CN116444651B - Porcine parvovirus monoclonal antibody and application thereof - Google Patents

Porcine parvovirus monoclonal antibody and application thereof Download PDF

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CN116444651B
CN116444651B CN202211680914.8A CN202211680914A CN116444651B CN 116444651 B CN116444651 B CN 116444651B CN 202211680914 A CN202211680914 A CN 202211680914A CN 116444651 B CN116444651 B CN 116444651B
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monoclonal antibody
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porcine parvovirus
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CN116444651A (en
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查银河
张稳涛
余晓玉
王芳
盖其静
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Zhejiang Hongsheng Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
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Abstract

The invention discloses a monoclonal antibody which can specifically bind porcine parvovirus VP2 protein, wherein the variable region sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO.1, and the variable region sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO. 2. The monoclonal antibody disclosed by the invention has the advantages of good specificity and good affinity, and can be used for detecting porcine parvovirus VP2 protein and developing related detection products.

Description

Porcine parvovirus monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a porcine parvovirus monoclonal antibody and application thereof.
Background
Porcine parvovirus disease (PPI) is also known as a porcine reproductive disorder. Is a reproductive disorder of pigs caused by Porcine Parvovirus (PPV). Pregnant sows are characterized by abortion, stillbirth and mummy. Pigs of different ages and sexes are susceptible to infection. The onset of disease is not obvious seasonally. The infectious agents mainly come from sows infected with cell viruses and boars with viruses, the replacement gilts are easier to infect than the multiparous sows, the viruses can be vertically transmitted through placenta, and live piglets produced by the pigs with viruses can carry the viruses and expel the viruses for a long time or even for life.
At present, the prevention and control of PPV are mainly whole virus inactivated vaccines. As indicated in CN 105349562B patent, whole virus inactivated vaccines have major drawbacks. With the development of biotechnology, researchers have found that PPV is VP2 protein as the main immunogenicity, and many scientific research units or companies have systematically studied VP2 protein and developed vaccines, and the subunit vaccine has a good immune effect. However, quantification of VP2 proteins is mainly performed by the BCA method in combination with SDS-PAGE; the quantitative method detects the total VP2 protein, and VP2 protein with good immunogenicity cannot be distinguished. Therefore, in order to better quantify VP2 protein, a good monoclonal antibody against VP2 protein is required.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a monoclonal antibody specifically combined with porcine parvovirus VP2 protein and application thereof.
The invention discloses a porcine parvovirus monoclonal antibody, which can specifically bind with porcine parvovirus VP2 protein, wherein the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO. 2.
Preferably, the heavy chain variable region sequence of the monoclonal antibody of the present invention comprises CDR-H1, CDR-H2 and CDR-H3, and the sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are respectively:
CDR-H1：TNYLIEE；
CDR-H2：YTCNPGSLRTKYNEFKC；
CDR-H3：DGPWSDAD。
preferably, the light chain variable region sequence of the monoclonal antibody of the present invention comprises CDR-L1, CDR-L2 and CDR-L3, and the sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are respectively:
CDR-L1：KSSQSSLNSFNQKKYLAS；
CDR-L2：GASTRQS；
CDR-L3：QNLHSYPFT。
preferably, the heavy chain variable region sequence of the monoclonal antibody of the present invention further comprises FR-H1, FR-H2, FR-H3 and FR-H4, and the sequences of the FR-H1, FR-H2, FR-H3 and FR-H4 of the heavy chain variable region are respectively:
FR-H1：QVQLQQSGQEEVRPGQTSGKVSCAASGYFAF；
FR-H2：WVKQPGQGLIWIG；
FR-H3：KAWLTADMCSGNSYMQHSSLKSDDADYFCAR；
FR-H4：WGQGTLVTVSA。
preferably, the light chain variable region sequence of the monoclonal antibody of the present invention further comprises FR-L1, FR-L2, FR-L3 and FR-L4, and the sequences of the FR-L1, FR-L2, FR-L3 and FR-L4 of the light chain variable region are respectively:
FR-L1：DIWMTQSVSSLRVSAGEKVTMAD；
FR-L2：FYQQKPGQPPQLLIY；
FR-L3：GVPERFEGGSGEGTDSTLTYSSVEAEDLAYVYC；
FR-L4：FHSGTKLEIK。
in still another aspect, the invention also discloses application of the monoclonal antibody in detecting porcine parvovirus VP2 protein.
The monoclonal antibody for resisting the porcine parvovirus VP2 protein has good specificity and sensitivity, and is suitable for preparing diagnostic reagents for different parvovirus VP2 proteins, such as detection kits of the wersterin blot, ELISA and the like.
Although the invention does not establish a detection kit for porcine parvovirus VP2 protein, the detection kit can be realized by using conventional technical means on the basis of the monoclonal antibody of the invention by a person skilled in the art. For example, the rabbit polyclonal antibody is prepared by using the porcine parvovirus VP2 protein, the prepared rabbit polyclonal antibody is used as a coating antibody, the monoclonal antibody is used as a detection antibody, and a quantitative or qualitative detection kit aiming at the porcine parvovirus VP2 protein can be established by combining the porcine parvovirus VP2 protein, and the kit can be an ELISA detection kit or a chemiluminescent detection kit.
Drawings
FIG. 1 shows the monoclonal antibody specificity test result (wersterin blot), wherein PPV-VP2 is porcine parvovirus VP2 protein (concentration is 1 mg/ml), and PEDV, PCV2 and PRV are total proteins extracted from whole viruses as samples.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
In the present invention, an "antibody" refers to a class of immunoglobulins that specifically bind to an antigen. Antibodies contain four heterologous polypeptide chains, of which the two chains with the larger molecular weight are called heavy chains (H) and the two chains with the smaller molecular weight are called Light chains (L). The amino acid compositions of the two H chains and the two L chains in the same antibody molecule are identical. By analysis of the amino acid sequences of the heavy and light chains of different antibodies, it was found that the amino acid sequences of about 110 amino acid sequences of the heavy and light chains near the N-terminus varied widely, with the other portions of the amino acid sequences being relatively constant. Thus, the regions of the antibody light and heavy chains that vary greatly near the N-terminal amino acid sequence are referred to as variable regions (V) and account for 1/4 and 1/2 of the heavy and light chains, respectively; the region of relatively stable amino acid sequence near the C-terminus is called constant region (C) and occupies 3/4 and 1/2 of the heavy and light chains, respectively. The V chains of the heavy and light chains are referred to as VH and VL, respectively. Each of VH and VL contains a region of highly variable 3 amino acid composition and arrangement sequence, called complementarity determining region (complementarity determining region, CDR), including CDRl, CDR2 and CDR3, wherein CDR3 varies to a greater extent. The 3 hypervariable regions of VH are located at amino acids 29-31, 49-58 and 95-102 respectively, while the 3 hypervariable regions of VL are located at amino acids 28-35, 49-56 and 91-98 respectively. The 3 CDRs of VH and VL together form the antigen-binding site of the antibody, which determines the specificity of the antibody and is the site where the antibody recognizes and binds to the antigen. In the V region, the amino acid composition and arrangement order of the regions outside the CDRs are relatively conserved, called Framework Regions (FR). VH or VL has four framework regions, denoted FR1, FR2, FR3 and FR4, respectively. The C chains of the heavy and light chains are referred to as CH and CL, respectively. CL lengths of different classes of antibodies (kappa or lambda) are substantially identical, but CH lengths of different classes of antibodies are different, e.g., igG, igA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CHl, CH2, CH3, and CH4.
Example 1: preparation of porcine parvovirus VP2 protein
The porcine parvovirus VP2 protein used in the invention is given away by Zhejiang Hailong biotechnology Co., ltd, and the specific preparation method can be seen in the embodiment of Chinese patent No. CN 105349562B.
Example 2: preparation of anti-porcine parvovirus VP2 protein monoclonal antibody
Monoclonal antibodies were prepared using conventional hybridoma cell techniques, briefly described as follows: the porcine parvovirus VP2 protein prepared in example 1 is used as antigen, 3 times of immunization are carried out on BALB/c female mice with the age of 6-8 weeks, and spleen cells are taken out and fused with myeloma Sp2/0 after 3 days of three-phase immunization. Detecting culture supernatant by an indirect ELISA method, screening positive hybridoma cells, and performing cell cloning on the positive hybridoma cells by a limiting dilution method to obtain 1 strain of better hybridoma cells, which is named as 6B2. After passaging and repeated cryopreservation and recovery, the cell strain can grow well and stably secrete antibodies. After the expansion culture, the strain is used for preparing ascites of mice, frozen and stored in liquid nitrogen for a long time.
Taking 6-8 week-old BALB/c female mice, injecting pristane, 0.5 ml/mouse, and injecting hybridoma cells 1×10 per mouse 7 days later 6 And each. The abdomen of the mice is obviously enlarged 7-10 days after injection, ascites is taken, centrifugation is carried out for 3min at 8000rpm at 4 ℃, and supernatant fluid is collected, thus obtaining the monoclonal antibody ascites. Taking 1-time volume of ascites, adding 2-time volume of acetate buffer (0.06 mol/L, pH value is 4.8), uniformly mixing, adding octanoic acid (30 mu L/ml of ascites) at room temperature under stirring, clarifying at 4 ℃ for 2 hours, centrifuging at 12000rpm at 4 ℃ for 20min, and collecting supernatant. Precipitating immunoglobulin with 50% saturated ammonium sulfate, standing at 4deg.C for 2 hr, centrifuging at 4deg.C at 3000rpm for 30min, and collecting precipitate. Dissolving the precipitate with 2 times volume PBS, dialyzing with PBS overnight to obtain purified ascites antibody, and storing at-70deg.C. The concentration of the monoclonal antibody is detected by using a BCA kit, the detection result of the monoclonal antibody is 2.25mg/ml, and the antibodies are respectively split into 0.5 ml/tube and stored below 70 ℃ for standby.
Example 3: performance determination of monoclonal antibodies
Antibody specific detection: the monoclonal antibodies were taken separately to perform the wersterin blot detection of the total proteins extracted from different viruses to judge the specificity thereof, and the results show (figure 1) that the monoclonal antibodies are negative to other swine diseases and the detection of the porcine parvovirus VP2 protein is positive, which indicates that the specificity of the monoclonal antibodies is good.
Class and subclass determination: the type and subclass of the monoclonal antibody are identified by ELISA kit, and the identification result shows that the heavy chain constant region of the monoclonal antibody is lgG1 type and the light chain constant region of the monoclonal antibody is Kappa type.
HRP labeling performance assay: monoclonal antibodies were HRP-labeled using either the classical sodium periodate method or a commercially available HRP-labeling kit, and the labeling titers were determined using a direct ELISA method (coating porcine parvovirus VP2 protein, coating amount 1 μg/ml) (positive when OD value was > 1.0), result 1:20000. The HRP labeling is successful, the labeling efficiency is high, and the kit development requirement is met.
Determination of the sequence of the variable region of the monoclonal antibody: the monoclonal antibodies prepared were subjected to the determination of the heavy chain variable region and the light chain variable region by the method of example 5 of chinese patent application (CN 111393525B), and the sequences of the heavy chain variable region and the light chain variable region were determined as shown in table 1.
TABLE 1 amino acid sequence information for monoclonal antibodies
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (5)

1. The monoclonal antibody is characterized in that the monoclonal antibody can specifically bind with porcine parvovirus VP2 protein, the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO. 2.
2. The monoclonal antibody of claim 1, wherein the heavy chain variable region sequence of the monoclonal antibody comprises CDR-H1, CDR-H2 and CDR-H3, and the sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are respectively:
CDR-H1：TNYLIEE；
CDR-H2：YTCNPGSLRTKYNEFKC；
CDR-H3：DGPWSDAD。
3. the monoclonal antibody of claim 1, wherein the light chain variable region sequence of the monoclonal antibody comprises CDR-L1, CDR-L2 and CDR-L3, and the sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are respectively:
CDR-L1：KSSQSSLNSFNQKKYLAS；
CDR-L2：GASTRQS；
CDR-L3：QNLHSYPFT。
4. the monoclonal antibody of claim 1, wherein the heavy chain variable region sequence of the monoclonal antibody comprises FR-H1, FR-H2, FR-H3 and FR-H4, and the sequences of FR-H1, FR-H2, FR-H3 and FR-H4 of the heavy chain variable region are respectively:
FR-H1：QVQLQQSGQEEVRPGQTSGKVSCAASGYFAF；
FR-H2：WVKQPGQGLIWIG；
FR-H3：KAWLTADMCSGNSYMQHSSLKSDDADYFCAR；
FR-H4：WGQGTLVTVSA。
5. the monoclonal antibody of claim 1, wherein the light chain variable region sequence of the monoclonal antibody comprises FR-L1, FR-L2, FR-L3 and FR-L4, and the sequences of FR-L1, FR-L2, FR-L3 and FR-L4 of the light chain variable region are respectively:
FR-L1：DIWMTQSVSSLRVSAGEKVTMAD；
FR-L2：FYQQKPGQPPQLLIY；
FR-L3：GVPERFEGGSGEGTDSTLTYSSVEAEDLAYVYC；
FR-L4：FHSGTKLEIK。
CN202211680914.8A 2022-12-27 2022-12-27 Porcine parvovirus monoclonal antibody and application thereof Active CN116444651B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101893633A (en) * 2010-08-06 2010-11-24 扬州大学 Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method for detecting porcine parvovirus
CN113150126A (en) * 2021-04-20 2021-07-23 开江县动物疫病预防控制中心 Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof
CN114149497A (en) * 2021-12-03 2022-03-08 河南省农业科学院动物免疫学重点实验室 Porcine parvovirus neutralizing monoclonal antibody, and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101893633A (en) * 2010-08-06 2010-11-24 扬州大学 Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method for detecting porcine parvovirus
CN113150126A (en) * 2021-04-20 2021-07-23 开江县动物疫病预防控制中心 Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof
CN114149497A (en) * 2021-12-03 2022-03-08 河南省农业科学院动物免疫学重点实验室 Porcine parvovirus neutralizing monoclonal antibody, and preparation method and application thereof

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Title
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猪细小病毒VP2蛋白单克隆抗体的制备及抗原捕捉ELISA方法的建立;田莉莉等;畜牧与兽医;第44卷(第07期);第13-17页 *
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