Porcine-murine chimeric antibody against porcine delta coronavirus and application thereof

Abstract

The application discloses a pig-mouse chimeric antibody against porcine delta coronavirus and application thereof, wherein the pig-mouse chimeric antibody against porcine delta coronavirus is composed of a pig-derived heavy chain constant region, a pig-derived light chain constant region, a mouse-derived heavy chain variable region and a mouse-derived light chain variable region. The pig-mouse chimeric antibody can effectively reduce the heterogenic reaction of the antibody in a pig body, avoid the generation of an immune side reaction, shorten the half-life of the antibody, reduce actual curative effect and possibly cause serious adverse reactions, improve the neutralization treatment effect of the antibody, provide a practical genetically engineered antibody for prevention and treatment of PDCoV, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to a pig-mouse chimeric antibody against porcine delta coronavirus and application thereof. BACKGROUND

[0002] Porcine delta coronavirus (PDCoV) is a newly discovered porcine enteric disease coronavirus belonging to the delta coronavirus genus. The virus is a single-stranded positive-sense RNA virus that is not segmented, has a full-length genome of about 25.4 kb, contains a 5' non-coding region and a 3' non-coding region, and encodes four major structural proteins: surface spike protein (S), small membrane protein (E), membrane protein (M), and nucleocapsid (N) protein. The virus mainly infects the small intestine of pigs, especially the jejunum and ileum, causing atrophy of small intestinal villus epithelial cells and causing severe atrophic enteritis. The main clinical symptoms are watery diarrhea, vomiting, and dehydration death in newborn piglets. In recent years, PDCoV has spread rapidly in China, seriously threatening the healthy development of the pig industry.

[0003] Monoclonal antibodies are antibodies produced by a single cell with high specificity and affinity. Since its advent, it has been rapidly applied to many fields of modern medicine due to its unique advantages, such as detection of pathogenic microorganism antigens and antibodies in medical diagnostic reagent development, detection of tumor markers, detection and identification of immune cells and their subsets, and determination of hormones and cytokines. Chimeric antibody technology, reconfigured antibody technology, and surface remodeling technology have been developed to overcome the immune side effects induced by the recognition of murine components in antibodies by the heterologous immune system. However, there is currently no chimeric antibody research targeting porcine delta coronavirus to obtain safe and efficient vaccines or antiviral drugs. SUMMARY

[0004] To solve the above problems, the present application provides a pig-mouse chimeric antibody against porcine delta coronavirus, which is composed of a heavy chain and a light chain. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 3, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 5, the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 6.

[0005] The present application also provides a gene fragment encoding the aforementioned pig-mouse chimeric antibody, which includes a gene fragment encoding the heavy chain with a nucleotide sequence shown in SEQ ID NO. 1, and a gene fragment encoding the light chain with a nucleotide sequence shown in SEQ ID NO. 2.

[0006] The application further provides a recombinant vector for expressing the aforementioned pig-mouse chimeric antibody, which is an expression vector into which a nucleotide sequence as shown in SEQ ID NO. 1 of a heavy chain coding gene fragment is inserted into Xba I and Not I enzyme cutting sites, and a nucleotide sequence as shown in SEQ ID NO. 2 of a light chain coding gene fragment is inserted into Hind III and Eco R I enzyme cutting sites.

[0007] The expression vector comprises a PKS001 plasmid vector.

[0008] The application further provides a recombinant cell for expressing the aforementioned pig-mouse chimeric antibody, which is a mammalian cell comprising the aforementioned recombinant vector, preferably ExpiCHO-S and CHO-K1 cells.

[0009] The application further provides a method for preparing the aforementioned pig-mouse chimeric antibody, comprising the following steps: taking the aforementioned recombinant vector, transfecting cells, culturing, taking supernatant for purification, and obtaining the product; the transfection comprises transient transfection and stable transfection.

[0010] Further, the method for transient transfection is as follows: mixing OptiPRO SFM and the recombinant vector in tube A; mixing OptiPRO SFM and ExpiFectamineCHO Reagent in tube B; mixing the solutions in tube A and tube B and standing for 5 min, and then adding into cell culture solution, culturing for 20 h, and then adding ExpiCHO Feed and ExpiCHO Enhancer, culturing until the 5th day, and then adding ExpiCHO Feed until the cell viability decreases to below 70%.

[0011] Further, the ratio of OptiPRO SFM and the recombinant vector in tube A is 2 ml:40 μg;

[0012] The ratio of OptiPRO SFM and ExpiFectamineCHO Reagent in tube B is 1.84 ml:160 μl;

[0013] The ratio of cell culture solution and the recombinant vector is 50 ml:40 μg;

[0014] The volume ratio of the cell culture solution, ExpiCHO Feed, ExpiCHO Enhancer added after culturing for 20 h, and ExpiCHO Feed added until the 5th day is 50 ml:8 ml:300 μl:8 ml;

[0015] The cells in the cell culture solution are ExpiCHO-S cells, and the culture medium is ExpiCHO Expression Medium.

[0016] The culture conditions are: rotation speed 95 rpm, temperature 37 DEG C, CO2 concentration 8%.

[0017] Further, the purification is carried out by using Protein A recombinant protein purification pre-packed column to purify the supernatant.

[0018] The application also provides a use of the aforementioned pig-mouse chimeric antibody in the preparation of a medicine for preventing and / or treating porcine delta coronavirus infection.

[0019] The application finally provides a vaccine for preventing porcine delta coronavirus, which is a vaccine prepared by using the aforementioned pig-mouse chimeric antibody as an antigen and adding a pharmaceutically acceptable carrier.

[0020] The application uses a previously prepared neutralizing mouse monoclonal antibody 1H10 which specifically recognizes the S protein of PDCoV, carries out genetic engineering recombination, constructs a recombinant expression vector for expressing a pig-mouse chimeric genetic engineering antibody, and obtains a pig-mouse chimeric antibody ch1H10 by expressing in CHO cells. Cell experiments prove that the ch1H10 antibody can specifically recognize PDCoV and has good virus binding activity and neutralizing activity. In vivo experiments show that the chimeric antibody has a strong antiviral therapeutic effect on PDCoV infected piglets. Since the recombinant pig-mouse chimeric antibody ch1H10 only contains a mouse-derived variable region sequence responsible for specific recognition of antigens, and the pig-derived FC fragment is homologous to the PDCoV host (pig), it can effectively reduce the heterologous reaction of the antibody in the pig body, avoid the production of immune side reactions, shorten the half-life of the antibody, reduce the actual therapeutic effect, and possibly cause serious adverse reactions, improve the neutralizing therapeutic effect of the antibody, provide a practical genetic engineering antibody for preventing and treating PDCoV, and have important clinical application value.

[0021] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical ideas of the application.

[0022] The above content of the application is further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the application to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Antibody recombinant schematic diagram;

[0024] Figure 2 Schematic diagram of construction and expression process of stable vector;

[0025] Figure 3In vivo treatment experiment process of piglets

[0026] Figure 4 ch1H10 transient vector schematic diagram

[0027] Figure 5 Antibody heavy chain and light chain amplification electrophoresis bands (A: antibody heavy chain amplification electrophoresis bands; B: antibody light chain amplification electrophoresis bands)

[0028] Figure 6 Antibody stable plasmid schematic diagram

[0029] Figure 7 ch1H10 purification peak diagram

[0030] Figure 8 SDS-PAGE verification of ch1H10 (A: antibody purification effect verification diagram; B: reduced / non-reduced verification diagram of purified antibody ch1H10)

[0031] Figure 9 Ch1H10 cytotoxicity experiment results

[0032] Figure 10 Ch1H10 EC50 experiment results

[0033] Figure 11 ELISA experiment results

[0034] Figure 12 IFA experiment results (magnification 40x)

[0035] Figure 13 IFA experiment results (magnification 100x)

[0036] Figure 14 Diarrhea score of piglets

[0037] Figure 15 Blank control group piglet anus surrounding situation diagram

[0038] Figure 16 Treatment group piglet anus surrounding situation diagram (A: treatment group ZL-1 piglet anus surrounding situation diagram; B: treatment group ZL-2 piglet anus surrounding situation diagram; C: treatment group ZL-3 piglet anus surrounding situation diagram; D: treatment group ZL-4 piglet anus surrounding situation diagram; E: treatment group ZL-5 piglet anus surrounding situation diagram)

[0039] Figure 17 Challenge group piglet anus surrounding situation diagram (A: challenge group GD-1 piglet anus surrounding situation diagram; B: challenge group GD-2 piglet anus surrounding situation diagram; C: challenge group GD-3 piglet anus surrounding situation diagram; D: challenge group GD-4 piglet anus surrounding situation diagram; E: challenge group GD-5 piglet anus surrounding situation diagram;)

[0040] Figure 18 Autopsy of blank control group piglets (A: Autopsy of MOCK-1 piglet in blank control group; B: Autopsy of MOCK-2 piglet in blank control group; C: Autopsy of MOCK-3 piglet in blank control group;)

[0041] Figure 19 Autopsy of challenge group piglets (A: Autopsy of GD-1 piglet in challenge group; B: Autopsy of GD-2 piglet in challenge group; C: Autopsy of GD-3 piglet in challenge group; D: Autopsy of GD-4 piglet in challenge group; E: Autopsy of GD-5 piglet in challenge group;)

[0042] Figure 20 Autopsy of treatment group piglets (A: Autopsy of ZL-1 piglet in treatment group; B: Autopsy of ZL-2 piglet in treatment group; C: Autopsy of ZL-3 piglet in treatment group; D: Autopsy of ZL-4 piglet in treatment group; E: Autopsy of ZL-5 piglet in treatment group;)

[0043] Figure 21 Viral copy number of piglet intestinal tissue (A: PDCoV viral copy number in duodenum tissue; B: PDCoV viral copy number in jejunum tissue; C: PDCoV viral copy number in ileum tissue; D: PDCoV viral copy number in cecum tissue; E: PDCoV viral copy number in colon tissue; F: PDCoV viral copy number in rectum tissue;)

[0044] Figure 22 Viral copy number of piglet anal swab (A: Viral copy number of daily anal swab of piglet in treatment group; B: Viral copy number of daily anal swab of piglet in challenge group;)

[0045] Figure 23 HE staining of intestinal tissue of treatment group (magnification 20x, 40x) (A: HE staining of jejunum and ileum of ZL-1 piglet in treatment group; B: HE staining of jejunum and ileum of ZL-2 piglet in treatment group; C: HE staining of jejunum and ileum of ZL-3 piglet in treatment group; D: HE staining of jejunum and ileum of ZL-4 piglet in treatment group; E: HE staining of jejunum and ileum of ZL-5 piglet in treatment group;)

[0046] Figure 24HE staining of intestinal tissue of the challenge group (magnification 20x, 40x) (A: HE staining of jejunum and ileum of GD-1 piglet in the challenge group; B: HE staining of jejunum and ileum of GD-2 piglet in the challenge group; C: HE staining of jejunum and ileum of GD-3 piglet in the challenge group; D: HE staining of jejunum and ileum of GD-4 piglet in the challenge group; E: HE staining of jejunum and ileum of GD-5 piglet in the challenge group;)

[0047] Figure 25 HE staining of intestinal tissue of the blank control group (magnification 20x, 40x) (A: HE staining of jejunum and ileum of MOCK-1 piglet in the blank control group; B: HE staining of jejunum and ileum of MOCK-2 piglet in the blank control group.)

[0048] Figure 26 Immunohistochemical staining of intestinal tissue of the challenge group (magnification 20x, 40x)

[0049] Figure 27 Immunohistochemical staining of intestinal tissue of the treatment group (magnification 20x, 40x)

[0050] Figure 28 Immunohistochemical staining of intestinal tissue of the blank control group (magnification 20x, 40x) DETAILED DESCRIPTION

[0051] Example 1 Development and application of genetically engineered chimeric antibody against porcine delta coronavirus (PDCoV)

[0052] 1. Materials and methods

[0053] 1.1 Experimental materials

[0054] 1.1.1 Cells, plasmids and strains ExpiCHO-S cells, pks001 eukaryotic vector, CHO-K1 cells, PDCoV isolate CHN-SC2015 (GeneBank accession number: KY398010) were preserved by the Pig Disease Research Center of Sichuan Agricultural University; TOP10 competent cells were purchased from Anhubio; a hybridoma cell expressing anti-PDCoV S protein neutralizing monoclonal antibody 1H10 was prepared and preserved by the Pig Disease Research Center in the early stage.

[0055] 1.1.2 Kits and main biochemical reagents

[0056] 1.1.2 Kits and main biochemical reagents

[0057] Max DNA polymerase, PrimeScript RT reagent Kit, Xba I, Not I, EcoR I, Hind III were purchased from Dalian Baobio Engineering Co., Ltd.; HRP-Goat anti Pig IgG was purchased from Solabio; FITC-Goat anti Pig IgG, HighGene transfection reagent were purchased from Abmole Biotechnology Co., Ltd.; 555-Donkey anti Mouse IgG was purchased from Biyun Tian; HiScript II Q RT SuperMix for qPCR(+gDNA wiper), ChamQ Universal SYBR qPCR Master Mix were purchased from Novogene Bioinformatics Technology Co., Ltd.; CHO cell culture medium CHO CD04 Medium was purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd.; Cellvento 4 CHO-C Cloning Medium was purchased from sigma-aldrich; Cell Boost 7a, Cell Boost 7b were purchased from Hyclone; Cck-8 reagent was purchased from Taotuo Biotechnology; SteadyPure RNA extraction kit was purchased from Aikangrui Biotechnology Co., Ltd.

[0058] 1.1.3 Experimental animals

[0059] 5-day-old suckling pigs were provided by Chenghua Pig Protection Research Base (Chengdu). After laboratory PCR detection, all piglets were free of PDCoV, CSFV, PCV2, PRRSV, TGEV and PEDV pathogens. The animal experiment scheme was approved by the Sichuan Agricultural University Experimental Animal Ethics and Welfare Committee, and the animal experiment operation procedures of Sichuan Agricultural University [license number SYSK (Chuan 2019-187)] were strictly followed.

[0060] 1.1.4 Main equipment

[0061] Low-temperature benchtop high-speed centrifuge (CF1524R, Scilogex, USA); PCR instrument, microplate reader (Bio-Rad, USA); CO2-resistant horizontal shaker (Jingqi, USA); Class II biological safety cabinet (Forma class II, A2 Biological Satety Cabinet Thereto electron corporation); 125ml cell shake flask, 500ml cell shake flask, purchased from Nunc; upright fluorescence microscope (BX53, OLYMPUS, Japan); cell counter (IE1000, Ruiyu Biotechnology Co., Ltd.).

[0062] 1.2 Methods

[0063] 1.2.1 Codon Optimization and Transient Carrier Synthesis

[0064] The variable region sequence of the laboratory-prepared neutralizing monoclonal antibody 1H10 specifically targeting the PDCoV S1 protein was determined. The variable region sequence was divided into VL and VH, and simultaneously analyzed by the National Center for Biotechnology Information (NCBI) in the United States. https: / / www.ncbi.nlm.nih.gov / nuccore / ab699686 The constant region of the heavy chain and the constant region of the light chain of porcine IgG were identified in the International Immunogenetic Database (IMGT) (https: / / www.imgt.org / ligmdb / view.action?id=FP312898). To improve the expression efficiency in CHO cells, codon optimization was performed after recombination of the murine variable region and the porcine constant region. The resulting sequences were then synthesized by Qingke Biotechnology Co., Ltd., and the synthesized sequences were constructed into the pcDNA3.4 transient transfection vector with XbaⅠ and AgeⅠ restriction enzyme sites for subsequent transient transfection expression. A schematic diagram of the recombination of the variable and constant regions is shown below. Figure 1 As shown.

[0065]

[0066] Nucleotide sequence of codon-optimized expressed recombinant light chain fragment (SEQ ID NO. 2): GCCACCATGGGTTGGTCTTGTATCATCTTGTTCTTGGTCGCTACCGCAACAGGTGTGCATAGCGACATCGTGTTGACTCAGAGTCCAGCTTCTTTGGCTGTCAGCCTGGGCCAGAGAGCCACCATCTCTTGCCGTGCTTCCAAGAGCGTGTCCATCAGCGGTCATAGCTATATGCACTGGTATCAGCAGAAGCCTGGTCAACCACCTAAGCTGCTGATTTACCTCGCCTCTAACTTGGAGAGCGGCGTGCCAGCTAGGTTTAGCGGCTCTGGCTCCGGCACAGATTTCACCTTGAACATTCATCCAGTGGAAGAAGAAGATGCCGCTACCTACTATTGCCAGCATTCACGGGAATTGCCACCTACCTTTGGCTCAGGCACTAAGTTGGAGATCAAGAGGGCTGATGCCAAGCCAAGCGTGTTCATCTTTCCACCAAGCAAAGAACAGCTGGAGACACAGACCGTGAGCGTGGTCTGTTTGCTGAACAGCTTCTTCCCAAGAGAGGTCAATGTTAAGTGGAAGGTCGATGGTGTGGTGCAGTCTAGTGGCATCTTGGACTCTGTCACCGAGCAAGACTCTAAGGACAGCACCTACAGCCTCAGCTCTACTCTGAGCCTGCCAACTTCTCAGTATCTGTCTCATAACCTGTATTCTTGCGAGGTGACTCACAAGACACTGGCCTCACCTCTGGTGAAGAGCTTCTCACGGAATGAGTGCGAAGCAtga

[0067] 1.2.3 Construction of chimeric antibody stable transfection vector

[0068] In order to obtain a monoclonal cell line stably expressing a chimeric antibody, it is necessary to reconstitute a stable transfection vector on the basis of a transient transfection vector, to perform homologous recombination of the amplified heavy chain fragment with the double-digested PKS001 vector, to continue to perform double digestion of the light chain site after single identification of transformation and picking, to perform homologous recombination with the amplified light chain fragment, to also perform single identification of transformation and picking after completion of the construction of the stable transfection vector; to transfer the constructed stable transfection plasmid into CHO-K1 cells, and to perform screening of stable cell lines and monoclonal cell lines, so as to finally obtain a CHO cell line stably expressing a chimeric antibody, and to obtain the final pig-mouse chimeric antibody by purifying cell culture supernatant, and the specific experimental process is shown in Figure 2 .

[0069] The pig-derived CH amino acid sequence of the pig-mouse chimeric antibody ch1H10 (SEQ ID NO. 3) is APKTAPSVYPLAPCGRDTSGPNVALGCLASSYFPEPVTMTWNSGALTSGVHTFPSVLQPSGLYSLSSMVTVPASSLSSKSYTCNVNHPATTTKVDKRVGTKTKPPCPICPGCEVAGPSVFIFPPKPKDTLMISQTPEVTCVVVDVSKEHAEVQFSWYVDGVEVHTAETRPKEEQFNSTYRVVSVLPIQHQDWLKGKEFKCKVNNVDLPAPITRTISKAIGQSREPQVYTLPPPAEELSRSKVTVTCLVIGFYPPDIHVEWKSNGQPEPEGNYRTTPPQQDVDGTFFLYSKLAVDKARWDHGETFECAVMHEALHNHYTQKSISKTQGK

[0070] The pig-derived CL amino acid sequence of the pig-mouse chimeric antibody ch1H10 (SEQ ID NO. 4) is

[0071] RADAKPSVFIFPPSKEQLETQTVSVVCLLNSFFPREVNVKWKVDGVVQSSGILDSV TEQDSKDSTYSLSSTLSLPTSQYLSHNLYSCEVTHKTLASPLVKSFSRNECEA

[0072] The mouse-derived VH amino acid sequence of the pig-mouse chimeric antibody ch1H10 (SEQ ID NO. 5) is

[0073] EVKLLESGGGLVQPGGSLKLSCAASGFDLSRYWMSWVRQAPGKGLEWIGEINPDSRTINYTPSLKDKFIISRDNAKNTLNLQMSKVRSEDTALYYCARQGRGFAYWGQGTLVTVSA

[0074] Porcine-mouse chimeric antibody ch1H10 murine VL amino acid sequence (SEQ ID NO. 6):

[0075] DIVLTQSPASLAVSLGQRATISCRASKSVSISGHSYMHWYQQKPGQPPKLLIYLASN LESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPPTFGSGTKLEIK

[0076] The recombinant heavy chain and light chain fragments are amplified by using the pcDNA3.4 transient transfection vector with the successfully inserted antibody heavy and light chain fragments at the enzyme cutting sites Xba I / Age I as the template. The PKS001 has a multiple cloning site, and the recombinant heavy chain fragment is inserted into the Xba I and Not I enzyme cutting sites, and the light chain fragment is inserted into the Hind III and Eco R I enzyme cutting sites. The specific steps are as follows:

[0077] 1) The heavy chain site to be connected is first subjected to double enzyme cutting by using the restriction endonuclease Xba I and Not I on the PKS001 vector, and the purified products of the recombinant heavy chain amplification fragment and the enzyme cutting vector are subjected to concentration determination, and 2x Seamless Cloning Mix is used for the connection of the two, and the reaction is performed at 50°C for 25 min. Finally, the stable transfection vector connection product of the antibody recombinant heavy chain is transferred into the TOP10 competent cells,

[0078] After the competent bacteria plate grows into colonies, random colonies are selected for identification. A certain number of colonies are resuspended and mixed uniformly in 50 μl Amp + liquid medium, and the bacterial solution is used as the amplification template for identification. After sequencing, the bacterial solution is cultured on a large scale, and the endotoxin-free plasmid is extracted. The plasmid is subjected to enzyme cutting by using the restriction endonuclease Hind III / Eco R I, and the enzyme cutting product is used for subsequent light chain connection.

[0079] 2) The purified products of the antibody recombinant light chain amplification fragment and the double enzyme cutting plasmid are subjected to concentration determination, and 2x Seamless Cloning Mix is used for the connection of the two, and the stable transfection vector connection product of the antibody light chain is transferred into the TOP10 competent cells. After the competent bacteria plate grows into colonies, random colonies are selected for identification. A certain number of colonies are resuspended and mixed uniformly in 50 μl Amp+ The liquid medium was resuspended and mixed, the bacterial solution was used as an amplification template for identification, and after sequencing, the bacterial solution was cultured, and the endotoxin-free plasmid PKS001-ch1H10 plasmid was extracted.

[0080] 3) Since the extracted plasmid may be contaminated with bacteria, and the concentration is relatively low, the ethanol precipitation method is used to remove bacteria and concentrate the extracted PKS001-ch1H10 plasmid. The steps are as follows: add 6 μl of 3M pH 3.5 NaAc solution to the PKS001-ch1H10 plasmid, mix gently; add 180 μl of anhydrous ethanol cooled at 4°C, mix gently; centrifuge, and gently aspirate the supernatant; add 1 ml of 75% ethanol, centrifuge, and gently aspirate the supernatant, repeat this step 6 times; aspirate the 75% ethanol used for washing, blow dry the plasmid in the biological safety cabinet for 10 min, add 30 μl of sterile ddH2O for dissolution, and finally determine the concentration of the plasmid.

[0081] 1.2.4 Expression and purification of chimeric antibodies

[0082] 1.2.4.1 Transient transfection of chimeric antibodies

[0083] Transient transfection requires ExpiCHO-S cells, which are recovered from liquid nitrogen. The cells are also cultured in suspension, and the culture conditions are 95 rpm, 37°C, 8% CO2, and the culture medium used is ExpiCHO Expression Medium. After cell recovery, the cells are continuously passaged at a seeding density of 2*10 5 cells / ml, and 1 day before transfection, 3*10 6 cells / ml are passaged in two shake flasks, each containing 50 ml, and are ready for use. The plasmid required is the PKS001-ch1H10 plasmid.

[0084] On the 2nd day, the ExpiCHO-S cells are adjusted to 6*10 6 cells / ml, 50 ml / flask; two sterile 15 ml centrifuge tubes are taken, labeled tube A and tube B, and the components are added according to Table 6.

[0085] Table 6 Transient transfection system

[0086]

[0087] After adding, transfer the liquid in tube B to tube A, slowly add while rotating the gun head, mix gently and incubate at room temperature for 5 min; take the cells ready for use, slowly add the mixed liquid to them while shaking the cells; after adding, mix the cells and count them, and put the cells back on the carbon dioxide shaker for further culture.

[0088] After 20h transfection, take out the cells, add 8ml ExpiCHO Feed and 300μl ExpiCHO Enhancer into each flask, mix well, take a small amount of cells for counting, put the cells back into the carbon dioxide incubator, 95rpm, 37℃, 5% CO2 for culture.

[0089] On the 5th day of culture, add 8ml ExpiCHO Feed into each flask, put it back into the carbon dioxide incubator, 95rpm, 37℃, 5% CO2 for culture, count and observe the cells every day until the cell viability decreases to below 70%, then collect the cell supernatant for verification.

[0090] 1.2.4.2 Purification and concentration of chimeric antibody

[0091] The cell supernatant was purified using a Protein A recombinant protein purification pre-packed column. The purification steps were as follows: wash the machine and purification column with distilled water; after the values are balanced, use the binding buffer (20mM phosphate buffer, 150mM NaCl, pH 7.4) to flush; after the values are balanced, filter the cell culture supernatant using a 0.45μm filter membrane, and then purify after filtration; after the sample is completely loaded, wash the purification column with binding buffer; after the values are balanced, use elution buffer (0.1M citric acid buffer, pH 4.0) to wash the column, collect the eluted liquid, and immediately adjust the pH to neutral. The chimeric antibody collected by elution was concentrated by ultrafiltration using a 50MWCO ultrafiltration tube, and the antibody concentration was measured.

[0092] 1.2.4.3 Stable transfection of chimeric antibody

[0093] Take the CHO-K1 cells that are being passaged to count and plate 6-well plates. Plate 1*10 6 live cells per well, put it into the incubator for shaking culture, and reserve for use.

[0094] On the 2nd day of cell plating, prepare for transfection experiment. The transfection steps are as follows: according to the cell volume, aspirate the corresponding mass of PKS001-ch1H10 plasmid, and perform transfection according to the ratio of 1 μg plasmid to 1 ml cells. First, dilute with CHO CD04 Medium, and add 100 μl of medium per 1 μg of plasmid. After mixing, add HighGene reagent according to the ratio of 3 times the mass of the plasmid, mix, and stand for 10 min. Add the mixture to the previously prepared 6-well plate cells. Place the cells in the incubator for continued shaking culture. After 3-5 days of transfection, take the stably transfected cells to culture the expressed chimeric antibody. Alternatively, take the stably transfected cells after pressure screening, perform single clone cell screening, and obtain high-expression single clone cells for seed preservation.

[0095] 1.2.5 In vitro activity identification of chimeric antibody

[0096] 1.2.5.1 SDS-PAGE identification of ch1H10

[0097] The non-reducing SDS-PAGE uses a protein loading buffer that does not contain mercaptoethanol, which can preserve the disulfide bonds between antibodies, allowing the antibody to maintain its complete molecular structure, which is used for the identification of complete multimeric proteins / antibodies. The reducing SDS-PAGE uses a protein loading buffer containing mercaptoethanol, which is used to observe the monomer size of the heavy chain and light chain of the antibody. The specific steps are as follows:

[0098] (1) Non-reducing SDS-PAGE:

[0099] Add 10 μl of 5x non-reducing protein loading buffer to 40 μl of the antibody to be tested at an appropriate concentration. React the mixture at 90°C for 1 min and cool. Add the sample and Marker to a 7.5% electrophoresis gel, and perform vertical gel electrophoresis according to the program of 80V for 25 min and 120V for 60 min. After electrophoresis, perform staining and decolorization using Coomassie Brilliant Blue. Take pictures and record them.

[0100] (2) Reducing SDS-PAGE:

[0101] Add 10 μl of 5x reducing protein loading buffer to 40 μl of the antibody to be tested. React the mixture at 100°C in a water bath for 5 min, and wait for the sample to cool completely. The remaining steps are the same as those for "non-reducing SDS-PAGE".

[0102] 1.2.5.2 Cytotoxicity assay of ch1H10

[0103] The ch1H10 used in this experiment is transiently transfected, with a concentration of 1.285 mg / ml. The 96-well plate ST cells are laid out and the cells are grown for about 24 h; 10 μl of different concentrations of chimeric antibodies are added to the 96-well plate, with 3 replicates for each concentration; after 48 h of co-incubation, 10 μl of CCK-8 solution is added to each well, and the incubation is continued for 2 h; the OD 450nm values are measured using an enzyme marker.

[0104] The cytotoxicity of the measured chimeric antibodies is calculated according to the formula cell viability (%) = [A (with drug) - A (blank)] / [A (0 with drug) - A (blank)] x 100, where A (with drug) refers to the OD value of the well with cells, CCK-8 and antibodies, A (blank) refers to the OD value of the well with only medium, CCK-8 and no cells, and A (0 with drug) refers to the OD value of the well with only cells, CCK-8 and no antibodies. The cell viability calculated according to the formula is used to determine whether the antibody has cytotoxicity.

[0105] 1.2.5.3 Determination of EC50 of ch1H10

[0106] The antibody concentration used in this experiment is the same as that in 1.2.5.2. The ST cells in the 96-well plate are laid out, and when the cells are nearly full (80% to 90%), the virus incubation is prepared; according to the previously determined virus TCID 50 data in the laboratory, the 107th passage of PDCoV virus is diluted by 330,000 times (serum-free DMEM as diluent); at the same time, the chimeric antibody is treated and gradient diluted (serum-free DMEM as diluent) according to the experimental requirements. Finally, the diluted virus solution is added to the antibody diluent with a ratio of 1:1, and the mixture is placed in a 37°C incubator for 1 h; the virus-antibody mixture after reaction is added to the pre-cultured ST cells, 100 μl per well, with 6 replicates for each experimental group, and the cells are further cultured, and the cell lesions are observed daily; when the cells in the experimental group appear obvious lesions, the lesion data of each experimental group well are recorded; when exactly half of the cell wells (3 wells) in the experimental group appear lesions, the antibody concentration under this condition is the EC 50 .

[0107] 1.2.5.4 Analysis of the binding ability of ch1H10 to virus S1 protein

[0108] The experiment uses the existing PDCoV S1 segment protein in the laboratory, and the antibody concentration used is the same as 1.2.5.2. Coating: Dilute the protein with coating solution and add it to the enzyme-labeled plate, 1 μg per well, 100 μl in volume. Incubate at 37°C for 1 h and then incubate overnight at 4°C; Wash the plate: Pour out the protein coating solution and pat dry, add 250 μl PBST to each well and shake to wash, a total of three times, 3 min each time. After the last washing, pat the plate dry; Blocking: Under room temperature conditions, use self-prepared 5% skimmed milk powder to block the ELISA plate, 100 μl / well, incubate at 37°C for 1.5 h; Wash the plate: as above; Primary antibody incubation: according to experimental requirements, add different dilution ratios of chimeric antibody solution, 100 μl / well, set 3 replicate wells, incubate at 37°C for 1 h; Wash the plate three times; Secondary antibody incubation: use HRP-goat anti-pig IgG for the experiment, dilute the secondary antibody 4000 times, 100 μl / well, incubate at 37°C for 0.5 h or at room temperature for 1 h; Wash the plate three times; Color development and termination: add color developing solution, 100 μl / well, avoid light at room temperature for 15 min, then add 100 μl of commercial H2SO4 termination solution to each well to terminate; Reading: use an enzyme-labeled instrument to read the OD 450nm of the enzyme-labeled plate.

[0109] 1.2.5.5 ch1H10 indirect immunofluorescence experiment

[0110] The antibody concentration used in the experiment is the same as 1.2.5.2. ST cells are plated in cell crawling sheets of 24-well plates, and when the cells grow to 90%, PDCoV is infected at MOI = 0.01; fixation: when the cells just begin to appear pathological, discard the virus liquid medium, add 4% paraformaldehyde, incubate at room temperature for 30 min; permeation: after fixation, wash 3 times with PBS solution, discard the water. Add an appropriate amount of 0.1% Triton X-100, cover the cells, incubate at room temperature for 30 min and wash again 3 times; blocking: prepare 2% BSA-PBS mixture, add to the 24-well plate to cover the cells for blocking, block at room temperature for 1 h; primary antibody incubation: after blocking, without washing the plate, dilute the chimeric antibody or the original mouse antibody as the primary antibody, 150 μl per well, and the blank control wells are added with the same amount of PBS solution. Incubate at 37°C for 1 h or at 4°C overnight; secondary antibody incubation: after incubation of the primary antibody, wash with PBST for 3 min each time, a total of 3 times, the chimeric antibody experimental wells and the blank control use FITC-goat anti-pig IgG as the secondary antibody, and the original antibody experimental wells use 555-donkey anti-mouse IgG as the secondary antibody, incubate at 37°C in the dark for 1 h; nuclear staining and result observation: after incubation of the secondary antibody, wash 3 times with PBST, pour out the liquid, take out the crawling sheet and pour it upside down into an anti-fluorescence decay mounting agent (containing DAPI), and finally observe the results using a fluorescence microscope and save the pictures.

[0111] 1.2.6 Animal in vivo treatment experiment of ch1H10

[0112] The whole treatment experiment process is shown in Figure 3 , and the antibody concentration used is the same as 1.2.5.2. One day after the stress is eliminated, the piglets are orally infected, and the antibody is injected intramuscularly 12 h later. The mental state and feces of the piglets are recorded daily after infection, and the situation near the anus of the piglets is photographed and recorded. Six days after infection, all experimental group piglets are dissected.

[0113] 1.2.6.1 Animal grouping and infection

[0114] Thirteen healthy 5-day-old piglets were prepared and randomly divided into three groups: 5 piglets in the challenge control group, 5 piglets in the antibody treatment group, and 3 piglets in the blank control group. After 1 day of pre-feeding to eliminate stress, anal swabs of all piglets were collected when they were healthy, and 1 ml of sterile PBS was added to the swabs, which were then stored in a -80°C refrigerator. The piglets were numbered and marked on their backs using crystal violet solution for identification. The piglets in the antibody treatment group were numbered ZL1-ZL5, the piglets in the challenge control group were numbered GD1-GD5, and the piglets in the blank control group were numbered MOCK1-MOCK3. The three groups of piglets were fed separately in different rooms. The specific grouping is shown in Table 7. After 1 day of pre-feeding, the piglets were orally challenged with the existing 21st generation PDCoV virus in the laboratory. The virus TCID 50 of this generation was about 10 5.7 / 100 μl. Based on this data, the piglets were orally challenged with 10 7 TCID 50 / mL for the challenge control group and the antibody treatment group, and the blank control group was given the same dose of DMEM medium. The specific dose is shown in Table 7.

[0115] Table 7: Grouping of piglets in the experiment

[0116]

[0117] 1.2.6.2 Injection of chimeric antibody

[0118] At 12 hours after challenge, the body weight of the piglets in the antibody treatment group was measured, and ch1H10 (1 mg / kg) was injected intramuscularly according to the body weight. The piglets in the challenge control group and the blank control group were injected with the same amount of DMEM (1 mg / kg) intramuscularly.

[0119] 1.2.6.3 Clinical observation and recording

[0120] After challenge, the mental state of the piglets was recorded every 8 hours to observe whether the piglets showed symptoms such as depression and decreased appetite. The feces of the piglets were also recorded, and a diarrhea score was given according to the diarrhea table (Table 8) to analyze the severity of diarrhea in the piglets during the experiment.

[0121] Table 8: Feces condition scoring table

[0122]

[0123] 1.2.6.4 Dynamic virus shedding monitoring

[0124] After challenge, anal swabs of the piglets were collected once a day, and 1 ml of sterile PBS was added to the swabs, which were then stored in a -80°C refrigerator for later analysis of the changes in virus shedding. At the same time, the anal area of the piglets was photographed for record, which facilitated the analysis of the changes around the anus caused by diarrhea in the piglets.

[0125] 1.2.6.5 Autopsy lesion observation and submission

[0126] On the 6th day after challenge, the piglets were anesthetized for autopsy, and the intestinal conditions of each piglet were recorded, mainly observing whether the piglet's intestine was congested, thinned, and transparentized, etc.; and observing the morphology of the intestinal contents. The small intestine segments (duodenum, jejunum, ileum) and large intestine segments (cecum, colon, rectum) of each piglet were collected in turn. The specific operation was as follows: about 1 cm of intestinal segment was cut with sterile surgical scissors and placed in 4% paraformaldehyde solution; an equal length of intestinal segment was cut and placed in a 2 mL centrifuge tube and stored in a -80°C refrigerator for standby. The fixed jejunum and ileum tissues were sent to Baoosi Biotechnology Co., Ltd. for HE staining and immunohistochemical detection analysis. The antibody used in the immunohistochemical experiment was a mouse monoclonal antibody 1H10 prepared by the laboratory, and the target was PDCoV S protein. The dilution multiple of the antibody was 1:600.

[0127] 1.2.6.6 Determination of enterovirus load

[0128] A centrifuge tube with small steel beads was used, 30 mg of intestinal tissue was weighed and placed in the centrifuge tube, and a grinder was used for crushing and grinding. 400 μl of Trizol lysis solution was added to the crushed tissue, and it was placed in a -80°C refrigerator for primary freeze-thawing. The freeze-thawed liquid was used for RNA extraction in the subsequent steps, and the extraction steps were performed according to the SteadyPure RNA Extraction Kit instructions of Aikewei Biological. RNA reverse transcription was performed immediately after extraction.

[0129] RNA extraction of anal swabs did not require the use of Trizol lysis solution. First, mix the liquid in each tube of anal swab thoroughly, and then extract an equal amount of 100 μl of suspension, and then follow the SteadyPure Rapid RNA Extraction Kit instructions of Aikewei Biological, and also immediately reverse transcribe the RNA obtained by extraction.

[0130] 1.2.6.7 Reverse transcription and fluorescent quantitative PCR

[0131] According to the instructions of the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit of Novozyme, the RNA extracted in the previous step was reverse transcribed.

[0132] The content of PDCoV in the intestinal tissue and anal swab of piglets was determined by fluorescent quantitative PCR. The detection primers used were PDCoV N-F: CTATGAGCCACCCACCAA, PDCoV N-R: TCCCACTCCCAATCCTGT. At the same time, PDCoV N plasmid was used for experiment, with a concentration of 740 ng / μl, dilution by ratio, and 10 -3 ~10 -10 The eight concentration gradients were used for experiment, and the standard curve was run for absolute quantification. The experiment was performed according to the instructions, and the total reaction system was 15 μl, including 7.5 μl of 2×ChamQ Universal SYBR qPCR Master Mix, 0.3 μl of upstream and downstream primers, 1.5 μl of cDNA template, and 5.4 μl of ddH2O. The reaction conditions were as follows: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, 39 cycles; 65℃ for 5 s, 95℃ for 5 s.

[0133] 2, Research results

[0134] 2.1 Codon optimization and synthesis of transient vector

[0135] After codon optimization and synthesis by GenScript, the recombinant antibody fragment was successfully constructed into the transient vector pcDNA3.4. The schematic diagram of the two transient vectors is shown in Figure 4 The antibody heavy and light chain fragments were inserted into the enzyme digestion site Xba I / Age I.

[0136] 2.2 Construction of stable transfection vector of chimeric antibody 1H10

[0137] The heavy chain fragment of antibody 1H10 was successfully amplified using primers 1H10-H-F (sequence: TCTAGAGCCACCATGGGCTGGTCT) and 1H10-H-R (sequence: GCGGCCGCTCACTTGCCCTGTGTCTTGGA), with a length of 1398 bp. The results are shown in Figure 5The amplified fragment was homologously recombined with the PKS001 vector digested with Xba I and Not I, and the PKS001-1H10-H vector was successfully constructed. The sequence was verified by GenScript. The light chain of the antibody 1H10 was amplified by PCR using the primers 1H10-L-F / 1H10-L-R (sequence: AAGCTTGCCACCATGGGTTGGTCTT / GAATTCTCATGCTTCGCACTCATTCCG), and the length of the fragment was 726 bp. Meanwhile, the heavy chain plasmid was digested with Hind III and EcoR I. After homologous recombination of the light chain fragment and the heavy chain plasmid and transformation, the PKS001 stable vector carrying the heavy chain and light chain fragments was successfully obtained. The size of the PKS001-ch1H10 plasmid was 11480 bp, and the schematic diagram of the plasmid is shown in Figure 6 .

[0138] 2.4 Expression and purification of the chimeric antibody

[0139] 2.4.1 Transient transfection

[0140] After the transient transfection of the EXPI-CHOS cells, the cells were normally cultured for expression. However, due to the limited conditions, the cells could not be cultured at 32°C, and the cell activity was reduced to less than 70% at the 10th day. The supernatant of the transiently transfected cells was centrifuged at the 10th day after transfection, and subsequent purification work was performed.

[0141] 2.4.2 Purification and concentration of the chimeric antibody

[0142] The supernatant of the CHO-K1 cell culture was purified using a recombinant protein A purification column. The elution peak was collected and neutralized. The peak chart of the purification process is shown in Figure 7 . The antibody concentration was low after purification, and concentration treatment was still needed. The antibody eluate was ultrafiltrated and concentrated using an ultrafiltration tube with a molecular weight cut-off of 50000. Finally, 8 ml of ch1H10 with a concentration of 1.285 mg / ml was obtained.

[0143] 2.4.3 Stable transfection

[0144] The constructed stable transfection plasmid PKS001-ch1H10 was transfected into the 6-well plate CHO-K1 cells prepared in advance. Four days after transfection, the cells were transferred to a T75 cell culture bottle for subsequent screening work.

[0145] 2.4.4 Establishment of the ch1H10 monoclonal cell line

[0146] After two weeks of pressure screening, the transfected ch1H10 CHO-K1 cells have recovered their growth activity, and at this time, the establishment of the single clone cell line was carried out. After expansion culture, 44 effective clones were obtained. The supernatant of the clones was verified using PDCoV S protein coated ELISA plate, and the supernatant adding order is shown in Table 9. According to the final OD 450nm reading value, 16 clones with relatively high reading value were selected for expansion culture, and finally preservation treatment was carried out, and the reading value results are shown in Table 10.

[0147] Table 9 supernatant adding order

[0148]

[0149] Table 10 supernatant ELISA verification reading value results

[0150]

[0151] 2.5 In vitro activity identification of ch1H10

[0152] 2.5.1 SDS-PAGE identification of ch1H10

[0153] The supernatant of ch1H10 cell culture, purified flow-through and purified antibody were verified by SDS-PAGE, and the results showed that under non-reducing conditions, ch1H10 was about 150 KDa, and in the supernatant of the cells, the antibody heavy chain band (about 50 KDa) and light chain band (about 23 KDa) could be seen; while in the flow-through, the heavy chain and light chain bands were obviously lighter, which proved that ch1H10 antibody was successfully expressed and purified, and correct assembly (heavy chain + light chain) also occurred. In the lane of the purified sample, in addition to the normal complete antibody band, there were two particularly light antibody heavy chain and light chain bands, which were speculated to be caused by the breakage of the disulfide bond of a small amount of antibody during sample processing, resulting in the splitting of the heavy chain and light chain. Details are shown in Figure 8 (A). Subsequently, the purified antibody sample was subjected to reduction / non-reduction SDS-PAGE verification alone, and the results are shown in Figure 8 (B), which showed that the purified and concentrated ch1H10 could be divided into heavy chain and light chain under reducing conditions, and the structure was correct.

[0154] 2.5.2 Cytotoxicity experiment of ch1H10

[0155] According to the neutralizing effect of the original mouse 1H10, the antibody concentrations of 24 μg / ml, 12 μg / ml, 6 μg / ml, 3 μg / ml and 1.5 μg / ml were selected for cytotoxicity experiment. After adding the antibody, the cells were placed in the incubator for 48 h to simulate the long-term presence of chimeric antibodies in animals. After adding CCK-8 reagent, continue to culture for 2 h, and analyze the results after color development, which is Figure 9 It can be seen that after incubation of ST cells with ch1H10 at various concentrations, the cell activity is higher than 100% after 48 h, and there is no obvious dose-dependent trend, indicating that ch1H10 has no cytotoxicity to porcine cells.

[0156] 2.5.3 Determination of EC50 value of ch1H10

[0157] According to the concentration of ch1H10 after purification and concentration, the antibody was diluted at a dilution ratio of 1:50, 1:75, 1:100, 1:150, 1:200, 1:300, 1:400, 1:600, 1:800, 1:1200. According to the existing virus titer data in the laboratory, 107 passages of PDCoV were diluted by 330,000 times, and then the diluted virus liquid was mixed with the antibody dilution liquid at a ratio of 1:1. According to the experimental procedure described previously, the cytopathic effect was observed and the data was arranged, and the results are shown in Figure 10 When the dilution ratio is 1:200-1:300, about half of the cell wells have obvious cytopathic effect, indicating that the EC 50 of ch1H10 is about in this dilution ratio interval, and the antibody concentration is about 3.125-4.16 μg / ml.

[0158] 2.5.4 Analysis of ELISA binding ability of ch1H10

[0159] ELISA plate was coated with PDCoV S1 protein. According to the ELISA experimental procedure, the antibody ch1H10 was diluted at a ratio of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, and the experimental results showed that when ch1H10 was diluted by 1:6400, it could still bind well with PDCoV S1 protein, and the antibody concentration was about 0.2 μg / ml, and the OD 450nm reading was about 0.59. Details are shown in Figure 11 , and the specific OD value data is shown in Table 11.

[0160] Table 11 ELISA experimental data

[0161]

[0162] 2.5.5 Identification of ch1H10 by indirect immunofluorescence experiment

[0163] According to the previous virus research of the laboratory, 33 million-fold diluted 107 generation PDCoV was inoculated into ST cell 24-hole cell slides, and after 24 h of constant temperature incubation at 37℃, the subsequent experiments could be carried out. After fixation, permeation, blocking and other steps, ch1H10 with a final concentration of 10 μg / ml was used for overnight incubation, and the same concentration of original mouse-derived 1H10 antibody was set as a control group, and PBS solution was set as a blank control group. As for the secondary antibody, both the experimental holes of ch1H10 and the blank control group used 200-fold diluted FITC goat anti-pig fluorescent secondary antibody, while the mouse antibody control holes used 200-fold diluted 555-donkey anti-mouse fluorescent secondary antibody. All experimental holes were operated consistently, and finally the cell slides were taken off and subjected to nuclear staining and fixation. After observation under a fluorescence microscope, obvious fluorescence could be observed on the cell slides of the experimental holes and the mouse antibody control holes, and the fluorescence position was cytoplasm, and the fluorescence intensity and quantity were comparable; the blank control holes had no fluorescence reaction. The results showed that ch1H10 could bind to PDCoV live virus, and the details were shown in Figure 12 (40x objective field), Figure 13 (100x objective field).

[0164] 2.5.6 ch1H10 in vivo challenge treatment experiment of piglets

[0165] One day before the challenge, the piglets were normally fed and ensured to be healthy. After 24 h of eliminating the stress caused by transportation, the piglets were orally challenged, and the feces and mental state of the piglets were recorded according to the plan. After 12 h of challenge, the body weight of the piglets was measured, and the injection amount of the antibody was arranged according to the body weight. The specific body weight data and antibody injection amount were shown in Table 12.

[0166] Table 12 Body weight and antibody injection amount of the treatment group

[0167]

[0168] 2.5.6.1 Analysis of mental state and feces of piglets

[0169] At the time of challenge, all piglets were normal, with a good spirit and normal feeding.

[0170] Control group: From 0 to 12 hours after challenge, piglets showed no obvious abnormalities or diarrhea. Starting at 16 hours post-challenge, piglets' appetite decreased to only 50% of normal levels, with no significant changes in fecal condition yet. From 24 to 32 hours, the piglets' appetite remained very poor, at its worst consuming only about 30% of their healthy levels. They were lethargic, frequently standing still, and developed severe watery diarrhea during this stage. From 40 hours onwards, the piglets' appetite began to improve, but the amount of feces outside the cage decreased. During anal swab collection, fecal splattering occurred as the swab was inserted. By the 72nd hour, most piglets showed significant improvement in their condition and diarrhea had subsided. However, piglets GD-1 and GD-5 continued to experience watery diarrhea, with watery feces overflowing during anal swab collection. By the 80th hour, piglet GD-1 exhibited lethargy, and several hours later, its limbs became cold, leading to its death. Piglet GD-5 also developed similar symptoms at the 96th hour and died at the 100th hour. Subsequently, the remaining three piglets were in good condition, and their feces gradually hardened. They were euthanized on the 6th day after infection.

[0171] Antibody treatment group: From 0 to 12 hours after challenge, piglets showed no obvious abnormalities or diarrhea. At 16 hours after challenge and 4 hours after antibody treatment, piglets also showed decreased appetite, but the situation was better than the challenge control group, with feed intake only decreasing by about 30% compared to the healthy period. At this time, most piglets had normal mental state and fecal condition; only piglets ZL-3 showed diarrhea and lethargy. From 24 to 72 hours after challenge and from 12 to 60 hours after antibody treatment, all piglets developed watery diarrhea with large fecal volumes. However, their mental state was much normal compared to the control group, with no lethargy or standing still. They were energetic and eager to eat during feeding, while piglets ZL-3 were relatively lethargic and had more severe diarrhea during this period. Subsequently, the diarrhea in the treatment group improved, and their feces began to dry and harden. Piglets ZL-3 also gradually returned to normal. About 108 hours after the challenge, piglets ZL-5 were observed to have diarrhea, but it was not severe. Their feces returned to normal after about 32 hours, which was speculated to be due to secondary infection or diarrhea caused by exposure to cold at night. All piglets were euthanized on the 6th day after the challenge.

[0172] Blank control group: The three piglets in this group showed no abnormal changes during the experiment, had normal diet, good mental state, and no obvious diarrhea symptoms.

[0173] Detailed scoring of piglet feces during the experiment can be found in [link to experiment]. Figure 14 As shown, the condition of the piglet's anus is as follows. Figure 15 , 16 17, of which Figure 16There were only 4 groups of pictures for ZL5 piglets in the treatment group due to the failure of shooting, resulting in the absence of pictures for 2 days. When the diarrhea time of piglets was longer and the degree of diarrhea was more severe, the amount of feces around the anus would be more, and the appearance would be more dirty. When the diarrhea improved, the sanitary condition around the anus would also improve.

[0174] In summary, the results showed that the diarrhea symptoms of piglets in the antibody treatment group were lighter than those in the challenge control group, and they could recover faster.

[0175] 2.5.6.2 Autopsy of piglets

[0176] In addition to GD-1 and GD-5 that died of disease, the rest of the piglets were autopsied on the 6th day after challenge.

[0177] There were no obvious abnormal conditions in the autopsy of the three piglets in the blank control group. MOCK-3 piglet had intestinal hyperemia due to insufficient blood loss, but the mesenteric lymph nodes were not congested. Details are shown in Figure 18 .

[0178] The GD-1 and GD-5 piglets that died of disease had obvious pathological changes, with obvious hyperemia of the intestinal tract and mesenteric lymph nodes, thinning of the intestinal wall, and obvious liquid and gas (black arrow) inside. The intestinal tissue was in a fragile and brittle state during sample separation.

[0179] In addition to GD-1 and GD-5, the other three piglets in the challenge control group had no severe hyperemia of the intestinal tract. The GD-2 and GD-3 piglets had thinning of the intestinal wall with obvious liquid (black arrow) inside, but their large intestine was normal, and moist feces were visible at the end of the rectum. The GD-4 piglet had normal intestinal tissue without obvious thinning or gas, and even formed yellow feces (red arrow) was visible. Details are shown in Figure 19 .

[0180] The five piglets in the antibody treatment group had no obvious characteristic pathological changes during autopsy. ZL-1 piglet had a firm intestinal tract (yellow arrow) without obvious symptoms of distension and thinning. In the colon of ZL-2 piglet, even formed dry feces (red arrow) was visible. ZL-4 piglet had slight transparent thinning symptoms (black arrow) in the jejunum, and the rest had no obvious abnormalities. Details are shown in Figure 20 .

[0181] In summary, the results showed that ch1H10 had a certain protective effect on piglets, and the degree of intestinal lesion was light, which was significantly improved compared with the challenge group.

[0182] 2.5.6.3 Determination of viral load in the intestinal tract of piglets

[0183] The RNA in the intestinal samples was extracted, and after reverse transcription, the detection of fluorescent quantitative PCR was carried out. The results show that PDCoV can be detected in each intestine. In the duodenum, the virus load of the challenged group piglets is significantly higher than that of the treatment group, except for GD2 and GD4. In the jejunum and ileum, the virus load of the challenged group is significantly higher than that of the treatment group, with significant difference. In the large intestine (colon, cecum and rectum), the difference is more significant. The virus load of GD2 and GD4 piglets in the intestine is always low, similar to that of some piglets in the treatment group. On the contrary, the virus load in the intestines of dead piglets GD1 and GD5 is always at the highest level, which can reach or even exceed 10 8 Copies / mL; while the virus level in the intestine of ZL1 piglet in the treatment group is the highest in the same group, with only a slight difference from the challenged group; in general, the virus copy number in the intestinal tissue of the antibody treatment group piglets is significantly lower than that of the virus control group, and the detailed virus copy number in the intestinal tissue of the three groups of piglets is shown in Table 2. Figure 21 .

[0184] In summary, the results show that ch1H10 can effectively neutralize PDCoV, so that the overall viral copy number in the intestinal tissue of the antibody treatment group is significantly lower than that of the challenged control group.

[0185] 2.5.6.4 Virus load determination of anal swabs

[0186] The results of fluorescent quantitative PCR detection of anal swabs of piglets show that the virus can be detected in the anal swabs before necropsy, and the highest virus shedding amount of both experimental groups of piglets is reached at 2-3 days after challenge, with virus copy number between 10 7 Copies / mL and 10 9 Copies / mL, but the virus shedding amount of the treatment group decreases rapidly, while the challenged group of piglets still has a high virus shedding amount (about 10 7 Copies / mL) at 5 dpi. The detailed information is shown in Table 3. Figure 22 .

[0187] The results show that ch1H10 can reduce the virus shedding amount of sick piglets in advance and help the piglets recover health.

[0188] 2.5.6.5 HE staining analysis of jejunum / ileum of piglets

[0189] After necropsy of experimental piglets, the tissues of jejunum and ileum were taken to make pathological sections and observe HE staining. The results of HE staining are shown in Figure 23 、 Figure 24 、 Figure 25The tissue samples from the blank control group were morphologically normal, thick overall, and had intact intestinal villi. The intestinal samples from the antibody treatment group were generally better, with no thinning or other abnormalities. Microscopically, the intestinal mucosa was normal in morphology, the intestinal villi were neatly arranged, the intestinal glands were regularly shaped, and there were few lymphoid follicles in the lamina propria, with only a few chronic inflammatory cell infiltrations. Mild intestinal edema and lymphoid follicles were observed in the intestines of piglets ZL-4 and ZL-5. The pathological changes in the samples from the challenge control group were more severe, especially in the samples from the two dead piglets GD1 and GD5. The jejunum and ileum of piglet GD1 showed extensive necrosis, mucosal epithelial sloughing, and significant thinning and even rupture of the intestinal wall, presumably due to the intestinal's fragility and potential damage during sampling. Extensive inflammatory cell infiltration was observed in the intestinal lamina propria, and vascular congestion and dilation were observed in multiple tissues. The samples from the other three piglets were relatively intact, but the intestinal villi showed slight atrophy and loss.

[0190] The results showed that ch1H10 could effectively protect small intestinal tissue and reduce the damage of PDCoV to intestinal cells.

[0191] 2.5.6.6 Immunohistochemical analysis of piglet jejunum / ileum

[0192] Immunohistochemical analysis of the jejunum and ileum was performed using the original mouse anti-1H10 antibody. A positive reaction was observed as brown granules. See details below. Figure 26 , 27 And 28. Except for the blank control group, viral particles were observed in the jejunum and ileum tissues of all experimental piglets. In the challenge control group, GD-1 and GD-5 had the highest number of viral particles, with a large number of brown particles clearly visible in the field of view, and a large number of inflammatory cells were also present. In the villi of the intestinal segments of the other piglets, varying numbers of viral particles were detected. The antibody treatment group samples all contained a small number of viral particles, with ZL-5 showing a significantly higher number of viral particles than the other piglets in the same group. However, except for GD-1 and GD-5, there was no significant difference in the number of viral particles in the piglet samples of the two experimental groups.

[0193] The results showed that ch1H10 could neutralize and reduce the number of viral particles in the jejunum and ileum of piglets to a certain extent.

[0194] 3. Conclusion

[0195] This study successfully constructed a recombinant vector expressing a porcine-mouse chimeric antibody using genetic engineering recombination technology, and obtained the chimeric antibody ch1H10 by expression in CHO cells. Cellular experiments demonstrated that this recombinant antibody possesses good viral binding and neutralizing activity. In vivo experiments showed that this chimeric antibody has antiviral therapeutic effects against PDCoV-infected piglets, providing a practical genetically engineered antibody that avoids heterologous reactions and exhibits excellent neutralizing therapeutic effects for the prevention and control of PDCoV, thus possessing significant clinical application value.

Claims

1. A pig-mouse chimeric antibody against porcine delta coronavirus, characterized in that: It is composed of heavy chain and light chain; the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 3, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5, the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

6.

2. A gene segment encoding the pig-mouse chimeric antibody of claim 1, characterized by: It includes a gene fragment encoding a heavy chain with a nucleotide sequence shown as SEQ ID NO. 1, and a gene fragment encoding a light chain with a nucleotide sequence shown as SEQ ID NO.

2.

3. A recombinant vector expressing the pig-mouse chimeric antibody of claim 1, characterized by: It is an expression vector with a gene fragment encoding a heavy chain with a nucleotide sequence shown as SEQ ID NO. 1 inserted into Xba I and Not I enzyme cutting sites, and a gene fragment encoding a light chain with a nucleotide sequence shown as SEQ ID NO. 2 inserted into Hind III and EcoR I enzyme cutting sites.

4. The recombinant vector of claim 3, wherein: The expression vector is a PKS001 plasmid vector.

5. A recombinant cell expressing the pig-mouse chimeric antibody of claim 1, characterized in that it is a mammalian cell comprising the recombinant vector of claim 3.

6. The recombinant cell of claim 5, wherein: The mammalian cell is an ExpiCHO-S cell or a CHO-K1 cell.

7. A method for preparing the porcine-mouse chimeric antibody of claim 1, characterized in that: The method comprises the following steps: The recombinant vector of claim 3 is used to transfect cells, which are cultured, and the supernatant is purified to obtain the product. The transfection is selected from transient transfection or stable transfection.

8. The method of claim 7, wherein: The method of transient transfection is: take OptiPRO TM SFM and recombinant vector in tube A; take OptiPRO TM SFM and ExpiFectamine TM CHO Reagent in tube B; mix the solutions in tube A and tube B and stand for 5 min, then add to cell culture medium, and add ExpiCHO after 20 h of culture TM Feed, ExpiCHO TM Enhancer, culture to the 5th day, and then add ExpiCHO TM Feed culture to the cell viability decreases to below 70%.

9. The method of claim 8, wherein: The OptiPRO in tube A TM The ratio of SFM and recombinant vector was 2 ml: 40 pg; OptiPRO in the tube B TM SFM and ExpiFectamine TM The ratio of CHO Reagent was 1.84 ml: 160 μΐ; the ratio of cell culture fluid to recombinant vector was 50 ml: 40 μg; The cell culture medium with ExpiCHO added after 20h of cultivation TM Feed, ExpiCHO TM Enhancer and ExpiCHO added to the culture until day 5 TM The volume ratio of Feed is 50ml: 8ml: 300μl: 8ml; The cells in the cell culture fluid are ExpiCHO TM - S cells, culture medium is ExpiCHO TM ExpressionMedium; The culture condition is 95 rpm of rotation speed, 37℃ of temperature, and 8% of CO2 concentration.

10. The method of claim 7, wherein: The method for stable transfection is as follows: the recombinant vector is diluted with CHO CD04 Medium, and then HighGene reagent is added and left for 10 min, and the obtained mixture is used to transfect CHO-K1 cells for 3-5 days; the ratio of the recombinant vector, CHO CD04 Medium and HighGene reagent is 1 μg: 100 μl: 3 μg. The culture condition is 95 rpm of rotation speed, 37℃ of temperature, and 8% of CO2 concentration. The purification is performed by using a Protein A recombinant protein purification pre-packed column to purify the supernatant.

11. Use of the pig-mouse chimeric antibody of claim 1 in the preparation of a medicament for preventing and / or treating porcine delta coronavirus infection.

12. A medicament for treating porcine delta coronavirus infection, characterized by: It is prepared by using the pig-mouse chimeric antibody of claim 1 as an active ingredient, and adding a pharmaceutically acceptable carrier.

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