LU503556B1 - Monoclonal antibody or derivative generated based on transgenic goat and application thereof - Google Patents

Abstract

This invention relates to a monoclonal antibody or derivative generated based on transgenic goat and application thereof. The NAb produced by the transgenic process of the present invention shows high neutralizing activity against the original strain, Alpha, Beta, Gamma, Delta and Omicron mutant strains of COVID-19. It has been found that tgNAb has higher broad spectrum when compared to neutralizing antibody isolated from peripheral blood of convalescent patients with COVID-19 (pbNAb).

Description

DESCRIPTION LU503556

MONOCLONAL ANTIBODY OR DERIVATIVE GENERATED BASED ON

TRANSGENIC GOAT AND APPLICATION THEREOF

TECHNICAL FIELD

The invention belongs to the field of cell immunology and molecular biology and relates to a monoclonal antibody or derivative generated based on transgenic goat and application thereof.

BACKGROUND

The International Committee on Taxonomy of Viruses has named the novel coronavirus SARS-CoV-2, and the World Health Organization has referred to pneumonia caused by the virus as COVID-19. The virus is highly infectious and has a wide spread.

The virus can quickly adapt to the human environment, and has the ability to spread in the incubation period after infection. At the same time, some asymptomatic infections have been reported, and even viral nucleic acids have been detected in a variety of animals. These factors make the prevention and control of the virus very complicated, and there are no effective therapeutic drugs and vaccines available.

SARS-CoV-2 belongs to the genus Coronavirus, which is a single-stranded positive-sense RNA virus with a size of about 30 kb. The similarity with SARS-CoV is 79%, and the highest similarity with Coronavirus (CoV) isolated from bats is about 88%.

SARS-CoV-2 has the typical characteristics of coronavirus, the virus envelope has typical spines, corona-like shape. Spike protein is the most important surface membrane protein of coronavirus, which determines the host range and specificity of the virus. It is an important site for host neutralizing antibodies and a key target for vaccine design.

A growing number of neutralizing antibodies are being developed for therapeutic and diagnostic applications; however, these neutralizing antibodies are difficult to scale production. The application of transgenic technology to the commercial production 6£/503556 neutralizing antibodies in the milk of transgenic animals offers significant advantages over traditional methods of neutralizing antibodies. That is the use of animal mammary gland bioreactor for the production of neutralizing antibodies, it is currently the only internationally proven commercial bioreactor. These advantages include the ability to obtain high-yield proteins with natural conformations, low cost, and broader application prospects.

The neutralizing antibody against SARS-CoV-2 is a receptor blinding domain RBD specific neutralizing antibody. Studies have shown that the SARS-CoV-2 binds to the angiotensin converting enzyme 2 (ACE2) of the host cell receptor through the receptor binding domain (RBD) on the S1 subunit, while the neutralizing antibody blocks the invasion of SARS-CoV-2 into the host cell by blocking the binding of RBD to the host cell receptor ACE2. The main epitope identified is located in the receptor binding motif (RBM) in the RBD. Through I-ELISA test, it was found that the neutralizing antibody against

SARS-CoV-2 produced by transgenic goat and the serum of patients infecting

SARS-CoV-2 could bind to the recombinant SARS-CoV-2 spike protein receptor binding region, wherein the neutralizing antibody against SARS-CoV-2 had stronger binding ability.

A need exists to develop a method to produce SARS-CoV-2-nAB without the inherent problems of the present process. Animal bioreactor is a technology that uses transgenic living animals to efficiently express a foreign protein in their organs or tissues, and to industrially produce functional proteins. Among them, mammary gland bioreactor is the only bioreactor that has been proved to be commercialized internationally. As a new production model, compared with mammalian cell expression systems that require specific culture environment and medium, its biggest advantage is that the protein can be expressed in high yield, and has a natural conformation protein, and the cost is low.

Although there are still some problems in the large-scale production of neutralizing antibodies using mammary gland reactors, it is still an ideal tool for the production of pharmaceutical proteins with great economic value and broad application prospects.

SUMMARY LU503556

The invention relates to neutralizing antibody against SARS-CoV-2 produced by transgenic technology. The SARA-CoV-2-nAB gene is integrated into the genome of

Saanen dairy goat by gene editing and somatic cell cloning technology, which utilizes mammary gland specific regulatory element of the milk protein gene PAEP to drive and is highly expressed in the mammary gland of dairy goats. Therefore, neutralizing antibodies against SARS-CoV-2 can be continuously obtained in milk, which lays foundation for large-scale production of neutralizing antibodies against SARS-CoV-2 and has important application and research value.

In one aspect, the present invention relates to a neutralizing antibody, particularly human neutralizing antibody that can specifically bind to the RBD of SARA-CoV-2 S protein. The antibody of the invention exhibits one or more desirable functional properties, such as binding to the RBD of SARS-CoV-2 S protein, thereby preventing the virus from invading human cells, achieving therapeutic effects and short-term preventive effects. In addition, the antibody of the invention contains specific structural features.

In another aspect, the invention relates to a mammary gland bioreactor for efficiently preparing neutralizing antibodies against SARA-CoV-2 by constructing a transgenic

Saanen dairy goat.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term “monoclonal antibody” refers to an immunoglobulin obtained from a pure line of cells having the same structural and chemical properties and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually with different antibodies against different determinants). Each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained by hybridoma or recombinant engineered cell culture and are not mixed with other immunoglobulins. The modifier 'monoclonal ‘represents the characteristics of antibodies and is obtained from a homogeneous antibody populatiohU503556 which should not be interpreted as requiring any special method to produce antibodies.

The term “antibody” and “immunoglobulin” are about 150,000 Daltons of isotetraglycan proteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isoforms is different. Each heavy chain and light chain also have regularly spaced intrachain disulfide bonds. There is a variable region (VH) at one end of each heavy chain, after that are several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is relative to the first constant region of the heavy chain, and the variable region of the light chain is relative to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of light and heavy chains.

The term “variable” used in this paper refers to the fact that some parts of the variable region of an antibody differ in sequence, forming the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the antibody variable region, which is concentrated in the light chain and heavy chain variable region as complementary determining region (CDR) or hypervariable region of the three fragments.

The more conserved parts of the variable regions are called framework regions (FR).

The variable regions of the antibody heavy chain and light chain each contain four FR regions, which are roughly in a B-sheet configuration and are linked by three CDRs that form a linker ring, in some cases forming a partially B-sheet structure. The CDRs in each chain are tightly linked through the FR region and form the antigen binding site of the antibody with the CDRs in the other chain (see Ka ba t et al, NIH Pu bl. No.91-3242, volume 1,647-669 pages (1991)). Antibody constant regions are not directly involved in antibody-antigen binding, but they exhibit different effector functions, Such as antibody-dependent cellular cytotoxicity (ADCC) or complemnt-dependent cytotoxicity (CDC).

The term “expression regulatory sequence” usually refers to the sequence involved/503556 in controlling gene expression. The expression regulatory sequence includes a promoter and a termination signal that are operatively connected to the target gene. The gene (DNA) sequence encoding the antibody of the invention can be obtained by conventional means known to the technical personnel in the field, such as artificial synthesis according to the protein sequence disclosed in the invention or amplification by PCR.

Subsequently, the DNA fragments obtained by synthesis or PCR amplification can be inserted into appropriate expression vectors by various methods known in this field. The expression vector used in the invention can be a commercially available expression vector known by technicians in this field, such as pCDNAS3.1 expression vector of

Invitrogen.

Suitable host cells for accepting expression vector transformation generally include prokaryotic and eukaryotic cells. Common examples of prokaryotic host cells include

Escherichia coli, Bacillus subtilis, etc. Common eukaryotic host cells include yeast cells, insect cells, mammalian cells, etc.

After the host cells transformed by expression vectors were cultured under suitable conditions (such as adherent or suspension culture in a cell culture flask or bioreactor with serum-free medium), the culture supernatant was harvested. The antibody of the invention is then purified by conventional separation steps or methods known to the technicians in this field, including protein-A affinity chromatography, ion exchange chromatography, filtration sterilization, etc.

Purified antibodies of the invention can be dissolved in an appropriate solvent such as sterile saline solution, the solubility can be prepared between 0.01 and 100 mg / ml, the ideal final solubility can be prepared between 1 and 20 mg / ml.

BRIEF DESCRIPTION OF THE FIGURES LU503556

FIG. 1 is the gene targeting donor vector map of the present invention.

FIG. 2 is CRISPR / Cas9 unitary vector map of the present invention.

FIG. 3 is a schematic diagram of monoclonal cell screening strategy of the present invention.

FIG. 4 is the junction PCR results of monoclonal cells of the present invention.

FIG. 5 is the Sanger sequencing results of T-A cloning of the present invention.

FIG. 6 is the typical Southern blot results of transgenic monoclonal cells of the present invention.

FIG. 7 is the karyotype detection results of positive clones for SCNT of the present invention.

FIG. 8 is the absolute quantitative PCR identification of exogenously inserted NeoR gene of the present invention.

FIG. 9A shows primary ear-derived fibroblasts, positive clone cells for SCNT and their transgenic cloned embryos; FIG. 9B shows photos of a one-month-old

SARS-CoV-2-nAB transgenic goat.

FIG. 10 shows validation of precise insertion of SARS-CoV-2-nAB gene in transgenic goat by junction PCR.

FIG. 11 shows validation of transgenic SARS-CoV-2-nAB bioreactor for precise insertion in Saanen dairy goat by Southern Blot.

FIG. 12 shows detection of relative expression level of PAEP gene neighboring genes.

FIG. 13A shows protein in whey, M: protein Marker; 1 and 2: non-transgenic goat milk supernatant; 3: Transgenic goat milk; 13B shows protein A purified neutralizing antibody against SARS-CoV-2, M: protein Marker; 1: non-transgenic goat milk supernatant; 2: Transgenic goat milk.

FIG. 14 is the SEC-HPLC analysis results of the present invention.

FIG. 15 shows detection of binding specificity of antibody to S-RBD by indirect

ELISA.

DESCRIPTION OF THE INVENTION

The invention is based upon discovery that neutralizing antibody against

SARS-CoV-2 by the transgenic method of the present invention is different from the neutralizing antibody isolated from the peripheral blood of convalescent patients with

COVID-19 (pbNAb). Compared with it, the neutralizing antibody against SARS-CoV-2 produced by the transgenic method has a higher broad spectrum. In particular, the

SARS-CoV-2-nAB gene can be efficiently expressed in the mammary gland of the transgenic Saanen dairy goat, and the binding ability to S-RBD is significantly stronger than that of pb-Nab.

Exemplification

Acquisition of Human Coronal Neutralizing Antibody Gene

The neutralizing antibody against SARA-CoV-2 used in this project was obtained by collecting peripheral blood of COVID-19 patients during recovery period and isolating the mononuclear cells (PBMC) from peripheral blood, then the RNA of PBMC cells was extracted using QIAGEN 's RNeasy Mini Kit, and the RNA was reverse transcribed into cDNA using Roche 's first strand synthesis kit. VK, VL and VH were amplified by PCR.

After digestion with Xba | enzyme and Sac | enzyme, VK and VL were ligated into pComb3H vector which was also digested with Xba | enzyme and Sac | enzyme. The ligation products were recovered and electroporated into XL1-Blue competent cells. The electroporation bacterial liquid was coated on a 15 cm large plate, and the next day the bacteria were scraped. The plasmid was extracted as a light chain library, and the recombinant plasmids were pComb3H-VK and pComb3H-VL. The light chain library pComb3-L and heavy chain Fd fragments were digested respectively by Xho | enzyme and Spe | enzyme, and ligated with pComb3H-VK and pComb3H-VL, which were also digested respectively by Xho | enzyme and Spe | enzyme. Then electroporated to get the antibody library. A total of 159 phage antibody fragments were obtained by phage library screening, which could bind to the extracellular domain of recombinant SARS-CoV-2 spike protein. The antibody fragments were human-derived Fab fragments, including full-length light chain and heavy chain Fd fragments. A total of 159 single colonies weté/503556 amplified and sequenced to obtain qualified sequences with complete light and heavy chains. Then the binding specificity of the antibody to S-RBD and S-ECD was detected by indirect ELISAJ and immunoprecipitation, respectively. Finally, A human antibody was selected for the preparation of transgenic cloned sheep, because it can bind to S-RBD and has higher activity, which is named Nab08.

Generation of the Gene Construct

A mammary gland-specific transgene was constructed by inserting the human

SARS-CoV-2-nAB cDNA into the PAEP gene on chromosome 11 of the goat genome, which is located in the downstream region of the first exon gene coding start site ATG.

The total length of the foreign gene sequence is about 3787bp, and its structure includes:

The 1-2127 bp is the open reading frame sequence of SARS-CoV-2-nAB gene, 2128-2347 bp is the 3'UTR sequence, 2348-2556 bp is the SV40 polyA sequence, 2557-2590 bp is the first LoxP element sequence, 2591-2660 bp is the HSV TK polyA sequence, 2661-3464 bp is the drug screening marker gene NeoR expression sequence, 3465-3753 bp is the PTK promoter sequence. 3754 - 3787 bp is the second LoxP element sequence. The obtained transgenic dairy goat genomes all added a heterozygote of the neutralizing antibody against SARS-CoV-2 initiated by tis own promoter, which could produce goat milk rich in the neutralizing antibody.

Assembly and Characterization of the Gene Construct

In this project, the commercial cloning vector pMD19-T Simple Vector (CN:D104A) of TaKaRa company was used as the skeleton. The vector was a fungal skeleton, a non-viral vector, non-pathogenic and no possibility of evolution into pathogenic. The T-A cloning method was used to connect the homologous arm sequence, the codon-optimized SARA-CoV-2-nAB gene and the subsequent clone with multiple cloning sites to the skeleton vector, and then the remaining elements of the PCR amplification were digested and ligated in turn. The constructed recombinant was subjected to Sanger sequencing, and the positive recombinant with correct sequencing was the gene targeting donor vector (Fig. 1). The main function of this vector in eukaryotic cells is to act as a donor vector for homologous recombination during the DNA double-strand bredkJ503556 repair process, providing homologous arm sequences to mediate exogenous gene integration. Another vector used in this project was pSpCas9 (BB) -2A-GFP (PX458,

Addgene plasmid # 48138). The eukaryotic expression vector contains both the complete elements required for hspCas9 protein expression and the hU6 promoter required for stable transcription of sgRNA (Figure 2). The main function is to specifically mediate DNA breakage at the targeting site and activate homologous recombination repair.

The promoter used in the vector of this project is the endogenous promoter of goat

PAEP gene, which guides the specific expression of the novel coronary neutralizing antibody gene downstream of the first exon gene encoding start site ATG of PAEP gene in mammary gland. The terminator is SV40 PolyA sequence with a total length of 209 bp, which is PolyA of monkey vacuolating virus. It has the function of transcription termination and adding PolyA tail to the transcribed mRNA. In addition, there is a marker gene (Neo), the size of 795 bp, for transgenic fibroblasts monoclonal screening. Among them, the nucleotide sequence and amino acid sequence of the neutralizing antibody against SARA-CoV-2 heavy chain-P2A-neutralizing antibody against SARS-CoV-2 light chain after gene editing is as follows.

Testing and Characterization of Gene Constructs

Transgene constructs are tested in a goat model system to assess their ability to direct high levels of expression and their ability to express in a tissue-specific manner.

Generation and Characterization of Transgenic Animals

A founder transgenic SARS-CoV-2-nAB goat is defined as a viable transgenic animal resulting from somatic cell nuclear transfer of 2-cell goat embryos that have been transfected two specified vectors by Electroporation. Twelve founder SARS-CoV-2-nAB goats were produced. The general methodologies that follow in this section were used to generate all transgenic goats.

Goat Species and Breeds

The transgenic saanen goats produced for SARA-CoV-2-nAB production are of

Shanxi province, Northwest Agriculture and Forestry University. Saanen dairy goat originated in Switzerland, is one of the world's best dairy goat breeds, is tH&J503556 representative type of dairy goat. In 1932, it was officially introduced in large quantities from Canada.After 40 years of domestication and breeding, and the introduction of high-quality breeding ram blood, Xinong Saanen dairy goat, a variety with excellent lactation performance, was finally bred.

Goat ear fibroblasts culture

The ear marginal fibroblasts used in this project were obtained by tissue block adherent method. In order to avoid bacterial contamination, the collected marginal tissue of dairy goat ear was washed more than three times with PBS buffer containing penicillin.

The stubble on the surface of the tissue block needs to be peeled off as much as possible on the sterile worktable, and the cartilage tissue in the middle of the tissue block is peeled off with ophthalmic scissors and tweezers. The remaining skin tissue is cut into 1mm3 tissue blocks, evenly attached to a petri dish with a diameter of 60mm and added with a small amount of DF12 culture fluids. The culture plate was inverted in a CO2 incubator and incubated at 37°C and 5 % CO2 for 4 h. Then 5 mL DF12 medium containing penicillin-streptomycin and 10 % FBS was added. About 3-4 days later, the fibroblasts precipitated around tissue blocks were removed for further culture until the cell confluence is greater than 90 %, and then discard the tissue, choose frozen cells or subculture.

Subculture and cryopreservation

The cell culture medium was discarded, and 3 mL PBS was added for gently washing. Then 1 mL PBS containing 0.25 % trypsin was added to digest the cells. After incubation at 37°C for 3 min, DF12 medium was added to terminate the digestion. The adherent cells were peeled off gently to form cell suspension, and the supernatant was discarded after centrifugation. DF12 culture medium was added to resuspend the cells, and the cells were subcultured at a ratio of 1: 3 to 1: 5; or to add the cell cryopreservation solution containing 10 % DMSO, completely resuspended the cells and transferred into the cryopreservation tube or the program cryopreservation box, and finally transferred to liquid nitrogen for cryopreservation. selecting transgenic clone cells

Targeting vector and donor vector were delivered into GEFs cells by electroporatiohU503556

GEFs were seeded in three 60mm culture dishes at a density of 5 x 10° cells dish 24h before transfection, washed twice with PBS and digested with trypsin, centrifuged before transfection. The prepared electrolyte (120mM KCI, 0.15mM CaCl, 10mM K2HP O4, 5SmM

MgClz; pH 7.6) and Opti-MEM culture medium were mixed at a volume ratio of 3: 1, and 20ug of target vector plasmid and 20ug of donor vector plasmid was added to construct a final volume of 400uL transfection system. The above transfection system was transferred to the cell mass centrifuged to discard the culture medium, and the resuspended cells were gently blown and mixed. After standing at room temperature for minutes, the cell suspension was transferred to a new BTX electric cup for standing for 5 minutes. The voltage of the electric converter was adjusted to 510 V, and the pulse was shocked twice with 1 ms. The cell suspension was gently inoculated into three 60 mm petri dishes and placed back in the incubator for further culture. After 48 hours, a dish of cells was taken to extract the genome for initial identification. After successful transfection, the remaining two dishes of cells were frozen for later use. One dish was subcultured to 20 dishes for culture (100 mm), and the culture medium was replaced with a culture medium containing a final concentration of 800 ng/mL G418 for drug screening.

The medium was changed every 2-3 days and the cell growth status was observed. After 8 days of screening, the cells in the genome that did not integrate the donor vector should all die, while the cells that stably integrated the donor vector gradually formed a monoclonal cell mass. When the vortex GEFs monoclonal cell mass grew to mung bean size, single colonies were picked and numbered under the inverted stereomicroscope 25 times field of view, inoculated in 48-well plates and part of the cells (about 10%) to the

PCR tube for subsequent junction PCR identification. After the cells in the 48-well plate were covered with the bottom of the dish, the cells were digested and passaged in the 24-well plate, and some cells (about 10%) were re-taken for junction PCR identification.

Site - specific integration of inserted genes identified by Junction PCR

The above monoclonal cells used for identification were centrifuged, the supernatant was discarded, and 20 uL of lysate (10 mM Tris-HCI, pH 8.5; 50 mM KCI; 1.5 mM MaClz; 0.5% NP-40; 0.5% Tween-20; 400 g/mL proteinase K) was added, fully resuspended in 65°C water bath for 30 minutes, 95. °C water bath for 15 minutes t&503556 complete cell lysis. A total of 5 uL cell lysate was used as a template to construct a 20 uL reaction system according to the EmeraldAmp (TaKaRa) instruction, and a standard amplification procedure was set up. The following junction PCR primers were used to identify positive clones. 5'-junction 5'j F (5'-CCCCTGTGATGAGTGGTTTAGTC-3) ; 5) R (5'-GGTGTTGGGTCGTTTGTTCG-3)) ; 3'-junction 3'j F (5'-GTTGTGCCCAGTCATAGCCG-3) ; 3'j R (5 -CGTTTCCAGGACATCTTGTTCAT-3') ,

Among them, the 5'-junction upstream primer matching sequence is the upstream genome sequence of the 5'-end homologous arm, and the downstream primer matching sequence is the SV40 polyA sequence on the targeting vector; the 3'-junction upstream primer matching sequence is the NeoR gene sequence on the targeting vector, and the downstream primer matching sequence is the downstream genome sequence of the 3'-end homologous arm (see FIG. 3.).

The primers 5'j F and 3'j R were located outside the homologous arm sequence, and 5'j R and 3'j F were combined on the target vector. The red lines represent the Southern blot probe binding region and the restriction site EcoRI is used to cleave the genome to form long fragment polymorphism (RFLP). The upstream primer matching sequence of the 5'-junction was the upstream genomic sequence of the 5'-end homologous arm, and the downstream primer matching sequence was the SV40 polyA sequence on the target vector. The product size was 3664 bp; the upstream primer matching sequence of 3'-junction is the NeoR gene sequence on the targeting vector, and the downstream primer matching sequence is the downstream genome sequence of the 3'-end homologous arm. The product size should be 1667bp. Positive clones identified by 3'-junction PCR were further identified by 5'-junction PCR. The PCR results of 3'junction (upper) and S'junction (lower) in this study (see FIG. 4.), where the positive results are marked with red numbers.

After the above junction PCR products were recovered, T-A cloning was constructédJ503556 and expanded for sequencing. Sanger sequencing was used to determine the precise insertion of foreign genes (see FIG. 5.).

Since the junction PCR results can only prove that the selected positive clones have a precise insertion of the foreign gene on the PAEP gene, it is not possible to explain whether the foreign gene has been integrated elsewhere in the bovine genome.

Therefore, Southern blot was performed on the positive clones that passed two rounds of junction PCR after expanded culture.

Southern Blot detection of copy number of inserted genes

Using the positive gene inserted monoclonal cell genome as a template, the probe 1 sequence targeting the SARS-CoV-2-nAB gene on the target vector and the probe 2 sequence targeting the downstream genome of the 3'end homologous arm were amplified respectively. The primers are as follows:

Probe 1 p1F (5'- GAATCTAACGGCCAGCCTGA -3)) ; p1R (5'- TCACGCTGCAAGAAAACACG -3) ;

Probe 2 p2F (5'- AGGCCCTGGAGAAATTCGAC -3) ; p2R (5'- GGAGGAGGAGGTCCTGGG -3') ©

The above PCR products were recovered by agarose gel electrophoresis and labeled with digoxin probe using DIG-High Prime DNA Labeling and Detection Starter Kit

II (Roche) kit. Take 1 ug gel recovery product with ddH2O constant volume of 16 pL, boiling water bath 10 minutes denaturation treatment immediately ice bath 10 minutes renaturation. Add 4 pL DIG-High Prime, mix well, incubate at 37°C for 14 hours for labeling, and stop the reaction in 65°C water bath for 10 minutes. Digoxin-labeled probes should be stored at -20°C and slowly thawed on ice before each use.

After expanding the positive clone cells identified by junction PCR, sufficient cells were taken to extract genomic DNA and digested overnight with restriction enzyme

EcoRI. Agarose gel with a thickness of 6 mm and a concentration of 1% (wt/vol) was prepared, and 15 ug of digested product was loaded into each lane. Electrophoresis was performed at 25 V (less than 1 V/cm) in 1 x TAE buffer at room temperature (ice bag temperature control to prevent overheating) for 14 hours, and the bands were separated.

The gel was then placed in a 10-fold gel volume of denaturation buffer (1.5 mol/L NaGlU503556 0.5 mol/L Tris, pH 7.4), low-speed shaking on a horizontal shaker for 15 minutes, repeated three times for DNA denaturation. After a brief immersion in deionized water, the gel was placed in a 10-fold gel volume of neutralizing buffer (1.5 mol/L NaCl; 0.5 mol/L NaOH), low-speed oscillation on a horizontal shaker for 30 minutes, and re-oscillation for 15 minutes to complete neutralization.

Transmembrane operation in strict accordance with the second edition of ' molecular cloning experiment guide ' to 20xSSC (3 mol/L NaCl; 0.3 mol/L sodium citrate) as the transfer buffer, using a positively charged nylon membrane (GE Healthcare) and downward capillary method. After the transfer operation, the nylon membrane was washed with 6xSSC for 15 minutes on a horizontal shaker, and then baked at 120°C for minutes between two 3mm thick filter papers for fixation. Carefully place the nylon membrane in the hybridization tube for subsequent hybridization and development operations. Southern hybridization and coloration were performed according to the instructions of DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche). The probe hybridization temperature was 42°C. and the hybridization time was 12 hours.

X-ray film was used to detect the nylon film after color treatment in darkroom.

Positive clones with precise gene insertion only at the PAEP target gene should hybridize a 6360 bp band (probe 1) and a 1368 bp band (probe 2). The PAEP gene positive clones without gene insertion should retain a wild-type normal chromosome when using probe 2, which contains only one and 3941 bp specific bands. Clones with foreign gene insertion in other parts of the genome should contain non-specific bands except 6360 bp when using probe 1. The Southern blot results of donor cells for subsequent nuclear transfer experiments (see FIG. 6.).

Karyotype analysis of positive clones

Before nuclear transfer, a small number of positive clone cells were taken for karyotype analysis. When most of the transgenic cells were in the logarithmic growth phase, colchicine with a final concentration of 0.4 ng/mL was added to the culture medium and continued to culture for 5 hours. The cells were digested with trypsin and enriched in a 15 mL centrifuge tube. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, 10 mL of cell hypotonic solution (0.075 M KCI) was adddd)503556 and the cells were gently and completely resuspended. After 30 min of water bath at 37°C, 1.5 mL of pre-cooled methanol and 0.5 mL of pre-cooled acetic acid were mixed for cell pre-fixation. After centrifugation at 1000 rpm for 10 minutes, the supernatant was discarded, and 10 mL of the above fixative solution was added again and the cells were gently resuspended, incubated at room temperature for 20 minutes for fixation. Repeat the above centrifugation and fixation steps 2-3 times, centrifugation to discard the supernatant, and add 100 uL pre-cooled stationary liquid gently blow hit re-suspended cells. The frozen slides were placed obliquely at 30°C and dropped at a certain height.

After natural air drying, the slides were immersed in 10% Giemsa dye solution for 10 minutes for staining, washed off the floating color and observed by oil immersion lens (see FIG. 7.).

Prior to subsequent nuclear transfer operations, this study performed absolute quantification of the NeoR gene in positive clones that were eventually used in transgenic animal production (RT-NeoR-F: ATATCACGGGTAGCCAACGC; RT-NoeR-R:

TGCCTGCTTGCCGAATATCA), The copy number of exogenous DNA inserted in the above clones was determined again. The corresponding standard curve and the copy number estimation results in each cell are shown (see FIG. 8.). This confirmed once again that all the positive clones for subsequent transgenic animal production were heterozygous clones inserted with a single copy of the SARS-CoV-2-nAB, retaining a wild-type chromosome without random integration.

Through the process of screening and identification of positive clones by the above various detection methods, it is considered that hybrid positive clones with typical dense spindle-forming fibroblast morphology, normal chromosome karyotype and faster cell proliferation rate are considered to be suitable for subsequent somatic cell nuclear transfer experiments.

Somatic cell nuclear transfer

The ovaries used in this study were collected from the slaughterhouse in Xi 'an, and stored in sterile saline containing penicillin (400 IU/mL penicillin + 400 g/mL streptomycin) at 37°C and transported to the laboratory. The ovaries were washed 3-5 times with normal saline containing penicillium streptomycin, and the mesangium and connectiv&/503556 tissue around the ovaries were cut off with sterilized scissors and tweezers, as clean as possible, and put into a new PBS. After that, the ovaries were quickly washed and disinfected with 75% alcohol, and then the ovaries were washed with normal saline to remove alcohol, so that the surface of the ovaries was washed clean, and placed in a 60 mm dish with 5 mL preheated PBS, each dish placed 5-7 ovaries. Follicles on the surface of the ovary were then pierced with a sterilized puncture needle, slightly squeezed to drain the follicular fluid containing the cumulus oocyte complexes (COCs), and the COCs with good oocyte uniformity were sorted under a stereomicroscope for maturation culture. The mature medium was TCM199 (Gibico) supplemented with 10%

FBS and 10 ng/mL epidermal growth factor. After cultured at 38.5°C and 5% CO2 for 21-24 hours, cumulus cells were removed with 0.2% hyaluronidase, and mature oocytes with good polar body morphology were selected for subsequent experiments.

When somatic cell nuclear transfer (SCNT) was performed, PBS containing 10%

FBS and 5 g/mL cytochalasin B (CB) was used as the micromanipulation solution. After incubating the oocytes for 15 minutes, the oocytes were fixed on the micromanipulator, and the first polar body and adjacent cytoplasm were extracted on the micromanipulator with the enucleation needle to complete the enucleation operation.

Hybrid positive clones with typical dense spindle fibroblast morphology and faster cell proliferation rate were strictly selected as nuclear donor cells (see FIG. 9A.). The clone number and source are shown in Table. The donor cells grown to contact inhibition state for 3 days were injected into the above spare oocyte zona pellucida with a denucleated needle. The nucleoplasm complex was incubated with electrofusion solution for 5 minutes, and the recombinant was arranged with the microelectrode tip connected to the micromanipulator to make the membrane contact surface perpendicular to the connection of the two electrodes. The electrofusion was performed twice with 20 um electric pulses at 30 V and 10 us interval. The fused recombinants were placed in preheated M199 buffer containing 10% FBS and cultured at 38.5°C, 5% CO2 and saturated humidity for 2 hours. The fully fused reconstructed embryos were selected and activated using mSOFaa medium containing 5 uM ionomycin or 2 mM dimethylaminopurine (6-DMAP). The embryos were cultured in mSOFaa medium &t503556 38.5°C, 5% CO2 and saturated humidity for 2 days, and the development rate of 2-cell embryos was examined.

The well-developed 2-cell embryos were transplanted into the oviduct ampulla of the estrus Saanen recipient dairy goat, and 10-15 2-cell embryos were transplanted into each recipient cow. After 35 days, the pregnancy test was performed on the recipient sheep with unreturned after transplantation by B-ultrasound. Thereafter a monthly check to observe the maintenance of pregnancy, until the successful preparation of transgenic cloned sheep. 1092 in vitro developed embryos were constructed by somatic cell nuclear transfer.

After 2 days of in vitro culture, 724 (66.3%) cloned embryos successfully developed into two-cell embryos and developed well. They were transplanted to a total of 64 recipient

Saanen dairy goats with estrus synchronization under natural conditions, and the pregnancy and estrus return of the recipient goats were counted. Finally, 12 transgenic sheep were born and grew normally (see FIG. 9B.).

Statistical table of in vitro development of nuclear transfer cloned embryos with positive clones

Nuclear

Typical colonies donor

Cellclone F4-50 F4-129 F4-184 F4-273 F4-283 F4-326 Total

Embryos 162 155 179 177 214 203 1092 obtained 2 cell 104 104 123 112 148 133 724 embryos (63.9) (66.9) (68.9) (67.7) (69.1) (65.4) (66.3) rate (%)

Recipients 9 10 11 10 12 12 64

Pregnancies 4 4 5 3 4 5 25

Calves at 3 2 4 2 2 3 16 birth

Calves 2 2 2 2 1 3 12 survived

Date of birth 2021.11.15 2021.11.18 2022.3.4 2022.38 2022.4.12 2022.4.16

Molecular Detection and Identification of Goats Expressing SARS-CoV-2-nAB

Neutralizing Antibody

Identification of SARS-CoV-2-nAB gene site-specific integration by Junction PCR

Peripheral blood samples of jugular vein were collected from 12 transgenic Saanen cloned sheep, which were fully mixed with an appropriate of sodium citrate anticoagulant, then whole blood genome extraction is performed strictly following the standard procedures of the blood genome extraction kit. Junction PCR was performed using the above PCR primers, DNA polymerase and reaction procedures. The peripheral blood genome of normal dairy goats without transgenic operation was used as a control to verify whether the transgenic dairy goats produced were accurate insertion of the

SARS-CoV-2-nAB gene. The expected specific target bands were observed in transgenic goats, but no corresponding bands were observed in the control group. This proved that these transgenic goat genomes included the site-specific integration bH503556

SARS-CoV-2-nAB gene (see FIG. 10.).

Identification of integrated copy number of SARS-CoV-2-nAB gene by Southern Blot

According to the standard procedure of the kit, the genome of peripheral blood cells of goats with SARS-CoV-2-nAB gene was extracted. After overnight digestion with restriction enzyme EcoRI, the probe (Probe 1) labeled with digoxin targeting the upstream genomic sequence of the 5' end homologous arm and the probe (Probe 2) targeting the NeoR gene sequence on the targeting vector were used for Southern blot detection to verify whether the transgenic dairy goats were SARS-CoV-2-nAB gene precise insertion and single allele gene targeting. When using probe Probe 1, the positive transgenic GEFs (P) and transgenic cloned goats (Swimming Channel 1-12) produced a 3941 bp wild-type band and a 6360 bp long transgenic recombinant band, while ordinary dairy goats (N) produced only a 3941 bp wild-type band. When using

Probe 2, both positive transgenic GEFs (P) and transgenic cloned goats (lane 1-12) produced only one transgenic recombinant band of 1368 bp, while ordinary dairy goats (N) did not produce any band (see FIG. 11.).

These results indicated that all the goats with SARS-CoV-2-nAB gene obtained in this study were site-specific insertions, only this site was inserted in the dairy goat genome, and the insertion was a single allele heterozygous knock-in, retaining a wild-type chromosome.

Real time RT-gPCR to detect the effect of gene insertion on adjacent gene transcription

Total RNA was extracted from peripheral blood of transgenic dairy goats and non-transgenic dairy goats using the Trizol method described in the second edition of the

Molecular Cloning Laboratory Guide, and cDNA was obtained by reverse transcription using the PrimeScript RT reagent. Reaction system: 1 uL PrimeScript RT Enzyme Mix I; 1 uL Oligo dT Primer (50uM); 1 uL Random 6 mers (100 uM); 1 ug Total RNA; 4 uL 5 x

PrimeScript Buffer, add RNA-Free ddH2O to a total volume of 20 uL. Reverse transcription reaction conditions: Step 1: 37°C incubation for 15 minutes; step 2:

Inactivation at 85°C for 5 seconds. Reverse transcription products were frozen at -80°C.

The adjacent genes on both sides of the PAEP gene targeting site are GL76D4503556 (glycosyltransferase 6 domain containing 1), LCN9 (lipocalin 9) and SOHLH1 (F spermatogenesis and oogenesis specific basic helix-loop-helix 1). Quantitative primers were designed as follows’

GLT6D1 Forward (5'- CTGGCTTGCTCCCATTGTCT -3') ;

Reverse (5'- CAAGCCTGCCAGAAACGAAG -3) ;

LCN9 Forward (5'- ATCATGTCCACACCACGACC -3') ;

Reverse (5'- AGGCTCGACCACAGTTTGTT -3") ;

SOHLH1 Forward (5"- TGGTGAGTGAGGAGACTCGG -3) ;

Reverse (5'- ATCTGCCACATCACATCCCG -3)) ;

GAPDH Forward (5- CTGCCCGTTCGACAGATAGC-3) ;

Reverse (5'- AACGATGTCCACTTTGCCAGT-3") .

The reaction system was constructed in strict accordance with the SYBR Premix Ex

Taq Il real-time PCR kit instructions: 0.4 uL Forward Primer (10uM); 0.4 uL Reverse

Primer (10uM); 1 uL Template (cDNA); 5 uL 2 x SYBR premix Ex Tag Il; 0.2 uL 50 x ROX

Reference Dye; add ddHzO to a total volume of 10 uL. Subsequently, the standard two-step PCR procedure was set up and the real-time PCR reaction was performed on the ABI StepOne Plus real-time PCR system. After the reaction, confirm the amplification curve and dissolution curve. The housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as an internal reference, and the expression levels of the above genes were evaluated using the 2 7 A4Ct algorithm, and grouped data statistics and comparison were performed.

According to the source of the samples, they were divided into normal dairy goat control group (n = 5) and transgenic dairy goat group (n = 12), and the data were summarized and analyzed. There was no significant difference in the expression of adjacent genes at the insertion site between transgenic cattle and ordinary cattle, suggesting that the insertion of SARS-CoV-2-nAB gene had no significant effect on the expression of adjacent genes (see FIG. 12.).

In summary, the peripheral blood of transgenic sheep was collected to extract thé/503556 genome, and junction PCR, Sanger sequencing and Southern blot were performed again. It was determined that the transgenic sheep obtained by SCNT technology were all accurate single copy insertion of PEAP locus of SARS-CoV-2-nAB gene, which was consistent with the expectation.

Generation and Selection of Production Herd

The procedures described above were utilized for production of the transgenic founder goats, as well as other transgenic goats in our herd. The transgenic

SARS-CoV-2-nAB founder goats, for example, were bred to produce milk, if female, or to produce a transgenic female offspring if it was a male founder.

This transgenic founder male, was bred to non-transgenic females, and produced transgenic female offspring.

Detection of SARS-CoV-2-nAB Expression in Transgenic Goat Milk

The fresh transgenic goat milk was collected from the experimental sheep farm and after centrifuged at 2000 rpm, The impurities and fat were discarded, and the casein was removed by acid precipitation method. The obtained transgenic goat milk sample was subjected to SDS-PAGE gel electrophoresis, and the protein gel was stained and decolored. The transgenic goat milk showed a new corona neutralizing antibody H chain with a protein size of 50 kDa and a new corona neutralizing antibody L chain with a protein size of 23 kDa, while the non-transgenic goat milk did not detect the corresponding size of the protein band (see FIG. 13A.). Protein A affinity chromatography was used to purify the neutralizing antibody in the transgenic goat milk supernatant. Protein A was coupled to the agarose matrix as an affinity ligand and could specifically bind to the fully human neutralizing antibody against SARS-CoV-2-nAB molecule in the transgenic goat milk supernatant sample without binding to the goat immunoglobulin molecule (see FIG. 13B.).

Protein A affinity chromatography has extremely high selectivity, and one-step affinity chromatography can achieve more than 95% purity. The purity of the purified neutralizing antibody against SARS-CoV-2 was analyzed by high performance liquld/503556 chromatography (see FIG. 14.).

Functional detection of neutralizing antibody against SARS-CoV-2

The Recombinant SARS-CoV-2 spike protein receptor binding region (S-RBD, purchased from Nanjing Baraode Biotechnology Co., Ltd., No. NCPOO29P) was coated on the ELISA plate with PBS at a concentration of 1ug/ml, and the concentration of the purified SARS-CoV-2-nAB was diluted to 1mg/ml, and then starting from 1 : 2500 multiple dilution 8 dilutions. The serum of patients with novel coronavirus pneumonia (Jiangsu Center for Disease Control and Prevention) was used as a positive control and healthy adult serum was used as a negative control, then starting from 1 : 100 multiple dilution 8 dilutions. After dilution, the specimens were incubated at 37°C for 30 minutes, then washed three times with PBST, added HRP-labeled anti-human Fc (1:5000), incubated at 37°C for 30 minutes, washed three times with PBST, colored with TMB, and read OD450 absorbance value after termination. Three batches were repeated under the same conditions, and the average absorbance value of each well was analyzed by

GraphPad software.

Both the SARS-CoV-2-nAB produced by transgenic sheep and the serum of patients with SARA-CoV-2 can bind to recombinant S-RBD, and the binding ability of the

SARS-CoV-2-nAB produced by transgenic sheep to recombinant S-RBD is significantly stronger than that of the serum of patients with novel coronavirus pneumonia (see FIG. 15.).

Equivalents

Although the above only describes the specific implementation examples of the invention, the technical personnel in this field should understand that these are only examples, and the scope of protection of the invention is limited by the attached claims.

Technicians in this field may make a variety of changes or modifications to these embodiments without departing from the principle and substance of the invention, but these changes or modifications fall within the protection of the invention.

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Atamp;F University</ApplicantNameLatin> <InventorName languageCode="en”>Xu Liu</InventorName> <InventionTitle languageCode="en”>MONOCLONAL ANTIBODY OR DERIVATIVE

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<SequenceData sequencelDNumber="4"> LU503556 <INSDSeq> <INSDSeq length>125</INSDSeq length> <INSDSeq moltype>AA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> <INSDFeature> <INSDFeature key>source</INSDFeature key> <INSDFeature location>l..125</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name>mol type</INSDQualifier name> <INSDQualifier value>protein</INSDQualifier value> </INSDQualifier> <INSDQualifier id="q8"> <INSDQualifier name>organism</INSDQualifier name> <INSDQualifier value>synthetic construet</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeq_sequence>EVQLLQQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGI IPILGIA

NYA

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<INSDFeature> LU503556 <INSDFeature key>source</INSDFeature key> <INSDFeature location>l..111</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name>mol type</INSDQualifier name> <INSDQualifier value>protein</INSDQualifier value> </INSDQualifier> <INSDQualifier id="ql4”> <INSDQualifier name>organism</INSDQualifier name> <INSDQualifier value>synthetic construet</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeq sequence>QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG

VPD

RFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGSVFGGGTKLTVL</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequence IDNumber="9”> <INSDSeq> <INSDSeq length>375</INSDSeq length> <INSDSeq moltype>DNA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> <INSDFeature> <INSDFeature key>source</INSDFeature key> <INSDFeature location>l..375</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name>mol type</INSDQualifier name> <INSDQualifier value>other DNA</INSDQualifier value> </INSDQualifier> <INSDQualifier id="q16"> <INSDQualifier name>organism</INSDQualifier name> <INSDQualifier value>synthetic construet</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeq sequence>gaggtgcagctgttgcagcagtcaggggctgagetgaagaagectgggtectcagtgaa get ctectgcaaggcttetggaggcaccttcagcagctatgctatcagctgggtgcgacaggecectggacaagggcttg agt ggatgggagggatcateectatecitggtatagcaaactacgcacagaagtteccagggcagagtcacgattaccgcg U503556 gac aaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacgggcgtgtattactgtgtgagaga acg tegatatagtggctacggggcggottactactttgactactggggccagggaaceetggtcaccgtetectca</IN

SDS eq _sequence> </INSDSeq> </SequenceData> <SequenceData sequence IDNumber="10"> <INSDSeq> <INSDSeq length>333</INSDSeq length> <INSDSeq moltype>DNA</INSDSeq moltype> <INSDSeq division>PAT</INSDSeq division> <INSDSeq feature-table> <INSDFeature> <INSDFeature key>source</INSDFeature key> <INSDFeature location>1..333</INSDFeature location> <INSDFeature quals> <INSDQualifier> <INSDQualifier name>mol type</INSDQualifier name> <INSDQualifier value>other DNA</INSDQualifier value> </INSDQualifier> <INSDQualifier id="q18"> <INSDQualifier name>organism</INSDQualifier name> <INSDQualifier value>synthetic construet</INSDQualifier value> </INSDQualifier> </INSDFeature quals> </INSDFeature> </INSDSeq feature-table> <INSDSeq sequence>cagtctgtgctgacgcagecgcectcagtetctggggceoccagggcagagggtcaccat ctc ctgcactgggagcagcectccaacatcggggecaggttatgatgtacactggtaccagecagettccaggaacagecceca aac tcctcatctatggtaacagcaatcggecctecaggggteecctgacecgattetetggetecaagtetggeacctecagee tcc ctggccatcactgggctecaggctgaggatgaggotgattattactgccagtectatgacagcagectgagtggtte ggt atteggcggagggaccaagetgacegtecta</INSDSeq sequence» </INSDSeq> </SequenceData> </ST26SequenceListing>

Claims (12)

1. CLAIMS LU503556

   1. A monoclonal antibody or a derivative, comprising an antibody Fab fragment, a single-chain antibody and a bispecific antibody, characterized in that the monoclonal antibody comprises a first variable region and a second variable region, wherein the first variable region is an antibody light chain variable region, and the antigen complementarity determining regions CDR1, CDR2 and CDR3 respectively comprise amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, wherein the second variable region is an antibody heavy chain variable region, and the antigen complementarity determining regions CDR1, CDR2 and CDR3 respectively contain amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the first variable region is an antibody light chain variable region und contains the amino acid sequence shown in SEQ ID NO: 8; the second variable region is an antibody heavy chain variable region und contains the amino acid sequence shown in SEQ ID NO:

   4.

   2. The monoclonal antibody or the derivative according to claim 1, characterized in that the monoclonal antibody comprises the antibody light chain variable region and the antibody light chain constant region, and the hinge region, CH1 region, CH2 region and CH3 region of the antibody heavy chain variable region and the antibody heavy chain constant region; the antibody light chain constant region comes from antibody kappa chain or lamda chain, and the antibody heavy chain constant region comes from human IgG1, 1gG2, lgG3 or 19G4 subtype.

   3. The monoclonal antibody or the derivative according to claim 1 or 2, wherein the coding sequence of the antibody light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 10, and the coding sequence of the antibody heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 9.

   4. The monoclonal antibody or the derivative according to claim 1 or 2, wherein LJ503556 gene targeting expression vector comprises the DNA molecule or/and nucleotide sequence and an expression regulation sequence operatively related to the sequence.

   5. The monoclonal antibody or the derivative according to claim 4, wherein the gene expression vector is a tissue-specific expression vector.

   6. The monoclonal antibody or the derivative according to claim 4, wherein the gene expression vector is a mammary gland specific expression vector.

   7. The monoclonal antibody or the derivative according to claim 4, wherein the promoter of the gene expression vector is a controllable promoter.

   8. The monoclonal antibody or the derivative according to claim 4, wherein a recombinant cell is transformed from the expression vector, and progeny cell can express the monoclonal antibody.

   9. Application of the monoclonal antibody or the derivative according to claim 1 or 2 in the detection of novel coronavirus or protein for non-diagnostic purposes.

   10. A composition or a kit, characterized in that the composition comprises the monoclonal antibody and derivative according to claim 1, a pharmaceutically acceptable carrier, any physiologically compatible solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, an isotonic and absorption delaying agent.

   11. Application of the monoclonal antibody or the derivative according to claim 1, characterized by comprising: (1) application in preparing novel coronavirus detection products or diagnostic products;

   (2) application in preparing medicines for preventing or treating novel coronavirus/503556 infection; (3) application in preparing medicines for preventing or treating diseases infected by novel coronavirus.

   12. Application of the composition according to claim 10, characterized by comprising: (1) application in preparing novel coronavirus detection products or diagnostic products; (2) application in preparing medicines for preventing or treating novel coronavirus infection; (3) application in preparing medicines for preventing or treating diseases infected by novel coronavirus.