Anti-gC1qcp antibody, preparation method and application thereof, and pharmaceutical composition

Abstract

The invention discloses an anti-gC1qcp antibody, a preparation method and application thereof, and a pharmaceutical composition, and belongs to the technical field of molecular biology. The invention also provides a preparation method. The anti-gC1qcp antibody provided by the invention is obtained by immunizing animals with gC1qcp molecules, and the amino acid sequence of the gC1qcp molecules is as shown in SEQ ID NO. 1. The antibody can specifically block the promoting effect of gC1qcp molecules on white spot syndrome virus replication, and white spot syndrome virus infection is effectively inhibited. By applying the antibody, the survival rate of prawns can be effectively increased, and economic loss caused by virus infection is reduced. Meanwhile, the antibody is high in specificity and safety, small in influence on other immune components of a host, low in side effect and suitable for long-term use.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an anti-gC1qcp antibody, a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Aquaculture, especially shrimp farming, is an important economic pillar in the global food supply chain. However, with the expansion of farming scale and the popularization of intensive farming methods, the outbreak of various diseases has become a major challenge in aquaculture, seriously affecting the production and health of aquatic animals. Among them, White Spot Syndrome (WSS) is a fatal disease caused by White Spot Syndrome Virus (WSSV), which has caused huge economic losses to the global shrimp farming industry. The virus can spread rapidly and cause high mortality. The lack of effective treatment has made the prevention and control of White Spot Syndrome a research focus in the field of aquatic disease prevention and control.

[0004] At present, the strategies for preventing and controlling white spot syndrome virus mainly include the use of antiviral chemical drugs to directly inhibit virus replication, vaccination to enhance resistance, and improving shrimp immunity through natural extracts, etc. However, these traditional methods have many limitations in effectiveness, specificity and safety, so there is an urgent need to develop new and efficient drugs for the prevention and control of white spot syndrome virus. Summary of the invention

[0005] In view of this, the present invention provides an anti-gC1qcp antibody and a preparation method and application thereof, and a pharmaceutical composition. The anti-gC1qcp antibody provided by the present invention has high specificity and low side effects, has no significant interference with the normal physiological processes of the host, can effectively block and inhibit the infection and spread of WSSV, is suitable for long-term use, and provides a sustainable safety prevention and control strategy for aquaculture.

[0006] In a first aspect, the present invention provides an anti-gC1qcp antibody obtained by immunizing an animal with a gC1qcp molecule, wherein the amino acid sequence of the gC1qcp molecule is shown in SEQ ID NO. 1.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned anti-gC1qcp antibody, comprising the following steps: Constructing a recombinant expression vector; the recombinant expression vector contains a nucleic acid molecule encoding a gC1qcp molecule; Transforming the recombinant expression vector into competent Escherichia coli cells to induce expression, thereby obtaining a recombinant gC1qcp molecule; The recombinant gC1qcp molecule is used as an antigen for animal immunization, and anti-gC1qcp antibodies are obtained through serum separation and purification.

[0008] In a third aspect, the present invention provides an application of the anti-gC1qcp antibody described in the first aspect or the anti-gC1qcp antibody prepared by the preparation method described in the second aspect, wherein the application is an application in preparing a drug for preventing and treating white spot syndrome in aquatic animals.

[0009] In a fourth aspect, the present invention provides a pharmaceutical composition, comprising the anti-gC1qcp antibody described in the first aspect or the anti-gC1qcp antibody prepared by the preparation method described in the second aspect, and a pharmaceutically acceptable carrier.

[0010] Compared with the prior art, the present invention has achieved the following beneficial effects: The present invention finds that the white spot syndrome virus interacts with the gC1qcp molecule through certain pathways, which may escape immune surveillance and accelerate its spread in the host. Therefore, the present invention provides an anti-gC1qcp antibody, which can specifically block the promotion of the gC1qcp molecule on the replication of the white spot syndrome virus and effectively inhibit the infection of the white spot syndrome virus. The application of the antibody can effectively improve the survival rate of shrimp and reduce the economic losses caused by viral infection. At the same time, the antibody has high specificity and safety, has little effect on other immune components of the host, has low side effects, and is suitable for long-term use. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0012] Figure 1 is the protein electrophoresis diagram of the anti-gC1qcp antibody of Example 1 of the present invention; Figure 2 is the specific recognition map of the anti-gC1qcp antibody of Example 1 of the present invention; Figure 3 is a graph of the relative expression of VP28 protein in the anti-gC1qcp antibody treatment group and the control antibody treatment group in Example 2 of the present invention, with β-actin as the internal reference protein; Figure 4is a graph of VP28 mRNA expression levels in the anti-gC1qcp antibody-treated group and the control antibody-treated group in Example 2 of the present invention; Figure 5 is a graph showing the number of WSSV copies in shrimps of the anti-gC1qcp antibody treatment group and the control antibody treatment group in Example 2 of the present invention; Figure 6 is a comparison chart of the survival rates of shrimps in the anti-gC1qcp antibody treatment group and the control antibody treatment group under WSSV infection in Example 3 of the present invention; Figure 7 3 are the hepatopancreatic cell morphology images of the shrimp in the untreated group, the control antibody treated group and the anti-gC1qcp antibody treated group of Example 3 of the present invention, wherein (a) is the hepatopancreatic cell morphology image of the shrimp in the control antibody treated group, (b) is the hepatopancreatic cell morphology image of the shrimp in the control antibody treated group 48 hours after WSSV infection, and (c) is the hepatopancreatic cell morphology image of the shrimp in the anti-gC1qcp antibody treated group 48 hours after WSSV infection; Figure 8 is a comparison chart of the survival rates of shrimps in the anti-gC1qcp antibody treated group and the untreated group in Example 4 of the present invention within 7 days; Fig. 9 3 are the morphological diagrams of hepatopancreatic cells of shrimp in the untreated group and the anti-gC1qcp antibody-treated group in Example 4 of the present invention, wherein (a) is the morphological diagram of hepatopancreatic cells of shrimp in the untreated group, and (b) is the morphological diagram of hepatopancreatic cells of shrimp after 48 hours of anti-gC1qcp antibody treatment. DETAILED DESCRIPTION

[0013] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0014] The present invention provides an anti-gC1qcp antibody, which is obtained by immunizing an animal with a gC1qcp molecule, and the amino acid sequence of the gC1qcp molecule is shown in SEQ ID NO. 1.

[0015] SEQ ID NO. 1: QGDRAAQSGGFQIAQAVHAAPPEYRCSGGFSVRKATSATSTTVRNSGTTRLRFQDVLTGEGGWSPSESDFLAPCRGLYYFSFHGVAQDRGDFTLALVKNGQYQVTAYGGKGNYLQGSNSALLLLDQGDLVHLELQQGSLYEHPSNEAYTSFTGFLVEAF.

[0016] The gC1qcp molecule described in the present invention is numbered LOC122244551 in the database of the National Center for Biotechnology Information (NCBI) of the United States. The gC1qcp molecule is a molecule containing a gC1q domain in shrimp. The present invention has found in research that the gC1qcp molecule may interact with the white spot syndrome virus through certain pathways, thereby allowing the white spot syndrome virus (WSSV) to escape immune surveillance and accelerate its spread in the host, so it has become one of the key targets for the prevention and treatment of white spot syndrome. Targeted intervention of the gC1qcp molecule is expected to block and inhibit the infection and spread of WSSV.

[0017] The anti-gC1qcp antibody of the present invention can specifically recognize and bind to the gC1qcp molecule, effectively blocking its promoting effect on the white spot syndrome virus, thereby reducing the susceptibility of shrimp, inhibiting the occurrence and spread of viral infection, providing effective immune protection for aquatic animals, and reducing the occurrence of viral infection. Compared with traditional prevention and control methods, the anti-gC1qcp antibody provided by the present invention has high specificity and low side effects, has no significant interference with the normal physiological process of the host, is suitable for long-term use, and provides a sustainable safety prevention and control strategy for aquaculture.

[0018] The present invention also provides a method for preparing the above-mentioned anti-gC1qcp antibody, comprising the following steps: Constructing a recombinant expression vector; the recombinant expression vector contains a nucleic acid molecule encoding a gC1qcp molecule; Transforming the recombinant expression vector into competent Escherichia coli cells to induce expression, thereby obtaining a recombinant gC1qcp molecule; The recombinant gC1qcp molecule is used as an antigen for animal immunization, and anti-gC1qcp antibodies are obtained through serum separation and purification.

[0019] In the present invention, the nucleic acid molecule encoding the gC1qcp molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA (heterogeneous nuclear RNA), etc. Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2: .

[0020] The present invention has no particular limitation on the type of E. coli competent cells used, as long as they are suitable for the effective expression of the recombinant expression vector in the cells. Preferably, in order to obtain a better expression effect, the E. coli competent cells are E. coli Rosetta (DE3) competent cells.

[0021] In the step of using the recombinant gC1qcp molecule as an antigen for animal immunization described in the present invention, the number of immunizations is 2 to 4 times, specifically: first, the recombinant gC1qcp molecule is mixed with a complete adjuvant for the first animal immunization, and then the recombinant gC1qcp molecule is mixed with an incomplete adjuvant for the second to fourth animal immunization. In the present invention, the immunization dose used when immunizing animals and the mixing ratio with the adjuvant can be carried out according to conventional methods in the art. The adjuvants described in the present invention include but are not limited to Freund's adjuvant, Montemi adjuvant or PLGA (polylactic acid-co-glycolic acid) microspheres, chitosan nanoparticles, etc., which can be reasonably selected according to the type of antigen, the required antibody titer, etc., and the present invention is not particularly limited here.

[0022] In the present invention, the immunized animals include but are not limited to New Zealand white rabbits, mice and rats. In a specific embodiment of the present invention, the immunized animals are selected from New Zealand white rabbits.

[0023] The present invention does not impose any particular limitation on the separation and purification method, and any separation and purification method commonly used by those skilled in the art may be used, such as affinity chromatography or ammonium sulfate precipitation. The present invention preferably uses affinity chromatography for separation and purification.

[0024] The present invention also provides the use of the anti-gC1qcp antibody or the anti-gC1qcp antibody prepared by the above preparation method, wherein the use is the use in preparing a drug for preventing and treating white spot syndrome in aquatic animals.

[0025] Furthermore, the medicine includes one or more of the following uses: (1) Specific binding to aquatic animal gC1qcp molecules; (2) Inhibit the replication of white spot syndrome virus in aquatic animals; (3) Improve the survival rate of aquatic animals infected with white spot syndrome virus; (4) Alleviate tissue damage caused by white spot syndrome virus infection in aquatic animals.

[0026] In the present invention, the aquatic animal is a crustacean aquatic animal, more preferably a prawn. In a specific embodiment of the present invention, the prawn is selected from Japanese mackerel.

[0027] The present invention also provides a pharmaceutical composition, comprising the anti-gC1qcp antibody or the anti-gC1qcp antibody prepared by the above preparation method, and a pharmaceutically acceptable carrier.

[0028] In the present invention, "pharmaceutically acceptable" ingredients are substances that are suitable for aquatic animals without excessive adverse side effects (such as toxicity, irritation, etc.), that is, substances with a reasonable benefit / risk ratio. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0029] In the present invention, the dosage form of the pharmaceutical composition is an injection dosage form or an oral dosage form, and can be applied to aquatic animals by direct injection, feed addition, etc. The present invention does not impose any special restrictions on the specific form of the pharmaceutical composition, for example, it can be a powder, a soluble powder, a granular premix, a composite preparation, microcapsule embedding, etc., and those skilled in the art can select according to actual needs.

[0030] In the present invention, the dosage of the anti-gC1qcp antibody in aquatic animals is preferably 4.5-5.5 µg / g body weight, at which a good anti-leukoplakia syndrome effect can be exerted.

[0031] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0032] Example 1 This example provides a process for preparing anti-gC1qcp antibodies.

[0033] The amino acid sequence of the gC1qcp molecule is shown in SEQ ID NO. 1, and its ID number in the National Center for Biotechnology Information (NCBI) database is LOC122244551.

[0034] TRIzol reagent was used to extract RNA from the intestinal tissue of Japanese shrimp, and the RNA was reverse transcribed into cDNA using the Novozyme reverse transcription kit (R323-01). Expression primers were then designed based on the nucleotide sequence encoding the gC1qcp molecule as shown in SEQ ID NO.2: Upstream primer F1: 5′-GCCATGGCTGATATCGGATCCCAGGGCGATCGCGCAGCC-3′ (SEQ ID NO. 3); Downstream primer R1: 5′-GTGGTGGTGGTGGTGCTCGAGAAAGGCCTCGACGAGGAATC-3′ (SEQ ID NO. 4).

[0035] After amplifying the sequence encoding the mature peptide of the protein using the above primers and recovering the gel, the pET-30a(+) plasmid was double-digested with BamHI and XhoI, and the target sequence was connected to the plasmid by homologous recombination. The recombinant vector was transformed into Rosetta(DE3) Escherichia coli and induced for expression at 28°C for 6 hours with 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside). 2+ ) to separate and purify the recombinant gC1qcp molecule by its strong affinity for the 6XHis tag carried on the recombinantly expressed gC1qcp molecule.

[0036] After the obtained recombinant gC1qcp molecule solution (1 mg / mL, 1 mL) was fully emulsified with an equal volume of Freund's complete adjuvant (F5881, purchased from Sigma-Aldrich, USA), the mixture was subcutaneously injected into New Zealand white rabbits for the first immunization. After 25 days, the complete adjuvant was replaced with an incomplete adjuvant (F5506, purchased from Sigma-Aldrich, USA) for the second immunization. After 7 days, the rabbit antiserum was collected and diluted with a buffer (0.15 M NaCl, 20 mM NaHPO4, pH=8.0) and then filtered through a 0.45 μm filter. Next, the antibodies in the serum were adsorbed using a 2 mL Protein A resin column (L00210, purchased from GenScript Biotech) and thoroughly washed with dilution buffer. Finally, the antibody on the resin was eluted with an elution buffer (0.1 M glycine, pH = 2.5), and the resulting antibody was neutralized with a 1 / 10 volume of a buffer (1 M Tris-HCl, pH = 8.5). The antibody was dialyzed overnight in a PBS buffer at 4°C to obtain the anti-gC1qcp antibody (polyclonal antibody), as shown in FIG. Figure 1 As shown in the figure, the anti-gC1qcp antibody includes two domains, heavy chain and light chain. Under protein electrophoresis, the two will be separated. The sizes of the heavy chain and light chain are around 55kDa and 30kDa respectively, which is consistent with the general size of antibodies. Western blot was used to verify that it can specifically recognize the gC1qcp molecule of shrimp, such as Figure 2 shown.

[0037] Example 2 This example provides an experiment on the inhibition of WSSV replication in shrimp by the anti-gC1qcp antibody of Example 1.

[0038] Experimental animal treatment: The shrimp were randomly divided into an experimental group and a control group, with at least 4 shrimps in each group. The experimental group was injected with the anti-gC1qcp antibody (50 µg / shrimp), and the control group was injected with an equal amount of nonspecific IgG (antibody that does not recognize any shrimp protein) as a negative control. 24 h after antibody treatment, the experimental group and the control group were injected with an equal amount of WSSV (1×10 5 virus particles / prawn).

[0039] 48 h after WSSV infection, hemolymph tissues of shrimp were collected and analyzed by Western blot ( Figure 2 ) and qPCR ( Figure 3 , Figure 4 ) to detect the level of WSSV in shrimp. The results showed that the WSSV level in the anti-gC1qcp antibody treatment group was significantly lower than that in the control group, which was specifically manifested in the following three aspects: (1) Western blot detection revealed that Figure 3 As shown, the expression level of WSSV specific envelope protein VP28 was significantly lower than that of the control antibody group after treatment with anti-gC1qcp antibody, and β-actin was used as the internal reference protein.

[0040] (2) qPCR detection using specific primers for VP28, such as Figure 4 As shown, the results showed that the VP28 mRNA level in the anti-gC1qcp antibody-treated group was significantly lower than that in the control group.

[0041] (3) At the genomic level, qPCR was performed using VP28-specific primers to detect the copy number of WSSV, such as Figure 5 As shown, the results showed that the WSSV load in the anti-gC1qcp antibody-treated group was much lower than that in the control group.

[0042] Based on the above three points, it can be concluded that anti-gC1qcp antibodies can effectively inhibit the replication of WSSV in shrimp.

[0043] Example 3 This example provides an experiment on using the anti-gC1qcp antibody of Example 1 to improve the survival rate of shrimp infected with WSSV and reduce tissue damage.

[0044] The experimental animals were treated as in Example 2, and the survival rate of the shrimps was recorded after treatment (30 shrimps in each group, and the shrimps lived in constant temperature and oxygen-rich seawater). Figure 6 As shown in the results, anti-gC1qcp antibody treatment significantly increased the survival rate of shrimp after WSSV infection. In addition, through the HE (hematoxylin-eosin) staining of hepatopancreatic tissue, it was found that compared with the control antibody treatment group, the hepatopancreatic damage of shrimp in the anti-gC1qcp antibody treatment group was effectively alleviated, and the hepatopancreatic tubule structure was still well preserved after WSSV infection (such as Figure 7 As shown by the yellow arrow in the middle, the untreated group shows the hepatopancreatic cell morphology of healthy shrimp).

[0045] Example 4 This example provides a safety assessment of the anti-gC1qcp antibody of Example 1.

[0046] To verify the safety of anti-gC1qcp antibodies in healthy shrimp and whether they have potential toxicity, healthy shrimp were randomly divided into two groups, 30 in each group. One group was injected with anti-gC1qcp antibodies (50 µg / shrimp, the same dose as in Examples 2 and 3), and the other group was not treated as a control group. The shrimp were raised in constant temperature and oxygen-sufficient seawater, and their survival rate was recorded. Figure 8As shown in the results, the survival rate of the anti-gC1qcp antibody-treated group was not significantly different from that of the untreated group at this dose, and did not lead to a significant increase in shrimp mortality. In addition, HE staining of hepatopancreas tissue was observed, such as Fig. 9 As shown, after 48 hours of treatment with anti-gC1qcp antibody, no hepatopancreatic damage was caused, and the hepatopancreatic tubule structure remained intact, with no significant difference from the untreated group. Combining the results of Example 2 and Example 3, it can be concluded that the anti-gC1qcp antibody in the present invention is a safe and effective product for the prevention and treatment of white spot syndrome. The anti-white spot syndrome drug prepared using the antibody provides an efficient and safe prevention and control strategy for the white spot syndrome that is widely present in shrimp farming, and has broad application prospects and significant economic benefits.

[0047] It is worth noting that the dosage of the anti-gC1qcp antibody in this example is 5 µg / g body weight, which has shown a good anti-white spot syndrome effect. Given that the antibody is an exogenous substance to shrimp, it is recommended to use a preferred dosage of 5 µg / g body weight in actual production, as excessive dosage may have potential toxic effects on shrimp.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-gC1qcp antibody, characterized in that It is obtained by immunizing animals with a gC1qcp molecule; the amino acid sequence of the gC1qcp molecule is shown in SEQ ID NO.

1.

2. The method for preparing an anti-gC1qcp antibody according to claim 1, characterized in that: The steps include: Constructing a recombinant expression vector; the recombinant expression vector contains a nucleic acid molecule encoding a gC1qcp molecule; Transforming the recombinant expression vector into competent Escherichia coli cells to induce expression, thereby obtaining a recombinant gC1qcp molecule; The recombinant gC1qcp molecule is used as an antigen for animal immunization, and anti-gC1qcp antibodies are obtained through serum separation and purification.

3. The preparation method according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule encoding the gC1qcp molecule is shown in SEQ ID NO.

2.

4. The preparation method according to claim 2, characterized in that: In the step of using the recombinant gC1qcp molecule as an antigen for animal immunization, the number of immunizations is 2 to 4 times.

5. The preparation method according to claim 4, characterized in that: The specific steps of using the recombinant gC1qcp molecule as an antigen for animal immunization are as follows: first, the recombinant gC1qcp molecule is mixed with a complete adjuvant for the first animal immunization, and then the recombinant gC1qcp molecule is mixed with an incomplete adjuvant for the second to fourth animal immunization.

6. Use of the anti-gC1qcp antibody according to claim 1 or the anti-gC1qcp antibody prepared by the preparation method according to any one of claims 2 to 5, characterized in that: The application is application in preparing medicine for preventing and treating white spot syndrome in aquatic animals.

7. The use according to claim 6, characterized in that The medicine includes one or more of the following uses: (1) Specific binding to aquatic animal gC1qcp molecules; (2) Inhibit the replication of white spot syndrome virus in aquatic animals; (3) Improve the survival rate of aquatic animals infected with white spot syndrome virus; (4) Alleviate tissue damage caused by white spot syndrome virus infection in aquatic animals.

8. The use according to claim 7, characterized in that The aquatic animal is a prawn.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-gC1qcp antibody according to claim 1 or the anti-gC1qcp antibody prepared by the preparation method according to any one of claims 2 to 5, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that The dosage form of the pharmaceutical composition is an injection form or an oral dosage form.

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