Antibodies to CGRP and SP and uses thereof in antiviral infection

Abstract

According to the CGRP and SP antibodies and the application thereof in virus infection resistance, CGRP polypeptide and SP polypeptide are optimized and combined respectively, the expression quantity and immunogenicity are greatly improved, the optimal CGRP and SP monoclonal antibodies are screened out through a phage display technology, the optimal antibodies are high in affinity and specificity, and the monoclonal antibodies can be used for resisting virus infection. When the polypeptide is used in combination, the polypeptide shows extremely high capability of resisting DNA / RNA virus infection in a synergistic manner, the expression of macrophage interferon and the expression of inflammatory factors are remarkably improved, and the polypeptide has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to antibodies of CGRP and SP and application thereof in anti-viral infection. BACKGROUND

[0002] Viruses are simple microorganisms that must parasitize in host cells to replicate and proliferate. Currently, drugs for treating viruses are mainly divided according to targets, such as drugs directly targeting viruses in the processes of virus adsorption, internalization, transport, membrane fusion and genome release, which can bind to viral proteins or nucleic acids to affect the entry, transcription, replication, assembly and release of viruses; and drugs directly targeting hosts, which can affect the entry, transcription, replication, processing, transport and signal pathway activity of viruses. Currently, the main molecular forms of anti-viral drugs include small molecules, polypeptides, neutralizing antibodies, interferons, CRISPR-CAS systems, si / shRNA and other nucleic acid polymers, etc. However, compared with the conservation of host cells, the nucleotide substitution rate in the replication process of viruses, especially RNA viruses and small DNA viruses, is much higher than that of host cells, because viruses are more likely to produce genetic material mutations during self-replication, resulting in the failure of the original drug treatment target. In addition, there is no drug on the market for broad-spectrum viruses, only drugs for single (class) viruses, and such drugs for single (class) viruses often face the challenge of drug resistance. Therefore, in-depth study of the common mechanisms of virus and host cell interaction and exploration of new anti-viral drug targets are key problems that need to be solved for the prevention and treatment of viral infectious diseases.

[0003] Calcitonin gene-related peptide (CGRP) is a kind of neuropeptide composed of 37 amino acids, which widely exists in central nervous system, peripheral nervous system, cardiovascular system, endocrine system and gastrointestinal system and other systems. CGRP has multiple physiological functions, including regulating vasodilation, neuroprotection, promoting osteoblast differentiation and inhibiting osteoclast formation. Existing studies have shown that neurons produce CGRP during acute viral infection, act on T cells expressing RAMP3, promote anti-viral TH1 cell response, thereby enhancing IFNγ production and controlling viral infection. SP is a kind of neuropeptide composed of 11 amino acids, belonging to the tachykinin family, encoded by tac1 gene. SP is widely distributed in the gastrointestinal system and central nervous system, and is involved in various physiological processes in the gastrointestinal system, and is mainly concentrated in the synapses of nerve endings in the nervous system. Current related researches mainly focus on the role of SP in immune regulation and inflammation reaction regulation, and SP indirectly affects viral infection through "regulating the body's immune function", and its role depends on the body's own immune function state.

[0004] Although existing studies suggest that CGRP and SP polypeptides have potential value in anti-viral infection, there is no research on the development of CGRP and SP for anti-viral infection drugs. SUMMARY

[0005] To solve the problems in the prior art, the application provides antibodies of CGRP and SP and their application in anti-viral infection, the heavy chain and light chain variable region nucleotide sequences of the monoclonal antibodies of the CGRP and SP polypeptides are obtained by screening and enrichment through construction of a murine phage library, the obtained heavy chain and light chain variable region nucleotide sequences are respectively subcloned into an expression vector containing an IgG1 constant region nucleotide sequence, an entire IgG1 antibody expression vector is constructed and introduced into eukaryotic cells for expression and purification, and the monoclonal antibodies of the CGRP and SP polypeptides are respectively obtained; the monoclonal antibodies with the strongest affinity are respectively screened out through ELISA detection, and the two monoclonal antibodies screened out are jointly applied to block the expression of the CGRP and SP polypeptides, thereby significantly improving the anti-viral function of macrophages.

[0006] Since the target molecular weight of the CGRP and SP polypeptides is small, it is difficult to produce immunogenicity when used as an immunogen, so in the preparation of the immunogen, the polypeptides of the CGRP and SP of human or murine origin are coupled with KLH / BSA tags, and the polypeptides coupled with different tags are cross combined as immunogens, so as to avoid non-specific binding to the KLH / BSA tag caused by single polypeptide immunization, and naked peptides are used as coating antigens in serum titer detection to further exclude these non-specific binding, so as to truly detect the titer level against the target.

[0007] In one aspect, the application provides an antibody of a CGRP polypeptide, comprising:

[0008] (1) CDR1 composed of the amino acid sequence of SEQ ID NO. 1, CDR2 composed of the amino acid sequence of SEQ ID NO. 2, and CDR3 composed of the amino acid sequence of SEQ ID NO. 3 in the heavy chain variable region, and

[0009] (2) CDR1 composed of the amino acid sequence of SEQ ID NO. 7, CDR2 composed of the amino acid sequence of SEQ ID NO. 8, and CDR3 composed of the amino acid sequence of SEQ ID NO. 9 in the light chain variable region.

[0010] The monoclonal antibody of the CGRP polypeptide provided by the present application is prepared by constructing a murine phage display library. Total RNA is extracted from the spleen of a mouse immunized with Hu-CGRP-BSA+Hu-CGRP-KLH, Hu-CGRP-KLH+Mu-CGRP-BSA, and Hu-CGRP-BSA+Mu-CGRP-KLH, and then cDNA is synthesized by using mRNA as a template through reverse transcription PCR (RT-PCR) technology, and the cDNA contains gene fragments encoding antibodies. The gene fragments encoding the variable regions (LC and HC) of the antibodies are connected with the coat protein gene of the phage to construct a fusion gene. Then, the fusion gene is inserted into a vector of the M13 phage to form a phage display library.

[0011] The constructed phage display library is transformed into an Escherichia coli (X-Blue competent) host bacterium, so that the phage can replicate and assemble in the host bacterium. In the process of growth and reproduction of the host bacterium, the antibody genes in the phage display library are amplified in large quantities along with the replication of the phage, and meanwhile, the antibody fragments are expressed in fusion with the coat protein of the phage and displayed on the surface of the phage. The target antigen is fixed on the surface of an enzyme-linked immunosorbent assay plate, so that the antigen can specifically bind to the corresponding antibody in the phage display library. The phage display library is incubated with the solid carrier coated with the antigen, so that the antibody fragments displayed on the surface of the phage specifically bind to the antigen. The unbound phage is removed through a washing step, and the phage specifically bound to the antigen is retained. The phage bound to the antigen is eluted by using a low-pH buffer, and then the eluted phage is used to infect fresh host bacteria for amplification culture. Through 2-4 rounds of screening-elution-amplification processes, the phage capable of specifically binding to the antigen is highly enriched.

[0012] The 8 phage clones screened are sequenced, and the antibody gene sequences are analyzed to determine the nucleotide sequences of the antibodies. The antibody genes with specificity screened are subcloned into a suitable expression vector and transformed into a mammalian cell expression system for expression. Then, the expressed antibodies are purified by affinity chromatography to obtain 8 monoclonal antibodies of CGRP with high purity.

[0013] The 8 monoclonal antibodies of CGRP purified are verified for functions by using enzyme-linked immunosorbent assay (ELISA) to detect the binding specificity and affinity of the antibodies to the antigen, and finally, 1-2 antibodies with the strongest specific binding capacity and the strongest affinity are selected. Compared with the traditional hybridoma technology, the CGRP monoclonal antibodies obtained by constructing the phage library have the characteristics of short preparation process period, small workload, easy gene modification, and higher specificity, affinity and stability of the antibodies.

[0014] Further, the amino acid sequence of the heavy chain variable 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. 10.

[0015] Further, the amino acid sequences of the constant regions of the heavy chain and the light chain are the amino acid sequence of the heavy chain constant region of murine IgG1 and the amino acid sequence of the light chain constant region of murine IgG1, respectively.

[0016] It can be understood that IgG has four subtypes, namely IgG1, IgG2, IgG3 and IgG4, and the heavy chain and light chain constant region sequences of all of them can be used as the heavy chain and light chain constant region of the CGRP and SP antibody, but their abilities to activate complement and bind to Fc receptors are different. The CGRP and SP monoclonal antibody prepared in the present application is used for detection, so the heavy chain and light chain constant regions of IgG1 are selected because the IgG1 constant region shows good adaptability in various detection environments, helps to maintain the conformational stability of the whole antibody molecule, whether it is used for detecting low concentration antigens or in complex biological samples (such as blood, tissue homogenate), so that the variable region can better play the function of recognizing and binding antigens, reduce cross-reactions with irrelevant substances in the sample, thereby improving the specificity of detection so as to accurately capture the target antigen in detection and reduce the occurrence of false negative results. For example, in a cell-based detection system, the IgG1 constant region can bind to the Fc receptor, amplify the detection signal through Fc receptor-mediated signal transduction, and improve the sensitivity of detection. For another example, in ELISA detection, the structure of IgG1 is relatively stable, and compared with other antibody subtypes, IgG1 is less likely to denature or degrade under different environmental conditions, such as different pH values, temperatures, etc., and can maintain its activity and antigen binding capacity in various operation steps of ELISA detection, ensuring the reliability of the detection results; in addition, IgG1 can be well combined with enzyme markers, fluorescent markers and the like commonly used in ELISA detection, and the combination does not significantly affect the antigen binding activity of itself and the activity of the marker, which is conducive to subsequent signal detection and amplification. In addition, current biological technology can efficiently produce IgG1 antibodies in various expression systems, such as Chinese hamster ovary cells (CHO cells), human embryonic kidney cells (HEK293 cells, HEK293T cells), etc. These expression systems are mature and stable, and can realize large-scale culture and high-yield expression of IgG1 antibodies

[0017] Further, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain is shown as SEQ ID NO. 11.

[0018] In another aspect, the present application provides a nucleic acid encoding the heavy chain and / or the light chain of the CGRP polypeptide antibody as described above.

[0019] Further, the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 6, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 12.

[0020] In another aspect, the present application provides a recombinant expression vector comprising the above-mentioned nucleic acid.

[0021] In another aspect, the present application provides a method for preparing an antibody of the CGRP polypeptide as mentioned above, characterized in that it comprises the following steps:

[0022] (1) immunizing an animal with a CGRP antigen;

[0023] (2) collecting the spleen of the immunized animal to construct a phage library;

[0024] (3) obtaining SCFV from the phage library by screening and enrichment, and sequencing to obtain the nucleotide sequence of the SCFV;

[0025] (4) cloning the heavy chain variable region and the light chain variable region nucleotide sequences of the SCFV into expression vectors respectively by subcloning;

[0026] (5) introducing the expression vectors into eukaryotic cells for expression and purification to obtain a monoclonal antibody of the CGRP polypeptide.

[0027] In another aspect, the present application provides a kit for detecting CGRP or predicting whether an individual is infected with a virus, comprising an antibody of the CGRP polypeptide as mentioned above.

[0028] In another aspect, the present application provides the use of an antibody of the CGRP polypeptide as mentioned above for preparing a reagent for improving the antiviral ability of cytomegalovirus.

[0029] In another aspect, the present application provides the use of an antibody of the CGRP polypeptide as mentioned above for preparing a reagent for predicting whether an individual is infected with a virus.

[0030] In another aspect, the present application provides an antibody of the SP polypeptide, comprising:

[0031] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 27, CDR2 consisting of the amino acid sequence of SEQ ID NO. 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 29 in the heavy chain variable region, and

[0032] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 33, CDR2 consisting of the amino acid sequence of SEQ ID NO. 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 35 in the light chain variable region.

[0033] Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 36.

[0034] Further, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 31, and the amino acid sequence of the light chain is shown as SEQ ID NO. 37.

[0035] The monoclonal antibody of the SP polypeptide provided by the present application is prepared by constructing a murine phage display library. Total RNA is extracted from the spleen of a mouse immunized with Hu-SP-BSA, Hu-SP-KLH, Hu-SP-BSA+Hu-SP-KLH, and then cDNA is synthesized by reverse transcription PCR (RT-PCR) technology using mRNA as a template, and these cDNAs contain gene fragments encoding antibodies. The gene fragments encoding the variable regions (LC and HC) of the antibodies are linked to the coat protein gene of the phage to construct a fusion gene. Then the fusion gene is inserted into the vector of the M13 phage to form a phage display library.

[0036] The phage library is then subjected to multiple rounds of panning to enrich specific monoclonal antibodies with high affinity round by round. The 26 phage clones obtained by screening are sequenced, and the antibody gene sequences are analyzed to determine the nucleotide sequence of the antibody. The specific antibody genes screened are subcloned into a suitable expression vector and transformed into a mammalian cell expression system for expression. Then the expressed antibodies are purified by affinity chromatography to obtain high-purity 5 monoclonal antibodies of SP. The 5 purified monoclonal antibodies of SP are subjected to functional verification by enzyme-linked immunosorbent assay (ELISA) to detect their binding specificity and affinity to antigens, and finally one antibody with the strongest specific binding ability and the strongest affinity is selected.

[0037] In another aspect, the present application provides a nucleic acid encoding the heavy chain and / or light chain of the SP polypeptide antibody as described above.

[0038] Further, the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 32, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 38.

[0039] In another aspect, the present application provides a recombinant expression vector comprising the nucleic acid as described above.

[0040] In another aspect, the present application provides a method for preparing the antibody of the SP polypeptide as described above, comprising the following steps:

[0041] (1) immunizing an animal with SP antigen;

[0042] (2) Collect the spleen of the immunized animal to construct a phage library;

[0043] (3) Obtain SCFV from the phage library by screening and enrichment, and sequence to obtain the nucleotide sequence of the SCFV;

[0044] (4) Clone the heavy chain variable region and the light chain variable region nucleotide sequence of the SCFV into an expression vector respectively by subcloning;

[0045] (5) Introduce the expression vector into eukaryotic cells to express and purify to obtain a monoclonal antibody of the SP polypeptide.

[0046] In another aspect, the present application provides a kit for detecting SP or for predicting whether an individual is infected with a virus, comprising the antibody of the SP polypeptide as described in any one of the above.

[0047] In another aspect, the present application provides the use of the antibody of the SP polypeptide as described above for preparing a reagent for improving the antiviral ability of macrophages.

[0048] In another aspect, the present application provides the use of the antibody of the SP polypeptide as described above for preparing a reagent for predicting whether an individual is infected with a virus.

[0049] The best monoclonal antibodies of the CGRP polypeptide and the SP polypeptide screened by the present application have good affinity and strong specificity, and when used for the treatment of RNA / DNA virus infection, can improve the survival rate, reduce lung injury, reduce viral load, promote the production of interferon Ifna and Ifnb and inflammatory factors IL-6 and TNF, and improve the antiviral ability of macrophages. When the two monoclonal antibodies of the CGRP polypeptide and the SP polypeptide are used in combination, the antiviral infection ability is significantly improved, and the effect is better.

[0050] In another aspect, the present application provides a kit for detecting CGRP and SP polypeptides, comprising an antibody of the CGRP polypeptide and an antibody of the SP polypeptide, wherein the antibody of the CGRP polypeptide comprises:

[0051] (1) CDR1 composed of the amino acid sequence of SEQ ID NO. 1, CDR2 composed of the amino acid sequence of SEQ ID NO. 2, and CDR3 composed of the amino acid sequence of SEQ ID NO. 3 in the heavy chain variable region, and

[0052] (2) CDR1 composed of the amino acid sequence of SEQ ID NO. 7, CDR2 composed of the amino acid sequence of SEQ ID NO. 8, and CDR3 composed of the amino acid sequence of SEQ ID NO. 9 in the light chain variable region;

[0053] The antibody of the SP polypeptide comprises:

[0054] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 27, CDR2 consisting of the amino acid sequence of SEQ ID NO. 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 29 in the heavy chain variable region, and

[0055] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 33, CDR2 consisting of the amino acid sequence of SEQ ID NO. 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 35 in the light chain variable region.

[0056] Further, the amino acid sequence of the heavy chain variable region of the antibody of the CGRP polypeptide is shown in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10; the amino acid sequence of the heavy chain variable region of the antibody of the SP polypeptide is shown in SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 36.

[0057] Further, the amino acid sequence of the heavy chain of the antibody of the CGRP polypeptide is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain is shown in SEQ ID NO. 11; the amino acid sequence of the heavy chain of the antibody of the SP polypeptide is shown in SEQ ID NO. 31, and the amino acid sequence of the light chain is shown in SEQ ID NO. 37.

[0058] In another aspect, the present application provides an antibody combination for predicting whether an individual is infected with a virus, comprising an antibody of a CGRP polypeptide and an antibody of a SP polypeptide, wherein the antibody of the CGRP polypeptide comprises:

[0059] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 1, CDR2 consisting of the amino acid sequence of SEQ ID NO. 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 3 in the heavy chain variable region, and

[0060] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 7, CDR2 consisting of the amino acid sequence of SEQ ID NO. 8, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 9 in the light chain variable region.

[0061] The antibody of the SP polypeptide comprises:

[0062] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 27, CDR2 consisting of the amino acid sequence of SEQ ID NO. 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 29 in the heavy chain variable region, and

[0063] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 33, CDR2 consisting of the amino acid sequence of SEQ ID NO. 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 35 in the light chain variable region.

[0064] Further, the amino acid sequence of the heavy chain variable region of the antibody to the CGRP polypeptide is shown in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10; the amino acid sequence of the heavy chain variable region of the antibody to the SP polypeptide is shown in SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 36.

[0065] In another aspect, the present application provides a use of an antibody combination for the preparation of a reagent for predicting whether an individual is infected with a virus, the antibody combination comprising an antibody to a CGRP polypeptide and an antibody to a SP polypeptide, the antibody to the CGRP polypeptide comprising:

[0066] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 1, CDR2 consisting of the amino acid sequence of SEQ ID NO. 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 3 in the heavy chain variable region, and

[0067] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 7, CDR2 consisting of the amino acid sequence of SEQ ID NO. 8, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 9 in the light chain variable region.

[0068] the antibody to the SP polypeptide comprising:

[0069] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 27, CDR2 consisting of the amino acid sequence of SEQ ID NO. 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 29 in the heavy chain variable region, and

[0070] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 33, CDR2 consisting of the amino acid sequence of SEQ ID NO. 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 35 in the light chain variable region.

[0071] In still another aspect, the present application provides a use of an antibody for the preparation of a reagent for improving an anti-viral ability of macrophages, the antibody including an antibody to a CGRP polypeptide and an antibody to an SP polypeptide, the antibody to the CGRP polypeptide including:

[0072] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 1, CDR2 consisting of the amino acid sequence of SEQ ID NO. 2, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 3 in a heavy chain variable region, and

[0073] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 7, CDR2 consisting of the amino acid sequence of SEQ ID NO. 8, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 9 in a light chain variable region;

[0074] the antibody to the SP polypeptide including:

[0075] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 27, CDR2 consisting of the amino acid sequence of SEQ ID NO. 28, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 29 in a heavy chain variable region, and

[0076] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 33, CDR2 consisting of the amino acid sequence of SEQ ID NO. 34, and CDR3 consisting of the amino acid sequence of SEQ ID NO. 35 in a light chain variable region.

[0077] Further, the amino acid sequence of the heavy chain variable region of the antibody to the CGRP polypeptide is shown in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10; the amino acid sequence of the heavy chain variable region of the antibody to the SP polypeptide is shown in SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 36.

[0078] In still another aspect, the present application provides a system for predicting whether an individual is infected with a virus, the system including a data analysis module for analyzing a detection value of an antigen detected by an antibody, the antibody including an antibody to a CGRP polypeptide and an antibody to an SP polypeptide, the antibody to the CGRP polypeptide including:

[0079] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 1, CDR2 consisting of the amino acid sequence of SEQ ID NO. 2 and CDR3 consisting of the amino acid sequence of SEQ ID NO. 3 in the heavy chain variable region, and

[0080] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 7, CDR2 consisting of the amino acid sequence of SEQ ID NO. 8 and CDR3 consisting of the amino acid sequence of SEQ ID NO. 9 in the light chain variable region;

[0081] The antibody of the SP polypeptide comprises:

[0082] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO. 27, CDR2 consisting of the amino acid sequence of SEQ ID NO. 28 and CDR3 consisting of the amino acid sequence of SEQ ID NO. 29 in the heavy chain variable region, and

[0083] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO. 33, CDR2 consisting of the amino acid sequence of SEQ ID NO. 34 and CDR3 consisting of the amino acid sequence of SEQ ID NO. 35 in the light chain variable region.

[0084] Further, the amino acid sequence of the heavy chain variable region of the antibody of the CGRP polypeptide is shown in SEQ ID NO. 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10; the amino acid sequence of the heavy chain variable region of the antibody of the SP polypeptide is shown in SEQ ID NO. 30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 36.

[0085] The present application has the following beneficial effects:

[0086] 1. A method for improving the immunogenicity of CGRP polypeptide and SP polypeptide is provided, after coupling the CGRP polypeptide and SP polypeptide to the macromolecular carrier BSA / KLH, and using the human-mouse cross immunogen as the immunogen, the problem of weak immunogenicity of small molecule polypeptide is solved, and immune deviation is avoided through carrier diversification, and finally high affinity, cross-species reaction antibodies are obtained.

[0087] 2. A new antibody sequence specifically recognizing CGRP polypeptide and SP polypeptide is provided, the antibody has high affinity and strong specificity for CGRP and SP, and does not produce non-specific binding with other irrelevant proteins;

[0088] 3. The combination of the monoclonal antibody with the highest binding activity and specificity to CGRP polypeptide and SP polypeptide can effectively block CGRP polypeptide and SP polypeptide, and improve the ability of macrophages to resist viral infection. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 G1, G2, G3 mouse spleen RNA electrophoresis map.

[0090] Figure 2 G1, G2, G3 mouse spleen RNA electrophoresis map.

[0091] Figure 3 PCR amplification of CCGP light and heavy chain electropherogram.

[0092] Figure 4 PCR amplification of SP light and heavy chain electropherogram.

[0093] Figure 5 Binding activity of antibody CGRP-A037 and antigen.

[0094] Figure 6 Binding activity of antibody SP-A080 and antigen

[0095] Figure 7 ELISA and RT-qPCR experiments to verify the effect of the combination of CGRP-A037 antibody and SP-A080 antibody on the anti-viral ability of macrophages, wherein αCGRP represents CGRP-A037 antibody, αSP represents SP-A080 antibody, and Vehicle represents PBS treatment.

[0096] Figure 8 HE staining, ELISA and RT-qPCR experiments to verify the anti-VSV viral infection ability of the combination of CGRP-A037 antibody and SP-A080 antibody, wherein αCGRP represents CGRP-A037 antibody, αSP represents SP-A080 antibody, and Vehicle represents PBS treatment.

[0097] Figure 9 HE staining, ELISA and RT-qPCR experiments to verify the anti-HSV-1 viral infection ability of the combination of CGRP-A037 antibody and SP-A080 antibody, wherein αCGRP represents CGRP-A037 antibody, αSP represents SP-A080 antibody, and Vehicle represents PBS treatment. DETAILED DESCRIPTION

[0098] The application will be described in further detail below with reference to the drawings and examples. It should be noted that the examples described below are intended to facilitate an understanding of the application and do not in any way limit the application.

[0099] Example 1: Antigen preparation, animal immunization and antibody screening

[0100] 1. Antigen preparation

[0101] Fusion of BSA or KLH tag at the C-terminal of human or mouse CGRP ectodomain to construct CGRP antigens (SEQ ID NO. 91) Human-CGRP, Human-CGRP-BSA, Human-CGRP-KLH, Mu-CGRP, Mu-CGRP-BSA, Mu-CGRP-KLH; Fusion of BSA or KLH tag at the C-terminal of human SP ectodomain to construct SP antigens (SEQ ID NO. 92) Hu-SP, Hu-SP-BSA, Hu-SP-KLH.

[0102] Verify the binding activity of CGRP antigen and Anti-CGRP antibody Anti-CGRP by ELISA, the steps are as follows:

[0103] 1. Coating: dilute Human-CGRP, Human-CGRP-BSA, Human-CGRP-KLH, Mu-CGRP, Mu-CGRP-BSA, Mu-CGRP-KLH to 2 μg / mL with 1xPBS, add 30 μL per well, incubate overnight at 4°C;

[0104] 2. Washing and blocking: wash the plate with PBST for 3 times, add 5% PBSM or 2% BSA, 180 μL per well, block at room temperature for 2 hours.

[0105] 3. Incubation of primary antibody: wash the plate with PBST for 3 times, dilute Anti-CGRP with 1% PBSM, 30 μL per well, incubate at room temperature for 60 minutes.

[0106] 4. Incubation of secondary antibody: wash the plate with PBST for 3 times, add Anti-Human-IgG-Fc-HRP (Jackson Immuno, 109-035-008) diluted 1:8000, 30 μL per well, incubate at room temperature for 50 minutes.

[0107] 5. Color development and reading: wash the plate with PBST for 6 times, add TMB color developing solution 30 μL per well, avoid light, react for 5-10 minutes, then add 2M H2SO4 stop solution 30 μL per well, immediately read the absorbance (OD450) at 450 nm. 450 ).

[0108] The results of the binding activity detection of the CGRP antigen and the anti-CGRP antibody are shown in Table 1 below.

[0109] Table 1 Binding activity of CGRP antigen and anti-CGRP antibody

[0110] Antigen name EC50 Human-CGRP 0.1219 Human-CGRP-BSA 0.0221 Human-CGRP-KLH 0.0220 Mu-CGRP 0.1456 Mu-CGRP-BSA 0.0273 Mu-CGRP-KLH 0.0631

[0111] As shown in the results of Table 1, the measured antigen and the anti-CGRP antibody all have binding activity, indicating that the antigen can be used for immunization.

[0112] Similarly, the binding activity of the human-derived Hu-SP, Hu-SP-BSA and Hu-SP-KLH antigens was detected in this embodiment, and the results showed that Hu-SP, Hu-SP-BSA and Hu-SP-KLH can be used for immunization.

[0113] 2. Animal immunization

[0114] The CGRP mouse immunization scheme is shown in Table 2, and according to different antigen combinations, a total of 3 groups are cross-immunized (A: Hu-CGRP-BSA, B: Hu-CGRP-KLH, C: Mu-CGRP-BSA, D: Mu-CGRP-KLH); the SP mouse immunization scheme is shown in Table 3, and 3 groups are immunized.

[0115] Table 2 CGRP mouse immunization scheme

[0116] Group Immunized antigen Mouse strain and number of immunized mice G1(A+B) Hu-CGRP-BSA+Hu-CGRP-KLH Balb / c, 3 G2(B+C) Hu-CGRP-KLH+Mu-CGRP-BSA Balb / c, 3 G3(A+D) Hu-CGRP-BSA+Mu-CGRP-KLH Balb / c, 3

[0117] Table 3 SP mouse immunization scheme

[0118] Group Immunized antigen Mouse strain and number of immunized mice GⅠ Hu-SP-KLH Balb / c, 3 GⅡ Hu-SP-BSA Balb / c, 3 GⅢ Hu-SP-BSA+Mu-SP-KLH Balb / c, 3

[0119] The CGRP mouse immunization is divided into primary immunization (antigen dose 100 μg), secondary immunization (antigen dose 50 μg), third immunization (antigen dose 50 μg), fourth immunization (antigen dose 50 μg) and booster immunization (antigen dose 100 μg). The first immunization is to make the antigen into an emulsifier with an equal amount of complete Freund's adjuvant, and to subcutaneously inject multiple points and intraperitoneally inject, with an interval of 2 weeks, 50 μg of immunogen and an equal amount of incomplete Freund's adjuvant to make an emulsifier, and after five times of immunization, the serum titer is determined by indirect ELISA, and the serum titer detection method is as follows:

[0120] (1) Take mouse serum: 0.7-0.8 mL of blood was taken from the eyeball 3 days after the fifth immunization, and after standing at 4°C for 1-2 h, the upper serum was separated by centrifugation at 4000 g for 5 min for detection;

[0121] (2) Coating: Human-CGRP, Mu-CGRP antigens were diluted to 2 μg / mL with 1x PBS, 30 μL was added to each well, and coated at 4°C overnight;

[0122] (3) Washing plate: The plate was washed with PBST at 180 μL / well for 3 times, and the plate was dried;

[0123] (4) Blocking: The plate was blocked with 5% PBSM at 160 μL / well, and the plate was blocked at room temperature for 1 h;

[0124] (5) Washing plate: The plate was washed with PBST at 180 μL / well for 3 times, and the plate was dried;

[0125] (6) Sample addition: The serum sample of step (1) was gradiently diluted with sample diluent and added at 30 μL / well;

[0126] (7) Addition of secondary antibody: 30 μL / well of detection antibody (goat anti-mouse IgG (1+2a+2b)-HRP, 1:5000) was added, and incubated at room temperature for 1 h;

[0127] (8) Washing plate: Then the plate was washed with washing solution at 180 μL / well for 6 times, and the plate was dried;

[0128] (9) Color development: 30 μL / well of color developing solution was added, and the plate was placed at room temperature for 5-10 min;

[0129] (10) Termination and detection: 30 μL / well of termination solution was added to terminate the reaction, and then the plate was detected by an enzyme-labeled instrument with a detection wavelength of 450 nm.

[0130] The immunization of SP mice included primary immunization, secondary immunization, third immunization, fourth immunization, fifth immunization, and booster immunization, and the antigen doses were 100 μg, 50 μg, 50 μg, 50 μg, 50 μg, and 50 μg, respectively, and the injection method was the same as above. After six times of immunization, the serum titer was determined by indirect ELISA, and the method was the same as above, wherein Human-SP antigen was used for coating in step (2).

[0131] The serum titer detection results of the CGRP mice are shown in Tables 4-5, and the serum titer detection results of the SP mice are shown in Table 6.

[0132] Table 4 Serum titer of Human-CGRP after five immunizations of CGRP mice

[0133]

[0134] Table 5 Serum titer of Mu-CGRP after five immunizations of CGRP mice

[0135]

[0136]

[0137] Table 6 Serum titer of Human-CGRP after six immunization of SP mice

[0138]

[0139] According to the data analysis of Tables 4-5, the serum titer of 9 mice after five immunization reached more than 1.65 times of the NC, and the results showed that the serum titer of 9 mice after five immunization reached more than 40,000 against human-CGRP, and the serum titer of 8 mice after five immunization reached more than 40,000 against mouse-CGRP, which can be used for subsequent phage library construction and screening.

[0140] According to the data of Table 6, the serum titer of 9 mice after six immunization reached more than 1.65 times of the NC, and the results showed that the serum titer of 9 mice after six immunization reached more than 40,000, which can be used for subsequent phage library construction and screening.

[0141] 3. Construction of phage library

[0142] The spleens of G1, G2, and G3 experimental mice and G1, G2, and G3 experimental mice were taken, and the spleen RNA was extracted using TriPure Isolation Reagent reagent, and the RNA electrophoresis map is shown in Figure 1 , 2 UV quantification of the extracted RNA, and the experimental results are shown in Table 7 below, and the results show that the purity and concentration of the extracted RNA are good and can be used.

[0143] Table 7 UV quantification results of spleen RNA

[0144] Detection item A260 / A280 Concentration (ng / μL) G1#1,2,3 spleen 1.92 790.29 G2#4,5,6 spleen 1.92 790.29 G3#7,8,9 spleen 1.92 790.29 G1#1,2,3 spleen 1.96 499.09 G2#4,5,6 spleen 1.97 471.23 G3#7,8,9 spleen 1.96 474.22

[0145] Further, commercially available reverse transcription kit (Takara, 6210A) was used for reverse transcription to obtain spleen cDNA, and the light chain variable region (LC) sequence and the heavy chain variable region (HC) sequence of the cDNA were amplified by PCR, and the electrophoresis results are shown in Figure 3 , 4 The coding sequence of the constant region was connected to the coding sequence of the pIII protein at the 3' end, and then cloned into the phage display vector. Then the vector was transformed into competent E. coli SS320 cells (Lucigen, MC1061 F) by an electroporator (Bio-Rad, MicroPulser), and after 1 hour of recovery, it was plated on 2-YT solid plates with ampicillin resistance. Gradient dilution spot plate was used to determine the capacity of the immune library, and the results showed that the capacity of the immune library reached 3.00x10 8cfu. The immune library was finally constructed and displayed on the coat protein of M13 phage in the form of Fab using the helper phage prepared by Zhiyou Bio.

[0146] 4. Antibody screening

[0147] The human or murine CGRP / CGRP-KLH / CGRP-BSA antigen was coated on the immunization tube or the biotinylated human or murine CGRP-BSA-Biotin antigen was connected to the magnetic beads through streptavidin, and the original antibody library was added. First, the non-specifically bound phage was washed away by washing solution, and then the phage bound to the antigen was eluted by elution solution, amplified in E. coli, prepared phage using helper phage auxiliary packaging, and subjected to the next round of enrichment screening. After three rounds of "adsorption-elution-amplification", the positive library combined with CGRP was screened. In the first round of screening, an antigen concentration of 30 μg / mL (100 nM) was used to capture as many potential binding clones as possible; in the second round, the antigen concentration was reduced to 6 μg / mL (20 nM) to increase the screening pressure and concentrate high-affinity clones; in the third round, the antigen concentration was further reduced to 1.2 μg / mL (40 nM) to screen clones with the highest affinity. Starting from the second round, the CGRP-KLH and CGRP-BSA pathways were subjected to 10-fold negative screening with Negative-BSA / KLH.

[0148] The binding of the positive library after multiple rounds of solid-phase screening to the CGRP antigen was detected by ELISA. The results showed that, at the phage level, 512 clones were selected, 215 positive clones with cross-binding activity to Human-CGRP, Human-CGRP-BSA, Mu-CGRP, and Mu-CGRP-BSA antigens were selected for sequencing analysis, 16 sequence unique molecules were obtained, and 8 molecules were selected for full-length construction after sequence diversity analysis.

[0149] The method for the positive library of multiple rounds of solid-phase screening SP was basically the same as described above. Hu-SP, Hu-SP-BSA, Hu-SP-KLH, and Hu-SP-BSA-Biotin antigens were coated, and the binding of the positive library to the SP antigen was detected by ELISA. The results showed that, through ELISA detection after binding to the SP antigen, at the phage level, 360 clones were selected, 102 positive clones (101 of which were Hu-SP-BSA / Hu-SP-KLH cross-binding, and 1 was single binding to Hu-SP) were selected for sequencing analysis, 40 sequence unique molecules were obtained, and 26 molecules could be subjected to full-length construction after sequence diversity analysis.

[0150] Example 2, Construction, Expression, and Purification of Anti-CGRP Antibody

[0151] The present embodiment first designs primers according to the heavy chain variable region sequences and light chain variable region sequences of the 8 lead molecules screened in Example 1, and then prepares and purifies the antibodies. Then, the binding ability of the 8 purified monoclonal antibodies to the antigen (Human / Mu-CGRP and its conjugates) is screened to select monoclonal antibodies with strong binding ability to the antigen. Then, the non-specific binding is excluded through Negative-BSA / KLH and 5% PBSM blocking verification. The specific process is as follows:

[0152] 1. Preparation of monoclonal antibodies

[0153] The light and heavy chain variable region fragments of the full-length antibodies are obtained by PCR amplification, and then recovered by gel electrophoresis. The recovered heavy and light chain variable region fragments are added to the membrane binding solution, mixed thoroughly, and then added to the purification column. After centrifugation at 13000 rpm for 1 min, the liquid is discarded. Then, 700 μL of rinse solution is added, and the liquid is discarded after centrifugation at 13000 rpm for 1 min. After air whirling for 5 min, the solution is left at room temperature for 5 min. Then, Nuclease-Free Water is added for elution, and the purified heavy and light chain variable region sequence fragments are obtained.

[0154] Further, the constant region sequences are added to the heavy and light chain variable region fragments by PCR method to construct complete heavy chain nucleotide sequences and light chain nucleotide sequences. The constant region sequence is the constant region sequence of murine IgG1. The heavy chain constant region amino acid sequence is shown in SEQ ID NO. 89, and the light chain constant region amino acid sequence is shown in SEQ ID NO. 90.

[0155] The complete heavy chain nucleotide sequence and the complete light chain nucleotide sequence are respectively connected to the pUC expression vector plasmid. The specific method is as follows: 4 μL of purified complete heavy chain nucleotide sequence / complete light chain nucleotide sequence, 1 μL of vector, and 5 μL of ligase are mixed thoroughly. After 50°C reaction for 20 min, it is added to 100 μL of competent cells. After ice bath for 30 min, heat shock for 90 s, and quickly placed on ice for 3 min, it is plated and cultured at 37°C overnight. The next day, the obtained monoclonal is subjected to PCR amplification and sequencing. Through software comparison, it is known that the expression vector plasmid has successfully connected the specific gene.

[0156] Further, the vector plasmid connected with the heavy chain and the vector plasmid connected with the light chain are paired and transferred to HEK 293 cells for transient expression. The specific method is as follows: HEK293 cells are subcultured in 293 serum-free CD medium. The plasmid DNA to be expressed is mixed with transfection reagent TF2 and added to the cells. After transfection, 293 serum-free feeding liquid is added on the 1st, 3rd and 5th day. The shaking flask culture conditions are as follows: 5% CO2, temperature 37°C, shaking bed speed 175 rpm.

[0157] Further, the cell supernatant was collected after 7 days of cell culture and the antibody was purified, specifically as follows:

[0158] (1) Sample preparation: centrifugation was performed using a desktop centrifuge at 4000g for 30 min, and 8 monoclonal cell supernatants were taken;

[0159] (2) Filtration: different filtration methods were selected according to the volume of the feed liquid, and 0.45 μm filter membrane was used for filtration;

[0160] (3) Connection of purification system: the appropriate size of affinity filler Protein A column was selected according to the expression amount, and was connected with the purification system;

[0161] (4) Water balance: 2 CV of ultrapure water was used for cleaning, and the 25% ethanol storage solution was replaced;

[0162] (5) Equilibrium chromatography column: AC Binding buffer was used for equilibrium of 3 CV to UV baseline stability;

[0163] (6) Sample loading: adjust the appropriate flow rate for sample loading;

[0164] (7) Elution: elution liquid was used for elution of 5-10 CV to UV baseline stability, and the flow rate was the same as the sample loading flow rate;

[0165] (8) Elution: elution liquid was used for elution, and the UV peak was collected;

[0166] (9) Neutralization: Tris, pH 8.0 was added to neutralize the eluted antibody;

[0167] (10) Equilibrium: AC Binding buffer was used for equilibrium of 3 CV to neutral;

[0168] (11) CIP cleaning: CIP regeneration liquid was used for cleaning of more than 5 CV;

[0169] (12) Alkali flushing: AC Binding buffer was used for alkali flushing until the outlet pH was neutral, and then ultrapure water was used for flushing of 3 CV;

[0170] (13) Preservation: 25% ethanol was used for equilibrium of 2 CV, and the column was preserved.

[0171] Through purification, 8 purified CGRP monoclonal antibodies were obtained.

[0172] 2. Detection of antibody purity

[0173] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the purity of the 8 purified antibodies. This method separates the proteins in the sample according to their molecular weights, thereby verifying the purity of the target antibody. The specific method is as follows:

[0174] (1) Treatment of samples: 5 μg of each of the 8 purified antibodies was added to 5 μL of 4x loading buffer (reduced) and 4x loading buffer (non-reduced), respectively, heated in a water bath at 100°C, and then centrifuged at 10000 rpm;

[0175] (2) Preparation of SDS-PAGE gels with concentrations of 13% and 7.5%;

[0176] (3) The SDS-PAGE gels prepared in (2) were fixed in the electrophoresis tank, and sufficient 1x glycine system electrode buffer was added to the electrophoresis tank. The sample solution treated with 4x loading buffer (non-reduced) in (1) and the protein molecular weight marker were added to the loading well of the 7.5% SDS-PAGE gel using a micropipette. The sample solution treated with 4x loading buffer (reduced) in (1) and the protein molecular weight marker were added to the loading well of the 13% SDS-PAGE gel using a micropipette;

[0177] (4) Electrophoresis: connect the power supply, first electrophorese at a constant voltage of 100 V until the bromophenol blue dye enters the separation gel from the concentrated gel, then adjust the current to 140 V and continue electrophoresis until the bottom of the gel plate, and then turn off the power supply;

[0178] (5) Staining: immerse the gel in Coomassie Brilliant Blue R-250 staining solution, heat in a microwave oven for 60 s, place on a gently shaking platform for room temperature staining for 2-4 h, replace the decolorizing solution to cover the gel, heat in a microwave oven for 60 s, place on a gently shaking platform for room temperature decolorization, and repeat the decolorization operation until blue bands and a clean background are obtained.

[0179] At the same time, the aggregation state of the antibodies was analyzed by size exclusion chromatography (SEC). The higher the monomer content, the better the purity and activity of the sample; the higher the high polymer content, the poorer the protein stability. The results are shown in the following table.

[0180] Table 8 Purity detection results of 8 antibodies

[0181]

[0182] As shown in the results in Table 8, the purity of the 8 purified antibody proteins was more than 95%, the HMW was less than 5%, and the monomer purity was more than 90%.

[0183] 3. Binding ability of purified antibodies to antigens

[0184] Further, the binding of the 8 purified antibodies to Human-CGRP / Mu-CGRP was detected by ELISA, and the specific steps were as follows:

[0185] (1) Coating: Dilute Human-CGRP, Mu-CGRP to 2 μg / mL with 1xPBS, add 30 μL per well, incubate at 4°C overnight.

[0186] (2) Washing and blocking: Wash the plate with PBST for 3 times, add 5% PBSM, 180 μL per well, block at room temperature for 2 hours.

[0187] (3) Incubation of primary antibody: Wash the plate with PBST for 3 times, dilute the antibody to be tested with 1% PBSM, 30 μL per well, incubate at room temperature for 60 minutes.

[0188] (4) Incubation of secondary antibody: Wash the plate with PBST for 3 times, add Anti-Human-IgG-Fc-HRP (Jackson Immuno, 109-035-008) diluted at 1:8000, 30 μL per well, incubate at room temperature for 60 minutes.

[0189] (5) Color development and reading: Wash the plate with PBST for 6 times, add TMB color developing solution, 30 μL per well, avoid light for 5-10 minutes, then add 2M H2SO4 stop solution, 30 μL per well, immediately read the absorbance (OD450) at 450 nm. 450 )。

[0190] The detection value is input into the GraphPad Prism software to calculate the affinity. The results show that the affinities of the 8 monoclonal antibodies to the antigen are very high, and they can exert their biological activity at a lower concentration. The 8 antibodies can be applied to immunodetection to assist in the diagnosis of diseases. The smaller the EC50 value, the higher the affinity. The affinities of the 8 monoclonal antibodies from high to low are CGRP-A037 > CGRP-A049 > CGRP-A193 > CGRP-A010 > CGRP-A069 > CGRP-A073 > CGRP-A184 > CGRP-A111, indicating that the antibody CGRP-A037 has the best binding activity to the antigen, followed by CGRP-A049. The results of the binding activity detection of the antibodies to the antigen are shown in Table 9, and the binding activity of the antibody CGRP-A037 to the antigen is shown in Table 9. Figure 6

[0191] Table 9 Detection results of the binding activity of CGRP antibodies to antigens

[0192]

[0193] 4. Evaluation of antibody specificity

[0194] Further, the specificity of the 8 antibodies screened is evaluated by using Negative-BSA / KLH and 5% PBSM as detection antigens. The specific method is as follows:

[0195] ​(1) Coating: ELISA plates were coated with NC-KLH, PBS-2% BSA blocking solution or PBS-5% PBSM blocking solution, respectively, 30 μL per well, 4°C overnight incubation.

[0196] (2) Blocking: PBST washing plates 3 times, 5% PBSM or 2% BSA was added, 180 μL per well, room temperature blocking for 2 hours

[0197] (3) Primary antibody incubation: PBST washing plates 3 times, the antibody to be tested was diluted with 1% PBSM, 30 μL per well, room temperature incubation for 60 minutes.

[0198] (4) Secondary antibody incubation: PBST washing plates 3 times, 1:8000 diluted Anti-Human-IgG-Fc-HRP (Jackson Immuno 109-035-008) was added, 30 μL per well, room temperature incubation for 60 minutes.

[0199] (5) Color development and reading: PBST washing plates 6 times, TMB color developing solution was added, 30 μL per well, avoid light reaction for 5-10 minutes, then 2M H2SO4 stop solution was added, 30 μL per well, immediately read the absorbance (OD450) at 450 nm. 450 )。

[0200] As shown in the results of Table 10, the OD450 values of the 8 antibodies were all less than 0.15, and there was no cross reaction with irrelevant proteins, indicating that the 8 antibodies all had high specificity.

[0201] Table 10 Specificity detection results of 8 antibodies

[0202]

[0203]

[0204] In summary, the two monoclonal antibodies CGRP-A037 and CGRP-A049 were preferred for subsequent experiments.

[0205] At the same time, the thermal stability Tm values of the CGRP-A037 and CGRP-A049 antibodies obtained by screening were determined by circular dichroism (CD) to evaluate the stability of the antibodies, and were compared with existing antibodies. The existing CGRP antibodies were purchased from Selleck, item number A2577. The detection results are shown in Table 11. The Tm represents the midpoint temperature of thermal denaturation of the protein, i.e. the temperature at which the protein unfolds by 50%, reflecting the trend of protein conformation change during temperature change, and the higher the Tm, the better the stability.

[0206] Table 11 Stability of CGRP antibodies

[0207] Antibody Tm (°C) CGRP-A037 61.7 CGRP-A049 60.2 Existing antibody 55.3

[0208] As shown in the above table data, the stability of the CGRP monoclonal antibody obtained by screening is obviously better than that of the commercially available CGRP antibody.

[0209] Example 3: Construction, expression and purification of anti-SP antibody

[0210] This example first designs primers according to the heavy chain variable region sequence and the light chain variable region sequence of the 26 lead molecules screened in Example 1 to prepare antibodies, and then screens the monoclonal antibodies with strong binding ability to the antigen from the 26 purified monoclonal antibodies by their binding ability to the antigen (Hu-SP and its conjugates), and evaluates the specificity of the antibodies. The preparation and purification process of the 26 SP monoclonal antibodies is basically the same as that of Example 2, and the detection process of the purity, affinity and specificity of the antibodies is as follows:

[0211] 1. Affinity evaluation of antibodies

[0212] The binding of 26 antibodies to Hu-SP and its conjugates was detected by ELISA, and the specific steps were as follows:

[0213] (1) Coating: Dilute Hu-SP, Hu-SP-BSA and Hu-SP-KLH to 2 μg / mL with 1xPBS, add 30 μL per well, and incubate overnight at 4°C.

[0214] (2) Washing and blocking: Wash the plate with PBST for 3 times, add 5% PBSM, 180 μL per well, and block at room temperature for 2 hours.

[0215] (3) Incubation of primary antibody: Wash the plate with PBST for 3 times, dilute the antibody to be tested with 1% PBSM, 30 μL per well, and incubate at room temperature for 60 minutes.

[0216] (4) Incubation of secondary antibody: Wash the plate with PBST for 3 times, add 1:8000 diluted Anti-Human-IgG-Fc-HRP (Jackson Immuno, 109-035-008), 30 μL per well, and incubate at room temperature for 60 minutes.

[0217] (5) Color development and reading: Wash the plate with PBST for 6 times, add TMB color developing solution 30 μL / well, avoid light, react for 5-10 minutes, then add 2M H2SO4 stopping solution 30 μL / well, immediately read the absorbance (OD 450 ) at 450 nm.

[0218] The detection value is input into the GraphPad Prism software to calculate the affinity, and the results are shown in Table 15.

[0219] Table 12 Detection results of SP antibody binding activity to antigen

[0220]

[0221]

[0222] SP-A011, SP-A035, SP-A037, SP-A080 and SP-A083 have good binding activity to Hu-SP antigen, and other antibodies have no binding or weak binding activity; to Hu-SP-BSA antigen, all antibodies have binding activity except SP-A102, and the EC50 is mostly between 0.003568 and 0.03339 μg / mL, and the affinity is high; to Hu-SP-KLH antigen, all antibodies have binding activity except SP-A102, and the EC50 is mostly between 0.003882 and 0.03652 μg / mL, and the affinity is high.

[0223] 2. Antibody specificity evaluation

[0224] Further, the specificity of the 8 antibodies screened is evaluated by using Negative-BSA / KLH and 5% PBSM as detection antigens, and the specific method is as follows:

[0225] (1) Coating: ELISA plates are coated with NC-KLH, PBS-2% BSA blocking solution or PBS-5% PBSM blocking solution, respectively, 30 μL per well, 4°C overnight incubation.

[0226] (2) Wash and block: wash the plate 3 times with PBST, add 5% PBSM or 2% BSA, 180 μL per well, room temperature blocking for 2 hours

[0227] (3) Primary antibody incubation: wash the plate 3 times with PBST, dilute the antibody to be tested with 1% PBSM, 30 μL per well, room temperature incubation for 60 minutes.

[0228] (4) Secondary antibody incubation: wash the plate 3 times with PBST, add 1:8000 diluted Anti-Human-IgG-Fc-HRP (Jackson Immuno 109-035-008), 30 μL per well, room temperature incubation for 60 minutes.

[0229] (5) Color development and reading: wash the plate 6 times with PBST, add TMB color developing liquid 30 μL per well, avoid light for 5-10 minutes, then add 2M H2SO4 stopping liquid 30 μL, immediately read the absorbance (OD 450 ) at 450 nm.

[0230] Table 13 Specificity detection results of 26 antibodies

[0231]

[0232]

[0233] As shown in the results of Table 13, only SP-A002, SP-A015, SP-A020, SP-A047, SP-A057, SP-A060 had non-specific binding (OD 450 >0.5) with NC-KLH, the rest of the antibodies had no binding with NC-KLH, 2% BSA, 5% PBSM

[0234] Therefore, therefore, the 5 high-affinity antibodies SP-A011, SP-A035, SP-A037, SP-A080, SP-A083 are preferred as candidate antibodies for further verification, and this embodiment uses sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SEC chromatography to analyze the purity of the 5 purified antibodies. The detection method is the same as the detection steps of the antibody purity in Example 2, and the detection results are shown in Table 14.

[0235] Table 14 Detection results of the purity of 5 antibodies

[0236]

[0237] As can be seen from the results of Table 16, the purity of the 5 antibodies is all above 95%, and the monomer purity is mostly above 90%, among which the monomer purity of SP-A035, SP-A080 is 100%, and the drugability is good.

[0238] The affinity and specificity of the 5 purified antibodies are verified again by ELISA, and the specific steps are the same as above, wherein the antigen coated in the affinity verification experiment is Hu-SP. The affinity detection results are shown in Table 18, and the specificity detection results are shown in Table 19.

[0239] Table 15 Detection results of the binding activity of 5 antibodies to antigens

[0240]

[0241]

[0242] Table 16 Specificity detection results of 5 antibodies

[0243] Antibody number NC-KLH 2% BSA 5% PBSM SP-A011 0.0716 0.1063 0.0976 SP-A035 0.0647 0.0998 0.082 SP-A037 0.0622 0.0672 0.0916 SP-A080 0.0609 0.0646 0.0769 SP-A083 0.0936 0.0922 0.0911 Mouse serum 1.1529 0.2246 0.1317 NC-Fc 0.0779 0.0635 0.0677 BC (1% PBSM) 0.069 0.0724 0.0792

[0244] As shown in the results, the 5 purified antibodies all have good binding activity to Hu-SP antigen, and have no non-specific binding with NC-KLH, 2% BSA, 5% PBSM. Among them, the EC50 value of SP-A080 monoclonal antibody is the lowest, indicating that its affinity is the best, and it is used as a preferred antibody for subsequent experiments.

[0245] Similarly, the thermal stability Tm value of the obtained SP-A080 antibody was determined by circular dichroism (CD) to evaluate the stability of the antibody, and compared with the existing antibody, wherein the existing SP antibody was purchased from Invitrogen, item number PA5-106886. The test results are shown in Table 17.

[0246] Table 17 Stability of SP antibody

[0247] Antibody Tm (°C) SP-A080 59.5 Existing antibody 54.8

[0248] As shown in the above table data, the stability of the screened SP monoclonal antibody is obviously better than that of the commercially available SP antibody.

[0249] Example 4: Combined detection of anti-CGRP and SP antibodies

[0250] In this embodiment, two candidate CGRP monoclonal antibodies CGRP-A037 and CGRP-A049 screened in Example 2 and the preferred SP monoclonal antibody SP-A080 screened in Example 3 were used to investigate the anti-viral infection ability of each antibody alone and the combination of the two antibodies. The test groups included:

[0251] 1. CGRP-A037 antibody alone (the amino acid sequence of the heavy chain is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain is shown in SEQ ID NO. 11);

[0252] 2. CGRP-A049 alone (the amino acid sequence of the heavy chain is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain is shown in SEQ ID NO. 16);

[0253] 3. SP-A080 antibody alone (the amino acid sequence of the heavy chain is shown in SEQ ID NO. 31, and the amino acid sequence of the light chain is shown in SEQ ID NO. 37);

[0254] 4. CGRP-A037 monoclonal antibody + SP-A080 monoclonal antibody;

[0255] 5. CGRP-A049 monoclonal antibody + SP-A080 monoclonal antibody;

[0256] The above 5 groups of antibodies were used for viral infection verification experiments.

[0257] I. Expression of macrophage interferon during viral infection

[0258] The macrophages were stimulated by adding CGRP (200 ng / mL) and SP (10 μM) at the same time. The control group of macrophages was added with PBS, and then the above-mentioned 5 groups of antibodies (10 μg / mL) and the corresponding volume of PBS (i.e. Vehicle) were added into the macrophage culture systems stimulated by CGRP & SP polypeptides and the control group of macrophage culture systems, respectively. After 12 hours, the above-mentioned macrophages were infected with VSV virus (MOI = 0.1), and the samples were collected after 6 hours or 12 hours. The expression levels of interferon Ifna and Ifnb of the macrophages and the contents of IFN-α and IFN-β were detected by RT-qPCR and ELISA.

[0259] Table 18 Expression fold of Ifna, Ifnb, IFN-α, IFN-β

[0260]

[0261] As shown in Table 18, the expression fold of Ifna, Ifnb, IFN-α and IFN-β of the macrophages treated with PBS were 174, 308, 292 and 306, respectively, after the virus interference. However, the expression levels of Ifna and Ifnb of the macrophages and the expression of IFN-α and IFN-β were significantly inhibited after the macrophages were treated with CGRP and SP at the same time. After adding a single antibody, the expression of interferon Ifna and Ifnb of the macrophages and the expression of inflammatory factors IFN-α and IFN-β were not restored, while the recovery effect was significantly improved when two antibodies were used in combination. The combination of CGRP-A037 monoclonal antibody and SP-A080 monoclonal antibody was better than the combination of CGRP-A049 monoclonal antibody and SP-A080 monoclonal antibody. The expression of Ifna, Ifnb, IFN-α and IFN-β was restored to the level comparable to the control, which could significantly block the response of CGRP and SP and improve the antiviral function of macrophages. The effects of CGRP-A037 monoclonal antibody, SP-A080 monoclonal antibody and the combination of CGRP-A037 monoclonal antibody + SP-A080 monoclonal antibody are shown in Table 18. Figure 7

[0262] II. RNA virus (VSV) infection

[0263] Further, the above-mentioned 5 groups of antibodies and PBS (i.e. the control group) were intraperitoneally injected into mice (10 mg / kg) at a time interval of one day. After two injections, the mice were infected with VSV virus (5 x 10 7 PFU g -1 ) through the tail vein. The survival status of the mice was observed and recorded, and the lung tissues of two groups of mice (2 days after virus infection) were fixed. The lung injury of the mice was detected by HE staining. The peripheral blood, liver and lung were taken, and the TCID -150 VSV viral load in serum, lung and liver was detected; RT-qPCR was used to detect the expression of Ifna, Ifnb, IL-6 and TNF in lung and liver tissues. The expression of Ifna, Ifnb, IL-6 and TNF in lung tissues is shown in Table 19.

[0264] Table 19 Expression fold of Ifna, Ifnb, IL-6 and TNF in lung tissues after RNA virus infection

[0265]

[0266] As Figure 8 and the results of Table 19 show that the use of CGRP-A037, CGRP-A049, SP-A080 antibodies alone cannot improve the survival rate of mice, reduce lung injury when infected with VSV virus, and the combination of CGRP-A037 monoclonal antibody + SP-A080 monoclonal antibody and CGRP-A049 monoclonal antibody + SP-A080 monoclonal antibody can improve the survival rate of mice, wherein the combination of CGRP-A037 monoclonal antibody + SP-A080 monoclonal antibody significantly improves the survival rate of mice, reduces lung injury, and reduces VSV viral load in mice. The higher the levels of interferons Ifna and Ifnb and inflammatory factors IL-6 and TNF, the better the body's ability to resist viral infection. According to the results, it can be seen that the combination of CGRP-A037 monoclonal antibody + SP-A080 monoclonal antibody significantly promotes the production of interferons Ifna and Ifnb and inflammatory factors IL-6 and TNF, which is better than the use of antibodies alone and the combination of CGRP-A049 monoclonal antibody + SP-A080 monoclonal antibody, and has obvious superior anti-VSV viral infection ability. The effect of the combination of CGRP-A037 monoclonal antibody + SP-A080 monoclonal antibody is shown in Figure 8 .

[0267] III. DNA virus (HSV-1) infection

[0268] Further, the above-mentioned 5 groups of antibodies and PBS (i.e. the control group) were injected intraperitoneally into mice (10 mg / kg), once every other day, and after two injections, HSV-1 virus (1 x 10 6 PFU g -1 ) was used to infect mice by retroinfusion through the tail vein. The survival status of mice was observed and recorded, and the injection of antibodies was continued, and the lung tissues of two groups of mice (7 days after viral infection) were fixed, and the lung injury of mice was detected by HE staining. The lung and liver were taken, and RT-qPCR was used to detect the expression of Ifna, Ifnb, IL-6 and TNF in lung and liver tissues. The expression of Ifna, Ifnb, IL-6 and TNF in lung tissues is shown in Table 20, and the effect of the combination of CGRP-A037 monoclonal antibody and SP-A080 monoclonal antibody is shown inFigure 9 As shown.

[0269] Table 20. Fold-up of expression of Ifna, Ifnb, IL-6, and TNF in lung tissue after DNA virus infection.

[0270]

[0271] As shown in Table 20 and Figure 9 The results showed that, compared with the control group, after injection of combined antibodies of CGRP-A037 and SP-A080 monoclonal antibodies and CGRP-A049 and SP-A080 monoclonal antibodies, the expression levels of IL-6, TNF, Ifna, and Ifnb in the serum and tissues of mice were significantly increased, lung injury was significantly alleviated, and the survival rate of mice was improved. This indicates that the use of combined antibodies improved the survival rate of mice infected with HSV-1 virus, reduced lung injury, promoted the production of interferon and inflammatory factors, and enhanced the body's ability to resist DNA virus infection. However, the use of antibodies alone did not significantly alleviate HSV-1 infection. The combined use of CGRP-A037 and SP-A080 monoclonal antibodies was more effective than that of CGRP-A049 and SP-A080 monoclonal antibodies, and had the best ability to resist HSV-1 virus infection.

[0272] In summary, the combination of CGRP-A037 monoclonal antibody and SP-A080 monoclonal antibody is preferred for improving the viral infectivity of tissues.

[0273] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

Claims

1. A kit for detecting CGRP and SP peptides, characterized in that, The antibodies include those containing CGRP peptides and SP peptides, wherein the CGRP peptide antibodies comprise: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.1, CDR2 composed of the amino acid sequence of SEQ ID NO.2, and CDR3 composed of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and (2) CDR1 composed of the amino acid sequence of SEQ ID NO.7, CDR2 composed of the amino acid sequence of SEQ ID NO.8, and CDR3 composed of the amino acid sequence of SEQ ID NO.9 in the variable region of the light chain; The antibody against the SP polypeptide includes: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.27, CDR2 composed of the amino acid sequence of SEQ ID NO.28, and CDR3 composed of the amino acid sequence of SEQ ID NO.29 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.33, CDR2 consisting of the amino acid sequence of SEQ ID NO.34, and CDR3 consisting of the amino acid sequence of SEQ ID NO.35 in the variable region of the light chain.

2. The kit according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody of the CGRP polypeptide is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10; the amino acid sequence of the heavy chain variable region of the antibody of the SP polypeptide is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

36.

3. The kit according to claim 2, characterized in that, The heavy chain amino acid sequence of the antibody of the CGRP polypeptide is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.11; the heavy chain amino acid sequence of the antibody of the SP polypeptide is shown in SEQ ID NO.31, and the light chain amino acid sequence is shown in SEQ ID NO.

37.

4. An antibody combination for predicting whether an individual is infected with a virus, characterized in that, The antibodies include those containing CGRP peptides and SP peptides, wherein the CGRP peptide antibodies comprise: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.1, CDR2 composed of the amino acid sequence of SEQ ID NO.2, and CDR3 composed of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and (2) CDR1 composed of the amino acid sequence of SEQ ID NO.7, CDR2 composed of the amino acid sequence of SEQ ID NO.8, and CDR3 composed of the amino acid sequence of SEQ ID NO.9 in the variable region of the light chain; The antibody against the SP polypeptide includes: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.27, CDR2 composed of the amino acid sequence of SEQ ID NO.28, and CDR3 composed of the amino acid sequence of SEQ ID NO.29 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.33, CDR2 consisting of the amino acid sequence of SEQ ID NO.34, and CDR3 consisting of the amino acid sequence of SEQ ID NO.35 in the variable region of the light chain.

5. The antibody combination as described in claim 4, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody of the CGRP polypeptide is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10; the amino acid sequence of the heavy chain variable region of the antibody of the SP polypeptide is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

36.

6. The use of an antibody combination in the preparation of a reagent for predicting whether an individual is infected with a virus, characterized in that, The antibody combination includes antibodies against CGRP peptides and antibodies against SP peptides, wherein the CGRP peptide antibody comprises: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.1, CDR2 composed of the amino acid sequence of SEQ ID NO.2, and CDR3 composed of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and (2) CDR1 composed of the amino acid sequence of SEQ ID NO.7, CDR2 composed of the amino acid sequence of SEQ ID NO.8, and CDR3 composed of the amino acid sequence of SEQ ID NO.9 in the variable region of the light chain; The antibody against the SP polypeptide includes: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.27, CDR2 composed of the amino acid sequence of SEQ ID NO.28, and CDR3 composed of the amino acid sequence of SEQ ID NO.29 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.33, CDR2 consisting of the amino acid sequence of SEQ ID NO.34, and CDR3 consisting of the amino acid sequence of SEQ ID NO.35 in the variable region of the light chain.

7. The use of an antibody combination in the preparation of a reagent to enhance the antiviral capacity of macrophages, characterized in that, The antibody combination includes antibodies against CGRP peptides and antibodies against SP peptides, wherein the CGRP peptide antibody comprises: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.1, CDR2 composed of the amino acid sequence of SEQ ID NO.2, and CDR3 composed of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and (2) CDR1 composed of the amino acid sequence of SEQ ID NO.7, CDR2 composed of the amino acid sequence of SEQ ID NO.8, and CDR3 composed of the amino acid sequence of SEQ ID NO.9 in the variable region of the light chain; The antibody against the SP polypeptide includes: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.27, CDR2 composed of the amino acid sequence of SEQ ID NO.28, and CDR3 composed of the amino acid sequence of SEQ ID NO.29 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.33, CDR2 consisting of the amino acid sequence of SEQ ID NO.34, and CDR3 consisting of the amino acid sequence of SEQ ID NO.35 in the variable region of the light chain.

8. The use as described in claim 7, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody of the CGRP polypeptide is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10; the amino acid sequence of the heavy chain variable region of the antibody of the SP polypeptide is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

36.

9. A system for predicting whether an individual is infected with a virus, characterized in that, The system includes a data analysis module for analyzing the detection value of the antigen. The antigen is detected by antibodies, which include antibodies against CGRP peptides and antibodies against SP peptides. The CGRP peptide antibody comprises: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.1, CDR2 composed of the amino acid sequence of SEQ ID NO.2, and CDR3 composed of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and (2) CDR1 composed of the amino acid sequence of SEQ ID NO.7, CDR2 composed of the amino acid sequence of SEQ ID NO.8, and CDR3 composed of the amino acid sequence of SEQ ID NO.9 in the variable region of the light chain; The antibody against the SP polypeptide includes: (1) CDR1 composed of the amino acid sequence of SEQ ID NO.27, CDR2 composed of the amino acid sequence of SEQ ID NO.28, and CDR3 composed of the amino acid sequence of SEQ ID NO.29 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.33, CDR2 consisting of the amino acid sequence of SEQ ID NO.34, and CDR3 consisting of the amino acid sequence of SEQ ID NO.35 in the variable region of the light chain.

10. The system as described in claim 9, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody of the CGRP polypeptide is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10; the amino acid sequence of the heavy chain variable region of the antibody of the SP polypeptide is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.36.

Images