CN116284352A - Bovine leukemia virus antibody and detection kit - Google Patents

Abstract

The invention relates to a bovine leukemia virus BLV P24 monoclonal antibody and an active fragment thereof, which target a P24 protein main neutralizing epitope of the bovine leukemia virus. The invention also relates to methods and compositions for preventing and treating bovine leukemia caused by bovine leukemia virus using the BLV P24 monoclonal antibody or active fragment thereof, and methods and kits for determining, identifying and/or quantifying bovine leukemia virus BLV in a sample or vaccine.

Description

Bovine leukemia virus antibody and detection kit

technical field

The invention relates to a virus antibody and a virus detection kit, in particular to the antibody and the detection kit for bovine leukemia virus.

Background technique

Bovine leukemia virus (BLV) is a retrovirus that can infect many ruminants. The main hosts of its infection are dairy cows and sheep, and can cause enzootic bovine leukemia (EBL). The main clinical symptoms are malignant proliferation of lymphocytes, decreased milk production of dairy cows, and progressive emaciation of beef cattle. The disease has been reported in many countries such as Europe, the United States and Japan, and has caused great harm to the cattle industry. In the 1970s in my country, after the disease was first discovered in Shanghai, it was reported in many provinces across the country. Recently, Yang Yet al conducted a survey of bovine leukemia in 15 provinces in my country, and found that BLV infection is relatively serious in clinical practice, and its infection can reduce the milk production of dairy cows, which has caused serious harm to my country's cattle breeding and dairy industries; Clinical tests in six major cities in the province showed that BLV infection was common, and some areas were seriously infected, with an infection rate of more than 50%. It is worth noting that recent reports of detection of BLV proviral DNA in human breast tissue have demonstrated that human breast cancer cases are associated with BLV infection. Therefore, the control of BLV infection is not only of great significance to animal husbandry production, but also to public biosecurity. Currently, there is no clinically effective vaccine for the prevention and treatment of BLV. The prevention and control of BLV in my country, the United States, Japan and other countries is mainly based on detection, and the hosts of patients are isolated and eliminated. The EU countries use detection and culling to avoid the spread of the disease.

Detection of BLV infection can be carried out by detection of viral nucleic acid and antigen as well as antibody detection of the virus in the host. Viral nucleic acid detection method is mainly to detect the proviral DNA of the virus in the host body, and nested PCR method and fluorescent quantitative PCR method can be applied. At present, there are many articles published on methods for the detection of BLV proviral nucleic acid. The problem is that these assays are acceptable for laboratory-wide application. For large-scale sample testing, these methods require a high professional level of clinical technical operators, and once the management and control are not strict, it is easy to cause DNA pollution in the small-scale testing environment, which has a great impact on the accuracy of the test results.

In the market, the established detection kits are all used to detect the antibody level of the virus in the host body, and there is no kit for directly detecting the virus antigen, partly because the virus replicates at a low level in the host body, and the amount of antigen is very low , which requires extremely high sensitivity for kit development. The antibody level against BLV GP51 envelope protein and P24 capsid protein in the host is the highest, which is easy for clinical detection. At present, the BLV GP51 antibody detection kit has been commercialized. It is mainly produced and developed by some foreign reagent companies. The detection effect is relatively satisfactory, but the detection cost is expensive. For long-term continuous detection of populations, certain economic strength is required. There is also a kit for simple detection of BLV GP51 antibody in China, which has a good effect on the initial screening of populations. However, there is still a long way to go to achieve the goal of population purification. In general, there is a lack of effective antibody detection kits for detecting BLV infection in my country. It should be pointed out that there is no effective and commercially available detection kit for BLV P24 protein at home and abroad. Although the detection accuracy of commercialized GP51 detection kits imported from abroad is relatively ideal, if they can be used in conjunction with efficient P24 detection kits, it may be more beneficial to achieve the purpose of purifying BLV infected populations.

Contents of the invention

Based on the above situation, the inventor intends to develop a high-efficiency antibody detection kit for BLV P24 protein. Specifically, the P24 protein was prepared by using the E. coli prokaryotic expression system. After the mice were immunized with affinity tags in vitro, two strains were successfully screened The monoclonal antibody against BLV P24 protein provides a good foundation for the subsequent development of the BLV antibody detection kit.

The invention relates to bovine leukemia virus BLV P24 monoclonal antibody and its active fragment, which targets the main neutralizing epitope of P24 protein of bovine leukemia virus. Further, the present invention also relates to methods and compositions for preventing and treating bovine leukemia caused by bovine leukemia virus using BLV P24 monoclonal antibody or its active fragment. And further relates to methods and kits for determining, identifying and/or quantifying bovine leukemia virus BLV in samples or vaccines.

Based on this, on the one hand, the present invention provides a monoclonal antibody specific to the main conformational epitope of bovine leukemia virus BLV P24 and its active fragment, namely antigen-binding fragment (also called antibody fragment). The monoclonal antibody or its fragment specifically binds to the conformational epitope of bovine leukemia virus BLV P24. One of the conformational epitopes is the 304th to 315th position in the full-length amino acid of the P24 protein, and its sequence is shown as QKLQACAHW, and the corresponding monoclonal antibody is a P24- 44-10. Further, the monoclonal antibody produced by mouse hybridoma 293T cells is P24-115-5, which recognizes the P24 protein epitope from the 216th to the 225th amino acid in the full-length amino acid, and its sequence is shown in GDLRSQYQN, and the corresponding Said monoclonal antibody is another strain P24-115-5 produced by murine hybridoma 293T cells. Since the two monoclonal antibodies recognize different regions of the BLV P24 protein and recognize different epitopes, the two antibodies may simultaneously bind to the same BLVP24 protein molecule, or may bind to different BLV P24 molecules.

Further, the present invention provides a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof, the nucleic acid encoding P24-44-10 or P24-115-5 or an antigen-binding fragment thereof. Further, the present invention provides a vector comprising the nucleic acid and a cell comprising and expressing the vector.

In yet another aspect, the present invention provides methods and compositions for preventing and treating bovine leukemia using P24-44-10 or P24-115-5 or fragments thereof. Further, the present invention provides a pharmaceutical composition, which comprises the monoclonal antibody described in the present invention and a pharmaceutically acceptable diluent or carrier, and the monoclonal antibody is P24-44-10 or P24-115-5. Further, the pharmaceutical composition comprises the antigen-binding fragment of the present invention and a pharmaceutically acceptable diluent or carrier, and the antigen-binding fragment is an antigen-binding fragment of P24-44-10 or P24-115-5. Still further, the pharmaceutical composition comprises a nucleic acid molecule encoding the antibody or antibody fragment and a pharmaceutically acceptable diluent or carrier. Further, the pharmaceutical composition comprises a carrier having the nucleic acid and a pharmaceutically acceptable diluent or carrier. Further, the pharmaceutical composition comprises cells expressing the vector and a pharmaceutically acceptable diluent or carrier.

Further, the present invention provides a method of reducing a subject's infection with bovine leukemia virus or reducing the risk of bovine leukemia virus infection in a subject. Wherein the method comprises administering to the patient animal a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule comprising a polynucleotide encoding the antibody or antibody fragment, a vector comprising the polynucleotide, or A cell expressing the vector. The monoclonal antibody is P24-44-10 or P24-115-5.

In a third aspect, the present invention provides methods and compositions for identifying, characterizing and/or quantifying P24 expression using said monoclonal antibodies or fragments thereof. In the present invention, the monoclonal antibody is P24-44-10 or P24-115-5, and the method includes detecting the combination of bovine leukemia virus and the monoclonal antibody or fragment thereof of the present invention. The present invention uses immunofluorescence assay (IFA), immunohistochemistry assay and other methods of binding protein, including ELISA, hemagglutination inhibition (HI) assay and virus neutralization (VN) assay.

In the present invention, the method and composition for identifying and/or quantifying the expression of BLV P24 are used for identifying and/or quantifying bovine leukemia virus immunogenic substances in vaccines. The immunogenic substance comprises a BLV P24 protein or an antigenic part thereof, or a nucleic acid encoding a P24 protein or an antigenic part thereof. Further, the antigenic portion includes an epitope of P24. In the present invention, the immunogenic substance is a virus comprising P24. Further, the virus is inactivated; further, the virus is an attenuated virus, or the virus is in the form of a virosome. Still further, the virus is obtained from eggs or from cell culture. In an aspect of the present invention, the immunogenic substance is a split virus comprising P24 or an antigenic preparation of a split virus. Further, the immunogenic substance is P24 or an antigenic part thereof. Furthermore, said P24 or an antigenic portion thereof has been isolated. In a further embodiment of the present invention, the P24 or its antigenic part is produced by an expression system, and the expression system is any expression system, such as a virus expression vector.

In the fourth aspect, the present invention provides kits and methods for detecting bovine leukemia virus in biological samples or detecting antibodies against bovine leukemia virus BLV P24 in biological samples. In one protocol for detecting bovine leukemia virus BLV in a biological sample, the method comprises contacting the sample with a primary antibody, said primary antibody being monoclonal antibody P24-44-10 or P24-115- 5 or antibody fragments thereof (known as capture antibodies). Further, the method further comprises contacting the sample with a second antibody or antibody fragment thereof specifically binding to bovine leukemia virus BLV P24 according to the present invention, wherein the second antibody contains or is conjugated with a detectable element (referred to as detection antibody). Furthermore, the method includes contacting the biological sample with a third antibody, and the third antibody refers to an antibody that binds to other immunogenic substances of the BLV virus except P24 protein, such as GP51.

In a specific embodiment of the present invention, the kit comprises a primary antibody and instructions for performing an assay for detecting bovine leukemia virus, and the primary antibody is a monoclonal antibody or an antibody fragment thereof described in the present invention. The monoclonal antibody is P24-44-10 or P24-115-5. Further, the kit further comprises a second antibody specifically binding to the epitope of bovine leukemia virus BLVP24, wherein the second antibody contains or is conjugated with a detectable element. The second antibody is P24-44-10 or P24-115-5, the second antibody contains a radioactive atom, is conjugated with a fluorescent molecule, or is conjugated with an enzyme. Furthermore, the kit contains antibodies specifically binding to other antigenic substances of bovine leukemia virus BLV, such as GP51.

The hybridoma cell line P44 that produces the monoclonal antibody P24-44-10 described in the present invention is taxonomically named as the mouse hybridoma cell line P44; the date of preservation is: October 12, 2018; the preservation unit is microbial strains in China Collection of General Microbiology Center of the Preservation Management Committee; deposit number: CGMCCNO.16682; deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

The hybridoma cell line P115 producing the monoclonal antibody P24-115-5 described in the present invention is taxonomically named as the mouse hybridoma cell line P115; date of preservation: October 12, 2018; depository unit: China Microbial Culture Collection General Microbiology Center of the Management Committee; deposit number: CGMCCNO.16683; deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Description of drawings

Figure 1: Expression and purification of BLV P24, in which A. expression and purification of P24-GST, B. expression and purification of P24-6×His recombinant protein, M, protein marker.

Figure 2: The serum antibody titer of mice immunized with P24 protein, where the OD value greater than 0.2 is the effective serum titer.

Figure 3: Multiple rounds of screening of hybridoma-positive strains against BLV P24 protein.

Figure 4: Cell-level verification of the ability of different hybridoma-positive strains to recognize and bind to BLV Gag protein.

Figure 5: Detection of monoclonal antibody subtypes secreted by hybridoma cells.

Figure 6: Monoclonal screening of hybridoma cell lines.

Figure 7: Epitope identification of two monoclonal antibodies. A: Epitope identification of monoclonal antibody No. 45-10, B: Epitope identification of monoclonal antibody No. 115-5.

Figure 8: Specific detection of two monoclonal antibodies. A: Indirect immunofluorescence detection of 44-10/115-5 and BLV P24, B: Indirect immunofluorescence detection of 44-10/115-5 and BVDV, C: ELISA detection of 44-10/115-5 and FMDV.

Detailed ways

The term "antibody" used in the present invention is a term recognized in the art, and refers to molecules or active fragments of molecules that bind to known antigens, especially immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, that is, containing the binding site of the molecule. An immunoglobulin is a protein comprising one or more polypeptides substantially encoded by the immunoglobulin kappa and lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Subclasses of heavy chains are also known.

"Specific binding" as used herein with respect to an antibody means that the antibody binds its target antigen with a higher affinity than it binds to a structurally different antigen.

Typical immunoglobulin structural units are known to comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light chain" (approximately 25 kD) and a "heavy chain" (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to these light and heavy chains, respectively.

Antibodies exist as full-length intact antibodies or as a number of well-characterized fragments produced by digestion with various peptidases or compounds. The "antibody" of the present invention includes chimeric monoclonal antibodies and active fragments thereof. Active fragments of molecules that bind known antigens include, for example, separate light and heavy chains, Fab, Fab/c, Fv, Fab' and F(ab') _fragments , including the products of Fab immunoglobulin expression libraries and any of the foregoing Epitope-binding fragments of antibodies and fragments.

The term "monoclonal antibody" is also well recognized in the art and refers to the product of a monoclonal antibody-producing cell. Monoclonal antibodies are typically produced by fusing normally short-lived antibody-producing B cells with fast-growing cells such as cancer cells (sometimes called "immortal cells"). The resulting hybrid cells, or hybridomas, multiply rapidly and antibody-producing clones are obtained.

The term "fragment" refers to a portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained by chemical or enzymatic treatment of intact or complete antibodies or antibody chains. Fragments can also be obtained recombinantly. Exemplary fragments include Fab, Fab', F(ab') ₂ , Fabc and/or Fv fragments. The term "antigen-binding fragment" refers to an antigen-binding or polypeptide fragment of an immunoglobulin or antibody that competes with intact antibody for antigen-binding (ie, specific binding). Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab') ₂ , Fabc, Fv, single chain and single chain antibodies.

As used herein, reference to "isolated" refers to a biomolecule free of at least some of the components with which it is naturally associated.

The invention is described by the following examples, which serve to illustrate the invention.

Example 1: Plasmid construction

After the BLV P24 capsid protein gene was synthesized, it was directional cloned into the prokaryotic expression p-coldIII-GST vector and pet-32a-6×His vector. The full-length gene of BLV Gag (amplified in a laboratory-preserved sample) was directional cloned into the eukaryotic expression vector pcDNA3.1(+) (C-terminal HA tag). After the P24 truncated gene fragment is connected with the pCAGGS-vector (C-terminal FLag tag), a series of P24 gene truncated series P24-1, P24-2, P24-3, P24-4, 1A, 1B, 1C, 3A, 3B, 3C, 3D, and prepared into corresponding recombinant vector pcDNA3.1-HA, used for epitope identification of monoclonal antibody. Vector construction was completed using Vazemy recombinant cloning technology. Wherein the p24-1 gene sequence (that is, the full-length P24 gene) and the protein sequence are shown in the sequence table SEQ ID NO: 1 and 2 respectively, and the p24-2 gene sequence and protein sequence are shown in the sequence table SEQ ID NO: 3 and 4 respectively Shown, the p24-3 gene sequence and protein sequence are respectively shown in the sequence table SEQ ID NO:5 and 6, the p24-4 gene sequence and protein sequence are respectively shown in the sequence table SEQ ID NO:7 and 8, p24-5 The gene sequence and protein sequence are shown in sequence table SEQ ID NO: 9 and 10 respectively, the 24-1A gene sequence and protein sequence are shown in sequence table SEQ ID NO: 11 and 12 respectively, and the p24-1B gene sequence and protein sequence are respectively As shown in the sequence listing SEQ ID NO: 13 and 14, the p24-1C gene sequence and protein sequence are respectively shown in the sequence listing SEQ ID NO: 15 and 16, and the p24-3A gene sequence and protein sequence are respectively shown in the sequence listing SEQ ID NO: As shown in 17 and 18, the p24-3B gene sequence and protein sequence are respectively shown in the sequence listing SEQ ID NO: 19 and 20, the p24-3C gene sequence and protein sequence are respectively shown in the sequence listing SEQ ID NO: 21 and 22, The gene sequence and protein sequence of p24-3D are respectively shown in SEQ ID NO: 23 and 24 in the sequence listing. It should be noted that the P24 protein is small, so the NC part of the GAG protein is fused at its N-terminus, which can increase the molecular weight of the protein, which is easy to detect in vitro, and does not affect the recognition of its sequence by the monoclonal antibody.

Example 2: Expression and detection of P24 protein and purification and detection of tagged protein

The 24-p-coldIII-GST vector was transformed into BL21(DE3) Escherichia coli competent cells, cultured overnight at 18°C, induced with 1mM IPTG, and the cells were collected after 18 hours. The P24-pet-32a-6×His vector was transformed into BL21(DE3) Escherichia coli competent cells, cultured overnight at 30°C, and the cells were collected after 18 hours. After the above Escherichia coli cells were lysed by supernatant, the supernatant samples and sediment samples were collected respectively, and then detected by SDS-PAGE electrophoresis. 293T cells were transfected with Gag-pcDNA3.1-HA, samples were collected 48 hours later for SDS-PAGE, WB was used to detect Gag gene expression and to verify the specificity of antibody recognition. 293T cells were transfected with P24 gene truncated serial recombinant vectors, and samples were collected 48 hours later for SDS-PAGE and WB to detect antibody epitope recognition. 293T cells were transfected with Gag-pcDNA3.1-HA. After 24 hours, 0.1% Triton was punched, and the ascites of P24 hybridoma was incubated. A simple indirect immunofluorescence assay (IFA) was performed to verify the specificity of the monoclonal antibody.

The P24-p-coldIII-GST recombinant plasmid was transformed into BL21(DE3) Escherichia coli, induced by IPTG, ultrasonically disrupted, and the supernatant was discarded by low-speed centrifugation. The supernatant was incubated overnight at low temperature with magnetic beads coupled with GST tag antibody, and then washed with the eluate and used for ELISA screening of hybridomas (SolarBio company). Similarly, P24-pet-32a-6×His obtained recombinant protein (SolarBio company) through Ni-column purification system, and immunized mice to prepare monoclonal antibody. Expression of anti-HA-tag HRP-tag antibody recombinant protein in 293T cells after sequencing verification of Gag-pcDNA3.1-HA recombinant plasmid.

The results are shown in Figure 1: the expression and purification of A.P24-GST 1, supernatant before induction; 2, precipitation before induction; 3, supernatant after induction 14 hours; 4, precipitation after induction 14 hours; 5, protein precipitation renaturation with urea Purification results; M, protein marker. B. Expression and purification of P24-6×His recombinant protein 1. Supernatant before induction; 2. Precipitation before induction; 3. Supernatant after induction for 1 hour; 4. Precipitation for 1 hour after induction; 5. Supernatant after induction for 2 hours; 6. , precipitation after induction for 2 hours; 7, supernatant after induction for 4 hours; 8, precipitation after induction for 4 hours; 9, protein precipitation and urea refolding purification results; M, protein marker. The results indicated that the expression vector of the P24 protein gene constructed by us coupled with GST-tag and His-tag was purified by GST-affinity resin and Ni column, and the P24 protein with higher purity was obtained.

Example 3: Immunization of recombinant proteins and preparation and screening of hybridoma cells

Recombinant P24-His protein purified by Ni particles was quantified and then immunized into mice (intraperitoneal injection). Each mouse was immunized with 100 μg of recombinant P24 protein each time, and a total of 5 mice were immunized. After the first immunization, the second immunization was performed 4 weeks later, and the third immunization was performed 6 weeks later. Serum was collected after the third immunization to detect the P24 antibody titer in the mice. 0 Three days before the fusion, booster immunization once.

After the immunization, select mice whose serum titers meet the fusion requirements, extract the spleen of the immunized mice and carry out cell fusion with Sp2/0 myeloma cells under the action of fusion agent (PEG3350), and divide the cells into 96-well cell culture after fusion Plates were selectively cultured with HAT medium, and clones were detected by ELISA method. After multiple rounds of detection, hybridoma-positive clones resistant to BLV P24 protein were obtained. Screening of hybridomas The prepared P24-GST recombinant protein was used to coat a 96-well plate, the supernatant of hybridoma cells was detected by EILSA method, and clones were screened out according to the positive clone standard. Positive cell lines were screened, and their supernatants were used for antigen recognition specificity detection. After 293T cells were transfected with Gag-pcDNA3.1-HA (the Gag gene contains the complete BLV P24 gene), the hybridoma cell supernatant obtained was used to test whether it could recognize the BLV P24 recombinant protein expressed by eukaryotic cells.

The results are shown in Figure 2 and Figure 3. Figure 2 shows that after immunization of 5 mice with His-tag purified recombinant P24 protein, 96-well plates were coated with GST-tag purified recombinant P24 protein, and the antibody in each mouse was detected by ELISA potency. The serum antibody titer of mouse No. 1 was the highest after immunization, and it still had a high antibody titer after 1:200,000 dilution. Therefore, the splenocytes of No. 1 mouse were further fused with Sp2/0 myeloma cells to prepare hybridomas. After splenocytes were fused with Sp2/0, they were planted in a 96-well plate, and HAT medium was pressurized for selection. Primary screening was performed after hybridoma cells were cultured for 10 years. In each screening, culture wells with higher antibody titers continued to the next round of screening, and a total of 3 rounds of screening were performed. As shown in Figure 3, a total of 14 hybridoma positive clones were obtained after multiple rounds of screening.

Example 4: Monoclonal Screening and Ascites Preparation of Hybridoma Positive Strains

Use the terminal dilution method to obtain monoclonal cell lines from the positive wells of hybridoma cells screened above, and verify the antibody titer of the obtained monoclonal cell lines by ELISA method. The verified positive monoclonal hybridoma cell lines continue to the downstream ascites preparation. One week before the preparation of ascites, the immune system of the mice was activated with Freund's incomplete adjuvant, and 500 μL of each mouse was injected intraperitoneally. After one week of activation, mouse peritoneal hybridoma cells were injected intraperitoneally, and each mouse was injected with 10 ⁶ hybridoma cells. After 10 days of injection, the abdominal cavity of the mice was observed every day, and after it swelled, the ascites could be collected for antibody titer detection.

Example 5: Activity and specificity analysis of monoclonal antibodies

The obtained supernatant of the P24 monoclonal hybridoma-positive strain was identified by an antibody type detection kit (Sigma), ie IgM, IgG and IgA. After the subtype identification of the antibody, proceed to identify the epitope of the antibody. The P24 series gene truncated recombinant protein constructed above was detected by WB method, and the epitope recognized by the monoclonal antibody was determined.

Specifically, the Gag gene containing the full-length P24 gene was expressed in 293T cells, and it was used to verify whether the obtained hybridoma-positive strain could recognize the BLV P24 protein expressed in mammalian cells. Firstly, the supernatants of 10 hybridoma cell lines with higher OD values were initially tested. As shown in Figure 4, all the supernatants tested could react with BLV GAG, indicating that the antibodies secreted by these hybridoma cell lines may recognize Wild-type BLV virus in its natural state. Antibodies produced by different hybridomas have different recognition abilities to BLVP24, such as No. 115, No. 173, No. 180, No. 55 and No. 358. weaker.

Example 6: Subtype identification of monoclonal antibodies

Preliminary subtype identification was carried out on the hybridoma-positive strains screened above, as shown in Figure 5, the types of monoclonal antibodies secreted by different hybridoma-positive strains were different. Among them, the types of monoclonal antibodies produced by clones No. 44, 115, 178 and 358 are relatively single, indicating that the hybridoma cells in the wells are relatively single; No. 36, No. 57, No. 173, No. 180 and No. It is not uniform, indicating that the cells in the wells are a mixture of different hybridoma cells, and they are cell wells containing IgM antibodies; No. 55 is also a mixture of hybridomas, secreting two subtypes of monoclonal antibodies, IgG1 and IgG3. No. 44 and No. 115 have strong reaction binding ability, and these two lines of hybridoma cells were selected for further monoclonal culture. As shown in Figure 6, No. 44-10 and No. 115-5 antibody titers are relatively high. These 3 cell lines were selected for ascites preparation for subsequent detection work.

Example 7: Ascites preparation and epitope identification

1. Antibody epitope identification of monoclonal antibody P44-10

Ascites of 44-10 and 115-5 were further prepared. As shown in Figure 7, a series of BLV P24 gene truncated expression plasmids were constructed. During the construction, because the P24 protein was small, the NC part of the GAG protein was fused at its N-terminus, which could increase the molecular weight of the protein and facilitate In vitro detection, and does not affect the recognition of its sequence by monoclonal antibodies. The fused protein is the fusion protein of NC-P24. A total of 5 proteins were constructed, named P24-1, P24-2, P24-3, P24-4 and P24-5, and the molecular weight of these five proteins decreased in turn, and each protein was higher than the C-terminus of the previous protein. There are about 40 amino acid residues less (the order of molecular weight is P24-1>P24-2>P24-3>P24-4>P24-5). The plasmids encoding P24-1, P24-2, P24-3, P24-4 and P24-5 five genes (i.e. p24-1, p24-2, p24-3, p24-4 and p24-5, see implementation Example 1) 293T cells were transfected, and 48 hours after transfection, cell samples were collected for Western blotting (western blotting, WB). Monoclonal antibodies can only bind and generate a signal to the protein they recognize. Monoclonal antibody P44-10 was used to detect these protein samples. The results showed that p44-10 could react with p24-1 protein. Therefore, it was preliminarily determined that the monoclonal antibody P44-10 recognized the C-terminus of the BLV P24 protein. On this basis, the p24-1 protein was further truncated and expressed, and it was gradually truncated every 10 amino acids from the C-terminus, and named p24-1A, p24-1B and p24-1C. After these genes were transfected into 239T cells, the cell samples were collected 48 hours later and continued to be detected by WB with monoclonal antibody P44-10. The results showed that the monoclonal antibody P44-10 could bind to the proteins P24-1A and P24-1B, but could not bind to the P24-1C protein. Therefore, it was preliminarily determined that the epitope region recognized by it was the C-terminal sequence of P24-1B. That is, the epitope GQKLQACAHW.

2. Antibody epitope identification of monoclonal antibody P115-5

Referring to the antibody epitope identification work of monoclonal antibody P44-10, the antibody epitope recognized by monoclonal antibody P115-5 was identified by the same means and method. 48 hours after the plasmids p24-1, p24-2, p24-3, p24-4 and p24-5 were transfected into 293T cells, the cell samples were collected for WB detection. The results showed that monoclonal antibody P115-5 could bind to protein P24-3, but could not bind to protein P24-4. Therefore, its recognized region should be at the C-terminus of protein P24-3. The C-terminus of protein P24-3 was sequentially truncated with a length of about 10 amino acids, and named as P24-3A, P24-3B, P24-3C and P24-3D, respectively. The results showed that monoclonal antibody P115-5 could recognize protein P24-3C, but not protein P24-3D. Therefore, the antigenic epitope it recognized was determined as the protein sequence GDLRSQYQN at the C-terminal of P24-3C.

Example 8: Specific detection of two monoclonal antibodies

In order to detect whether the obtained monoclonal antibody has antigen recognition specificity, bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) and foot-and-mouth disease virus (Foot and mouth disease virus, FMDV) were used to detect the 44-10 and 115-5 two specificity of monoclonal antibodies. As shown in Figure 8, the two monoclonal antibodies could recognize BLV P24 protein, but could not recognize intracellular BVDV virus. ELISA test showed that the two monoclonal antibodies 44-10 and 115-5 could not recognize the Asian I, A and O FMD viruses, which proved that the obtained two monoclonal antibodies had good specificity. As shown in Figure 8, after BVDV proliferates in the cells, the BVDV-specific antibody can recognize the virus in the cell, but the two monoclonal antibodies of the present invention cannot recognize the virus, that is, there is no green fluorescent signal in the cell. Similarly, detect monoclonal antibodies using the FMDV antigen detection kit. Find that two strains of monoclonal antibodies of the present invention all can not recognize the foot-and-mouth disease virus antigen of O type, A type and Asia I type, and positive control antibody all can recognize these antigens, show that the antibody specificity of the present invention is good, and generally popular FMDV has no cross-reactivity.

Claims (10)

1. A monoclonal antibody or active fragment thereof that specifically binds to a neutralizing conformational epitope of bovine leukemia virus BLV P24, wherein the neutralizing conformational epitope: (a) Comprising amino acid GQKLQACAHW, or (b) comprising amino acid GDLRSQYQN.

2. The monoclonal antibody or active fragment thereof according to claim 1, wherein the neutralizing conformational epitope of bovine leukemia virus BLV P24: (a) Is an epitope specifically bound by the murine monoclonal antibody P24-44-10, or (b) is an epitope specifically bound by the murine monoclonal antibody P24-115-5, and the hybridoma cell line P44 producing the monoclonal antibody P24-44-10 is deposited with the China general microbiological culture Collection center with the accession number: the CGMCC is NO.16682, the hybridoma cell strain P115 for producing monoclonal antibody P24-115-5 is preserved in China general microbiological culture Collection center with the preservation number of: the CGMCC No. is 16683.

3. The monoclonal antibody or active fragment thereof according to claim 1, wherein said monoclonal antibody is: (a) The murine monoclonal antibody P24-44-10, or (b) the murine monoclonal antibody P24-115-5.

4. Monoclonal antibodies P24-44-10 or P24-115-5 produced by hybridoma 293T cells, hybridoma cell line P44 producing the monoclonal antibodies P24-44-10 is deposited with China general microbiological culture Collection center with the accession number: the CGMCC is NO.16682, the hybridoma cell strain P115 for producing the monoclonal antibody P24-115-5 is preserved in China general microbiological culture Collection center with the preservation number of: the CGMCC No. is 16683.

5. A method of detecting bovine leukemia virus BLV in a biological sample ex vivo comprising contacting the sample with a first antibody, wherein the first antibody is the monoclonal antibody or active fragment thereof of any one of claims 1-4, and determining the presence or absence of bovine leukemia virus BLV.

6. The method of claim 5, further comprising contacting the sample with a second antibody that specifically binds to the P24 epitope of bovine leukemia virus BLV, wherein the second antibody contains a detectable element or is conjugated to a detectable element, wherein the contacting with the second antibody occurs prior to the determining.

7. The method of claim 6, wherein the second antibody is the monoclonal antibody or active fragment thereof of any one of claims 1-4, wherein the first antibody is one of monoclonal antibody P24-44-10 or monoclonal antibody P24-115-5, and the second antibody is the other of said monoclonal antibody P24-44-10 or monoclonal antibody P24-115-5.

8. A kit for detecting bovine leukemia virus BLV in a biological sample comprising a first antibody, wherein the first antibody is the monoclonal antibody or antibody fragment of any one of claims 1-4.

9. The kit of claim 8, further comprising a second antibody that specifically binds to the P24 epitope of bovine leukemia virus BLV, wherein the second antibody contains or is conjugated to a detectable element.

10. The kit of claim 9, wherein the second antibody is the monoclonal antibody or active fragment thereof of any one of claims 1-4, and the second antibody is the other of said monoclonal antibodies P24-44-10 or monoclonal antibodies P24-115-5.

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