtagagccct gaggactgca ggacggacgg 60

gaaggtgtgc acaccgctgg tcaaggagcc ggaattccag gacacagtta caggctcggg 120

gaagtagcct gacaccaggc aggccagggc caccgtggag ccggaagtgg acccgcagct 180

gggggccagt gggaaaaccg agggggccgt ggtggaggct gaggagacgg tgaccagggt 240

tccctggccc cagtagtcac agtaaggggg gcctaggcca tatatatata cgcggggacc 300

cttcgcacag taatacacag ccgtgtcctc ggctctcagg ctgttcatct gcagatacac 360

tgtgttcctg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt cagtgtagta 420

tgtgctactt ccatcatagc taatagctgc gacccgctga agccccttcc cggtgggtgt 480

ggttctggtg gtggtggttc tggcggcggc ggctccggtg gtggtggatc cgctgctgag 540

gctgtaggta ctgtccttgt cctgctctgt gacactttcc tggatgcctg tgtcttggat 600

gacaccatcc actttccact tgacattgat gtctttgggg tagaagctat tcagcaaaca 660

cacaacagag gcacttcctg tgtaccttgc ccgcagtaat aaactccagt atcgtcagcc 720

tccact 726

<210> 26

<211> 876

<212> DNA/RNA

<213> Artificial Sequence

<400> 26

cccgattcag tgatcaggac acaacaaaga caaaccacca tggagtctgt gctcagctgg 60

gtttttcttg tcgctatttt acaaggtgtc cagggtgagg tgcagctggt ggagtctggg 120

ggagacctgg tgaagcctgg ggggtccctg agactctcct gtgtagcctc tggattcacc 180

ttcagtgact tcgacatgag ctgggtccgc caggctcctg ggaaggggct gcagtgggtc 240

gcagctattg cctatgatgg aagtagcaca tactacactg acgctgtgaa gggccgattc 300

accatctcca gagacaatgc caggaacacg gtgtatctgc agatgaacag cctgagagcc 360

gaggacacgg ccgtgtatta ctgtgcgagt cccactacgg tccctactat tgactggttc 420

tactactggg gccaagggac cctggtcacc gtctccggtg gtggtggttc tggtggtggt 480

ggttctggcg gcggcggctc cggtggtggt ggatccatgc tctggatccc aggatccact 540

ggggaggccg tgatgacgca gaccccactg tccctggccg tcacccctgg agagctggcc 600

actatctcct gcagggccaa tcagagtctc ctgcgcagtg atggaaaatc ctatttgaat 660

tggtacctgc agaagccagg ccagactcct cggccgctga tttatgaggc ttccaagcgt 720

ttctctgggg tctcaggcag gttcagtggc agcgggtcag ggacagattt cacccttaaa 780

attaccaggg tggaggctga ggatgttgga gtttattact gccagcaagg tctacatttt 840

cctcctacgt tcggccaagg gaccaaggtg gagatc 876

<210> 27

<211> 804

<212> DNA/RNA

<213> Artificial Sequence

<400> 27

ggagggcact gtcaccatgc tgctgaggga gtagagccct gaggactgca ggacggacgg 60

gaaggtgtgc acaccgctgg tcaaggagcc ggaattccag gacacagtta caggctcggg 120

gaagtagcct gacaccaggc aggccagggc caccgtggag ccggaagtgg acccgcagct 180

gggggccagt gggaaaaccg agggggccgt ggtggaggct gaggagacgg tgaccagggt 240

tccctggccc cagtagtcaa agttgaaggt tttcgcacag taatacacag ccgtgtcctc 300

ggctctcagg ctgttcatct gcagatacac tgtgttcctg gcgttgtctc tggagatggt 360

gaatcggccc ttcacagcgt cagtgtagga tgtgctactt ccatcatagc taatagctgc 420

gacccgctga agccccttcc cggtgggtgt ggttctggtg gtggtggttc tggcggcggc 480

ggctccggtg gtggtggatc cagcagagtg gaggctgacg atactggact ttattactgc 540

gggcaaggta tacagcttcc gttcactttt ggccaaggga ccaaactgga gatcaaacgg 600

aatgatgccc agccagccgt ctatttgttc caaccatctc cagaccagtt acacacagga 660

agtgcctctg ttgtgtgttt gctgaatagc ttctacccca aagacatcaa tgtcaagtgg 720

aaagtggatg gtgtcatcca agacacaggc atccaggaaa gtgtcacaga gcaggacaag 780

gacagtacct acagcctcag cagc 804

<210> 28

<211> 852

<212> DNA/RNA

<213> Artificial Sequence

<400> 28

cccgccggaa acagagaccc accatgggag tctgtgttct gctgggtttt ccttgtcgct 60

attttaaaag gtgtcctttg tgaggtgcag ctggtggagt ctgggggaga cctggtgaag 120

cctgcggggt ccctgagact gtcctgtgtg gcctctggat tcacctccag tagctacagt 180

atgagctggg tccgccaggc tcctgagaag gggctgcagt tggtcgcagg tattaacagc 240

ggtggaagtt acacatacta cacagacgct gtgaagggcc gattcgccat ctccagagac 300

aatgccaaga acacgctgta tctgcagatg agcagcctga gagccgagga cacggctatg 360

tattactgtg caaaggactc gctgtttggt tcttttgact actggggcca gggaaccctg 420

gtcaccgtct ccggtgggtg tggttctggt ggtggtggtt ctggcggcgg cggctccggt 480

ggtggtggat ccatgctctg gatcccagga tccggtgggg atattgtcat gacacagacg 540

ccaccgtccc tgtctgtcag ccctagagag ccggcctcca tctcctgcag ggccagtcag 600

agcctcctgc acagtaacgg gaacacctat ttgaattggt acctgcaaaa gccaggccag 660

tctccacagc ttctgatcta cttggtttcc aaccgcttca ctggcgtgtc agacaggttc 720

agtggcagcg ggtcagggac agatgtcacc ctgagaatca gcagagtgga ggctgacgat 780

actggagttt attactgcgg gcaaggtaca cagcttcctc ctacgttcgg ccaagggacc 840

aaggtggaga tc 852

<210> 29

<211> 867

<212> DNA/RNA

<213> Artificial Sequence

<400> 29

cctgattcac tgaccaggac acaacagaga caaaccacca tggagtgtgt gcttggctgg 60

gttttccttg tcgctatttt aaaaggtgtc cagggagagg tacagctggt ggaatctggg 120

ggagacctcg tgaagcctgg gggttccctg agactctcct gtgtggcctc gggattcacc 180

ttcagtagct actacatgag ctggatccgc caggctcctg ggaaggggct gcagtgggtc 240

gcagatatta gtgatagtgg aggtagcaca ggctacgcag acgctgtgaa gcgccggttc 300

accatctcca gagagaacgc caagaacaag ctgtatcttc agatgaacag cctgagagcc 360

gaggacacag ccgtgtatta ctgtgcgaac tacggtagct actccccttt tgactactgg 420

ggccagggaa ccctggtcac cgtctccggt gggtgtggtt ctggtggtgg tggttctggc 480

ggcggcggct ccggtggtgg tggatccatg ctctggatcc caggatccgg tggggatatt 540

gtcatgacac agacgccacc gtccctgtct gtcagcccta gagagccggc ctccatctcc 600

tgcagggcca gtcagagcct cctgcacagt aacgggaaca cctatttgaa ttggtacctg 660

caaaagccag gccagtctcc acagcttctg atctacttgg tttccaaccg cttcactggc 720

gtgtcagaca ggttcagtgg cagcgggtca gggacagatg tcaccctgag aatcagcaga 780

gtggaggctg acgatactgg agtttattac tgcgggcaag gtacacagct tcctcctacg 840

ttcggccaag ggaccaaggt ggagatc 867

Claims

1.A method of making a recombinant caninized antibody scFv-Fc directed against H3N2 subtype CIV, characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

extracting peripheral blood of a dog infected by H3N2 subtype CIV which generates clinical symptoms, separating peripheral lymphocytes, extracting RNA, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification to obtain heavy chain and light chain variable region genes of an antibody and heavy chain constant region Fc genes; the heavy chain variable region and the light chain variable region flexible peptide are connected into scFv gene; the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 16; the amino acid sequence of the connecting peptide is GGGGSGGGGSGGSGGGGS, and the amino acid sequence of the heavy chain constant region Fc fragment is shown as SEQ ID No. 17; the sequence of the scFv is shown as SEQ ID No. 26;

prokaryotic expression vector pET-32a is subjected toEcoR I andHand III, HandIII andXhoi, performing double enzyme digestion to obtain linear vectors, and then respectively connecting the linear vectors with the scFv genes and the Fc genes by using a homologous recombination method to construct pET-32a-scFv and pET-32a-Fc expression vectors;

transforming Rosetta competent cells, sequencing the positive bacteria correctly, selecting relatively optimal pET-32a-scFv by analysis, extracting plasmid, specifically amplifying scFv gene, and performing PCR amplificationEcoR I andHindIII, after the pET-32a-Fc plasmid is cut by double enzymes, the scFv gene is connected to the pET-32a-Fc plasmid by utilizing a homologous recombination method to construct a pET-32a-scFv-Fc expression vector; the amino acid sequence of the scFv-Fc is shown as SEQ ID No. 18;

and (3) inducing expression of the selected positive bacterium pET-32a-scFv and pET-32a-scFv-Fc with correct sequencing by using IPTG, and purifying by using a His-labeled Ni column to obtain scFv and scFv-Fc antibodies.

2. A primer for the preparation of a recombinant caninized antibody scFv-Fc against CIV subtype H3N2 in the method of claim 1, characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

the sequence of the VH-F is shown as SEQ ID NO.l;

the sequence of the VH-R is shown as SEQ ID NO. 2;

the sequence of the VL-F is shown as SEQ ID NO. 3;

the sequence of the VL-R is shown as SEQ ID NO. 4;

the sequence of the Fc-F is shown as SEQ ID NO. 5;

the sequence of the Fc-R is shown as SEQ ID NO. 6;

the sequence of the CIV-VH-F is shown as SEQ ID NO. 7;

the sequence of the CIV-VH-R is shown as SEQ ID NO. 8;

the sequence of the CIV-VL-F is shown as SEQ ID NO. 9;

the sequence of the CIV-VL-R is shown as SEQ ID NO. 10;

the sequence of the CIV-Fc-F is shown as SEQ ID NO. 11;

the sequence of the CIV-Fc-R is shown in SEQ ID NO. 12;

the sequence of the CE-scFv-F is shown as SEQ ID NO. 13;

the sequence of the CE-scFv-R is shown as SEQ ID NO. 14.

3. Use of a recombinant caninized antibody scFv-Fc prepared according to the method of claim 1, characterized in that: the application in preparing medicine or immune preparation for preventing or controlling influenza virus.