Avian leukosis virus poly-subtype antigen epitope fusion protein and application thereof

Abstract

The application provides an avian leukosis virus multi-subtype antigen epitope fusion protein, a multi-subtype recognition antibody, a detection kit and application thereof. The amino acid sequence of the avian leukosis virus multi-subtype antigen epitope fusion protein is shown as SEQ ID NO:1. Experiments show that the avian leukosis virus multi-subtype antigen epitope fusion protein is reactive to avian leukosis virus multi-subtype serum and is not reactive to other common chicken infectious virus or bacterial serum. The avian leukosis virus multi-subtype antigen epitope fusion protein can be used for preparing multi-subtype recognition antibodies by immunizing animals. The multi-subtype recognition antibody can be specifically recognized with multi-subgroup avian leukosis specific antigen epitopes, a detection kit is developed, and the detection kit is used for rapid screening and purification of chicken population avian leukosis virus, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparations, specifically an avian leukosis virus antigen epitope fusion protein and its application. Background Technology

[0002] Avian leukosis is a collective term for various neoplastic diseases in poultry caused by avian leukosisvirus (ALV), a member of the genus Alpha Retrovirus in the family Retroviridae. Based on the specificity of the envelope glycoproteins, host range, and cross-linking of ALV, it can be divided into 11 subgroups (AK), among which subgroups AJ and K are primarily pathogenic to chickens. Subgroups A, B, and J are exogenous viruses occurring in field chickens. Subtype J ALV can spread horizontally and vertically; vertical transmission can lead to immune tolerance in offspring, resulting in negative antibody tests. Simultaneously, affected chickens develop acute tumors leading to death, requiring immediate treatment upon diagnosis. Subtype AB ALV mainly affects chick quality through vertical transmission, causing slow weight gain and reduced egg production in infected chickens. Subtype K virus has been found in different chicken flocks, indicating an increasing trend in K subgroup infection, potentially becoming a new major potential threat to my country's poultry industry after subgroup J ALV.

[0003] This disease can be transmitted both horizontally and vertically. Congenitally infected, immune-tolerant chickens are the most important source of infection, and cross-contamination during mating is a major pathway for horizontal transmission among flocks. There is no available vaccine; flock eradication is currently the only way to prevent infection. Although existing technologies for detecting specific subgroups of ALV are relatively mature and can distinguish between different subgroups, this distinction is not very meaningful during flock eradication. Even if the test results show the absence of a particular subgroup, infection of other subgroups cannot be ruled out. Therefore, there is an urgent need for a detection method for ALV that does not require distinguishing specific subtypes, but rather provides a quick and accurate veto result for rapid screening and eradication of the chicken flock. Summary of the Invention

[0004] The purpose of this invention is to provide a fusion protein containing multiple subtype antigenic epitopes of avian leukosis virus, wherein the fusion protein contains specific recognition antigenic epitopes of subgroups A, B, J, and K, which can simultaneously achieve rapid screening of common ALVs.

[0005] The amino acid sequence of the fusion protein described in this application is RKLLVSCLKLGGGSSPRSDWSIDTLSRRYCGNEFTSARGGSSDILKSRTILANSGICFQDCLSRTGGSQSREINETEPFS FMNLVLCVTGEV (SEQ ID NO.1).

[0006] Furthermore, the present invention provides a DNA molecule encoding the aforementioned fusion protein. Due to the degeneracy of codons, many nucleotide sequences can exist that can encode the specific antigenic epitopes described in this invention.

[0007] The present invention also provides a recombinant expression vector containing the DNA molecule described herein.

[0008] On the other hand, the present invention also provides an engineered bacterium containing the recombinant expression vector described in the present invention.

[0009] In this invention, the "engineered bacteria" are obtained by heat-shock conversion of a recombinant expression vector into a host cell. The "host cell" expressed in this invention includes prokaryotic or eukaryotic cells.

[0010] The host cell is selected from Escherichia coli, yeast, insect or mammalian cells.

[0011] The present invention also claims protection for multi-subtype recognition antibodies against avian leukosis virus, which are monoclonal or polyclonal antibodies prepared by immunizing animals with the fusion protein.

[0012] The multi-subtype recognition antibody described in this invention can specifically recognize avian leukosis of subgroups A, B, J, and K, and does not cross-react with other common chicken viruses or bacteria.

[0013] Furthermore, the present invention provides a detection kit for rapid purification of chicken flocks.

[0014] Furthermore, the test kit contains:

[0015] (1) The avian leukosis virus multi-subtype antigenic epitope fusion protein of the present invention;

[0016] and / or

[0017] (2) The multi-subtype recognition antibody described in this invention.

[0018] Preferably, the multi-subtype recognition antibody can be a monoclonal antibody or a polyclonal antibody.

[0019] Preferably, the kit described in this invention also includes an enzyme-labeled plate.

[0020] Those skilled in the art will understand that the kit provided by the present invention can immobilize the multi-subtype antigenic epitope fusion protein of avian leukosis virus onto an enzyme-linked immunosorbent assay (ELISA) plate for the detection of avian leukosis virus antibodies, or can coat the antibody onto an ELISA plate for the detection of avian leukosis virus.

[0021] Furthermore, as a preferred embodiment, the kit of the present invention further includes a coating solution, a blocking solution, a secondary antibody, a chromogenic solution, and a stop solution.

[0022] Furthermore, the present invention provides an application of avian leukosis virus multi-subtype antigenic epitope fusion protein, multi-subtype recognition antibody, and detection kit, wherein the application is for rapid screening and purification of avian leukosis virus in chicken populations.

[0023] Preferably, the rapid screening and purification of avian leukosis virus in chicken flocks is for non-diagnostic and non-therapeutic purposes.

[0024] Beneficial effects

[0025] This invention provides a multi-subtype epitope fusion protein of avian leukosis virus, multi-subtype recognition antibodies, a detection kit, and their applications. The amino acid sequence of the avian leukosis virus multi-subtype epitope fusion protein described in this invention is shown in SEQ ID NO: 1. Experiments show that this avian leukosis virus multi-subtype epitope fusion protein is reactive to avian leukosis virus multi-subtype sera but unreactive to sera from other common chicken infection viruses or bacteria. Immunizing animals with the avian leukosis virus multi-subtype epitope fusion protein of this invention can prepare multi-subtype recognition antibodies. The multi-subtype recognition antibodies of this invention can specifically recognize avian leukosis-specific epitopes of multiple subgroups. Developing a detection kit for rapid screening and purification of avian leukosis virus in chicken populations has promising application prospects. Attached Figure Description

[0026] Figure 1 This invention provides a high-order structural prediction of the avian leukosis virus multi-subtype antigenic epitope fusion protein.

[0027] Figure 2 To detect the specificity and sensitivity of antigenic epitopes. Detailed Implementation

[0028] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0029] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0030] Example 1: Screening of antigen epitope fusion peptides

[0031] First, the amino acid sequences of the gp85 and p27 proteins of ALV-A, ALV-B, ALV-J, and ALV-K viruses, which are prevalent in my country in recent years, were preliminarily analyzed using ClustalX2 combined with MEGA 5.1 software, resulting in the identification of 12 candidate polypeptide sequences. Then, candidate antigen fragment regions were screened using bioanalytical methods, and some amino acid residues were replaced or optimized based on bioinformatics analysis. Finally, the higher-order spatial structure was fine-tuned using flexible linkers or direct ligation (see [link to relevant documentation]). Figure 1 The amino acid sequence of the antigen epitope fusion peptide was ultimately determined to be RKLLVSCLKLGGGSSPRSDWSIDTLSRRYCGNEFTSARGGSSDILKSRTILANSGICFQDCLSRTGGSQSREINETEPFS FMNLVLCVTGEV (SEQ ID NO.1). It was synthesized using a peptide solid-phase synthesizer and then preliminarily purified by desalting (BGI Genomics).

[0032] An antigen epitope fusion peptide expression cassette was constructed using overlap PCR. The cassette was then ligated into PET-28a (purchased from Invitrogen, catalog number A11499) using double enzyme digestion. Single clones were selected to identify the insertion orientation. Plasmids with correct insertion orientation were sent to Invitrogen for sequencing. The correctly sequenced plasmid was named PET-28a-ALV. The PET-28a-ALV recombinant positive plasmid was expressed, and after verification by SDS-PAGE, the soluble fusion protein was purified by nickel column chromatography.

[0033] Example 2: Specificity and sensitivity detection of antigenic epitopes

[0034] The selected fusion proteins were identified using positive sera from ALV subgroups A, B, J, and K, Newcastle disease virus (NDV), avian influenza virus (AIV) H5 subtype, infectious bursal disease virus (IBDV), reticuloendotheliosis virus (REV), Eimeria tenella, and Vibrio cholerae. The fusion protein efficacy was detected using an indirect ElISA method. The specific detection method was as follows: The synthesized fusion protein was resuspended in DMSO to 20 mg / ml, diluted to 20 μg / ml with pH 9.6 carbonate buffer (coating solution), and coated onto CORNING ELISA plates. The plates were incubated overnight at 4°C, with the uncoated group serving as a control for positive / negative determination. The plates were then blocked with 2% skim milk at 37°C for 2 hours. Each well was washed with 300 μl PBST, and the plates were shaken dry after each wash, then air-dried on clean absorbent paper. The washing process was repeated 5 times. 100 μl / well of positive serum or negative control for various viruses / bacteria, diluted 50-fold, 250-fold, and 1000-fold, incubated at 37°C for 1 hour, and washed 5 times; 100 μl / well of horseradish peroxidase-labeled goat anti-chicken IgY (IgG) enzyme-labeled secondary antibody diluted 1:5000, incubated at 37°C for 1 hour, and washed 5 times; 100 μl / well of TMB chromogenic reagent, allowed to stand for 10-15 minutes, and 50 μl of stop solution were added before detecting the D450 value using an ELISA reader. The result was judged as positive if OD450 > the mean of the standard negative samples + 2 × the standard deviation of the standard negative samples. Statistically, this judgment result has a 95% confidence interval.

[0035] The results showed that the avian leukosis virus multi-subtype epitope fusion protein obtained by screening in this invention had significant positive reactions to ALV subgroups A, B, J, and K, but showed virtually no reaction to positive sera from Newcastle disease virus (NDV), avian influenza virus (AIV) H5 subtype, infectious bursal disease virus (IBDV), reticuloendotheliosis virus (REV), Eimeria tenella, and Vibrio cholerae. These results confirm that the avian leukosis virus multi-subtype epitope fusion protein obtained by screening in this invention has high specificity and sensitivity. Figure 2 )

[0036] Example 3: Preparation of specific antibodies against avian leukosis virus multi-subtype antigenic epitope fusion protein

[0037] Healthy female large-eared white rabbits weighing approximately 2 kg (purchased from the Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army; rabbits with natural antibodies were culled before the experiment) were selected. The avian leukosis virus multi-subtype epitope fusion protein obtained in Example 1 was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple sites. Supplementary injections of the aqueous immunizing agent were administered on days 20, 30, and 40 after the initial immunization, using the same dosage and route as the initial immunization. Blood samples were collected 10 days after the final immunization, and serum titers were determined using a slide agglutination test. Titers greater than 1:640 were obtained. Blood was collected from the carotid artery and centrifuged to collect the antiserum. The specific antibodies against the avian leukosis virus multi-subtype epitope fusion protein in the antiserum were purified using a conventional saturated ammonium sulfate precipitation method (33% saturated ammonium sulfate precipitation three times, followed by dialysis desalting and antibody collection). The antibody concentration was determined using a UV spectrophotometer. The method involved appropriately diluting the purified antibody and measuring the A value at 280 nm and 260 nm wavelengths. The protein concentration was then calculated using the formula: Protein content (mg / mL) = (1.45 × A280nm - 0.74 × A260nm) × dilution factor. The result showed that the antibody protein concentration obtained by the above method was approximately 10 mg / mL.

[0038] Example 4: Detection of antibody specificity

[0039] Using an indirect ELISA method, gp85 protein of ALV subgroups A, B, J, and K, HN antigen of Newcastle disease virus (NDV), NS1 non-structural protein of avian influenza virus (AIV) H5 subtype, VP2 antigen of infectious bursal disease virus (IBDV), envelope protein of chicken reticuloendotheliosis virus (REV), lysate of Eimeria tenella, and Vibrio cholerae culture medium were coated at 100 ng / well. The antibodies against these multiple subgroups were detected (Table 1). The results showed that the above antibodies were positive for ALV subgroups A, B, J, and K, but negative for other viruses, bacteria, or parasites. The antibody was identified as a multi-subtype specific antibody against avian leukosis virus.

[0040] Table 1. Results of antibody type specificity detection

[0041] ALV-A +++ NDV - Chicken tender Eimeria coccidia - ALV-B +++ AIV - Vibrio cholerae - ALV-J ++ IBDV - ALV-K +++ REV -

[0042] Example 5: Identification of neutralizing activity and stability of specific antibodies

[0043] The assay was validated in the laboratory using a microcell neutralization test. The antibodies obtained from the screening were serially diluted with physiological saline from 1:2 to 1:128. One sample of immune serum was used as a positive control and also serially diluted from 1:2 to 1:128, with five replicates for each dilution. 100 mg TCID₂ was added to each well of the culture plate containing the diluted test sample. 50 The virus solution was prepared, and serial dilution wells for negative serum controls were also included, with an equal volume of diluent added. All culture plates were placed in a 37°C cell culture incubator for 2 hours for neutralization. After neutralization, Vero cell suspension was added to each well, and the plates were incubated at 37°C for 5 days. Normal cell controls were also included. After 5 days, the presence of cytopathic effects was observed and counted in each well, indicating that the plate could protect 50% of the cells from 100 TCID50. 50 The highest dilution of the virus-infected serum was used to determine the antibody titer, and the entire test was repeated three times. The final antibody titer was 1:64, and the neutralizing titer of the positive control serum was 1:32 (≥1:4 was considered positive). The neutralizing activity test results showed that the above antibody has a high neutralizing titer (1:64), and the coefficient of variation of the three repeated tests was <3%, indicating that the antibody has good stability. This provides a good foundation for the next step of antibody identification.

[0044] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A fusion protein comprising multiple subtype antigenic epitopes of avian leukosis virus, wherein the fusion protein contains specific recognition antigenic epitopes of subgroups A, B, J, and K, enabling rapid screening of common ALVs simultaneously, characterized in that, The amino acid sequence of the multi-subtype antigenic epitope fusion protein is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the multi-subtype antigenic epitope fusion protein as described in claim 1.

3. A recombinant expression vector, characterized in that... The recombinant expression vector contains the DNA molecule as described in claim 2.

4. An engineered bacterium, characterized in that... The engineered bacteria contain the recombinant expression vector as described in claim 3.

5. A test kit, characterized in that... The test kit contains the avian leukosis virus multi-subtype antigenic epitope fusion protein as described in claim 1.

Images