CN115873077A - Reagent for detecting bovine nodular skin disease virus antibody and polypeptide used by reagent - Google Patents
Reagent for detecting bovine nodular skin disease virus antibody and polypeptide used by reagent Download PDFInfo
- Publication number
- CN115873077A CN115873077A CN202211265005.8A CN202211265005A CN115873077A CN 115873077 A CN115873077 A CN 115873077A CN 202211265005 A CN202211265005 A CN 202211265005A CN 115873077 A CN115873077 A CN 115873077A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- conjugate
- bovine
- nucleic acid
- acid molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a reagent for detecting bovine sarcoidosis virus antibody and a polypeptide used by the same. The polypeptide provided by the invention is an eA27-1 polypeptide or a complete set of polypeptide consisting of the eA27-1 polypeptide and an eA33-1 polypeptide; the eA27-1 is polypeptide with an amino acid sequence shown as SEQ ID No. 1; the eA33-1 is a polypeptide with an amino acid sequence of SEQ ID No. 5. The antibody detection kit prepared by taking the conjugate of the polypeptide as the coating antigen has the advantages of strong specificity, high sensitivity, high accuracy, simple and quick operation, and is suitable for quick and large-scale screening detection of bovine sarcoidosis infected serum antibodies by various veterinarian departments at the basic level and entry and exit inspection and quarantine bureaus.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a reagent for detecting bovine nodular skin disease virus antibodies and polypeptides used by the reagent.
Background
Sarcoidosis (LSD) first occurred in nipponia in 1929, followed by endemic prevalence primarily in african countries and regions, sahara and madagaska, and in israel in 1989 and 2006, followed by continued spread to iran, pakistan, saudi arabia, and other countries. In 2015, the traditional Chinese medicine has outbreaks in Greece and Russia, and is spread in China and Europe including Russia, kazakhstan, turkey, syria and Israel, and the like, thereby constituting a serious threat to China. The occurrence of LSD has been reported in the middle east, europe and the west asia during the last decade (OIE, 2017). Outbreaks of sarcoidosis tend to occur sporadically, depending on animal activity, immune status, and wind and rain changes that affect vector insect populations. The primary mode of transmission is arthropod-mediated mechanical transmission. 8, 12 months in 2019, the disease is confirmed to be developed in the cattle nodular skin disease epidemic situation in Ili of the Uygur autonomous region of Xinjiang through the research center of exotic animal epidemic diseases in the China center of animal health and epidemiology, which is the first diagnosis of large-scale development of the disease in China.
Bovine sarcoidosis virus (LSDV) and Capripoxvirus (GPV), sheeppox virus (SPV) belong to the Poxviridae (Poxviridae), capripoxviruses (capropivirus, caPV) families as well. Observed under an electron microscope, the virus particles are in a brick shape or a short tube shape, consist of 1 nucleus, 2 lateral bodies and 2 layers of lipid outer membranes, have the size of about 290nm multiplied by 270nm, and belong to small poxvirus virus particles. The virus has only 1 serotype, and the representative strain is a Neethling strain in south Africa. The virus can proliferate and generate cytopathic effect in primary cells such as lamb, calf kidney or innocent pill cells, sheep embryonic kidney and lung cells, chicken embryonic fibroblasts and the like, and passage cells such as bovine kidney cells, african green monkey kidney and the like. Infected cell cultures were stained with hematoxylin and eosin or hematoxylin and rose bengal, and cytoplasmic inclusions were visible. The virus can grow and propagate on the chorioallantoic membrane of chick embryos and cause pocks, but the chick embryos do not die. The virus has cell binding property, and can be released to the outside of cells by destroying cells with ultrasonic wave. The virus particles can survive for a long time in an environment with pH = 6.6-8.6. Survival was 4-6 months in 4 ℃ glycerol saline and tissue culture medium. The virus survived in the dry lesions for more than 1 month and survived for several months in the dry houses. The virus is freeze-thaw resistant, and can be preserved at-20 deg.C for several years and at-80 deg.C for 10 years. Sensitive to heat, and can be inactivated at 55 deg.C for 2 hr or 65 deg.C for 30 min. Sensitive to direct sunlight, acids, bases and most commonly used disinfectants (alcohol, mercuric chloride, iodine tincture, lysol, formalin, carbolic acid, etc.), and also sensitive to chloroform and diethyl ether.
The natural host of the LSDV is mainly cattle, and each cattle is susceptible to infection and has no obvious variety specificity. Rabbits, sheep, goats, giraffes, black antelopes, and the like may also be infected, but there is no evidence that LSDV can infect humans. LSDV can infect all cows, the morbidity rate is 5-85%, the mortality rate can reach 20%, the incidence of the lactating cows is the most serious, and the milk yield of the lactating cows is obviously reduced. The bull can be caused to be permanently or temporarily sterile due to orchitis and testis atrophy, and can be detoxified for a long time through semen.
Clinical and subclinical sick cattle are the major source of spread, with live cattle movement being an important cause of widespread spread, such as grazing or marketing. LSDV is widely found in skin, dermal lesions, colonic, saliva, nasal juice, milk, semen, muscle, spleen, lymph nodes, etc. When ulcers form on eyes, nose, mouth, rectum, mammary gland and genitals, the nodules are rapidly ulcerated, and the secretions of the nodules contain virus particles, such as aerosol generated by exposing the infected animals to the virus particles, so that the infected animals can be infected.
The disease has a latent period of 2-5 weeks of natural infection, with 4-12 days, usually 7 days of experimental infection. The body temperature of sick cattle can rise to more than 41 ℃ and is in a continuous heat retention type. The initial manifestations are rhinitis, conjunctivitis, keratitis. On the 4 th to 12 th days after experimental infection, the skin of the body surface appears hard, round and raised nodules with the diameter of 1-5 cm, and the skin feels painful especially to the head, the neck, the chest, the perineum, the breasts and the limbs. The skin nodules are located in the epidermis and dermis and can aggregate into irregular masses and eventually necrose. The lymph nodes on the body surface of the sick cattle are swollen, and the lymph nodes on the front shoulder, the outside of the groin, the front thigh, the hind limb and the lower ear are the most prominent. The mucous membranes in the eyes, nose, mouth, rectum, breast and external genitalia, etc. can also form nodules and ulcers. Mastitis can occur to a lactating cow, the lactation amount is reduced, the pregnant cow is aborted, the beef cattle production performance is reduced, necrotic nodules are easy to be bitten by flies, deep holes are left on the skin after the necrotic nodules fall off, and skin can not be utilized. The subcutaneous tissue was dissected and was seen to be infiltrated with a grayish red slurry. The nodules were dissected and contained cheesy gray necrotic tissue in the cavity, with purulent blood in some cases, and were deep reaching the subcutaneous or costal tissue. Similar nodules may be present on the surface of the body, pharynx, trachea, lungs, rumen, abomasum, kidneys. Eosinophilic inclusions are visible in the damaged cells at the beginning of the disease. Initial diagnosis is possible based on these clinical symptoms and pathological changes, but confirmed diagnosis requires further laboratory pathogenic and serological tests.
Primarily to identify pseudoLSD as a result of bovine herpes virus type 2 (BoHV-2). This is usually a milder clinical symptom, characterized by superficial nodules, which only resemble the early stages of LSD. Intranuclear inclusions and viral syncytia are histopathological features of BoHV-2 infection not seen in LSD. Other differential diagnoses (for skin lesions) include: dermatosis, dermatophytosis, psoriasis, photosensitivity, actinomycosis, urticaria, insect bites, belleville filariasis, nocardia disease, demodex, onchocerciasis, pseudovaccinia and vaccinia. The differential diagnosis of mucosal lesions comprises: foot and mouth disease, bluetongue, bovine viral diarrhea, malignant catarrhal fever, bovine infectious rhinotracheitis, and bovine epidemic stomatitis.
Disclosure of Invention
The invention aims to solve the technical problem of how to improve the sensitivity and specificity of the bovine sarcoidosis virus antibody detection, thereby diagnosing the bovine sarcoidosis more accurately.
In order to solve the technical problems, the invention provides a polypeptide kit for preparing a bovine nodular skin disease virus antibody detection reagent or a polypeptide kit for preparing a bovine nodular skin disease diagnostic reagent.
In a first aspect, the invention claims a polypeptide or a set of polypeptides.
The polypeptide claimed by the invention is an eA27-1 polypeptide.
The claimed polypeptide set consists of the said eA27-1 polypeptide and eA33-1 polypeptide.
The eA27-1 polypeptide is shown as the following P11, P12 or P13:
p11, polypeptide with the amino acid sequence of SEQ ID No.1,
p12, the polypeptide with the amino acid sequence of SEQ ID No.1 from the 2 nd to the 28 th,
p13, and a polypeptide which is obtained by connecting amino acid residues to the amino terminal or the carboxyl terminal of the polypeptide shown in P12 and can be coupled with a carrier protein.
The eA33-1 polypeptide is shown as the following P21, P22 or P23:
p21, polypeptide with the amino acid sequence of SEQ ID No.5,
p22, a polypeptide of which the amino acid sequence is the 2 nd to the 33 th positions of SEQ ID No.5,
p23, and a polypeptide which is obtained by connecting amino acid residues to the amino terminal or the carboxyl terminal of the polypeptide shown in P22 and can be coupled with a carrier protein.
Wherein, SEQ ID No.1 consists of 28 amino acid residues, the 1 st cysteine residue is a connecting arm added for connecting with a carrier protein, and other amino acid residues are derived from a protein A27 of the bovine sarcoidosis dermatosis virus; SEQ ID No.5 consists of 33 amino acid residues, the cysteine residue at position 1 is a connecting arm added for connecting with a carrier protein, and other amino acid residues are derived from a bovine sarcoidosis virus A33 protein.
In a second aspect, the invention claims a conjugate or a conjugate kit.
The claimed conjugates of the present invention are eA27-1 conjugates.
The claimed set of conjugates consists of the eA27-1 conjugate and the eA33-1 conjugate.
The eA27-1 conjugate is a complete antigen derived from the conjugation of the eA27-1 polypeptide and a carrier protein as described in the first aspect above; the eA33-1 conjugate is a complete antigen derived from the conjugation of the eA33-1 polypeptide and a carrier protein as described in the first aspect above.
In one embodiment of the invention, the weight ratio of the eA27-1 conjugate to the eA33-1 conjugate in the set of conjugates is 1:1.
wherein the carrier protein can be bovine serum albumin, hemocyanin, human serum albumin, ovalbumin, mouse serum albumin, thyroglobulin, rabbit serum albumin, etc.
In one embodiment of the invention, the carrier protein is Bovine Serum Albumin (BSA).
In a third aspect, the invention claims the use of a polypeptide or a polypeptide set as hereinbefore described in the first aspect or a conjugate set as hereinbefore described in the second aspect in any one of:
(A1) Preparing a reagent or a kit for detecting the bovine sarcoidosis virus antibody;
(A2) Preparing the bovine nodular skin disease virus diagnostic antigen.
In a fourth aspect, the invention claims a kit comprising a polypeptide or a polypeptide set as described in the first aspect above or a conjugate set as described in the second aspect above.
Further, the kit is used for detecting bovine sarcoidosis virus antibodies; in the kit, a polypeptide or a polypeptide set as described in the first aspect or a conjugate set as described in the second aspect is used as the coating antigen.
Still further, the kit may be a chemiluminescent immunoassay kit; the kit can also contain a horseradish peroxidase (HRP) labeled secondary antibody, a coating buffer solution, a washing solution, a secondary antibody diluent and/or a calibrator; the secondary antibody is an antibody capable of resisting the bovine sarcoidosis virus antibody.
In a fifth aspect, the invention claims any one of the following biomaterials:
(B1) A nucleic acid molecule, which is a nucleic acid molecule 1 or a nucleic acid molecule 2; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eA27-1 polypeptide of the first aspect hereinbefore; the nucleic acid molecule 2 is a nucleic acid molecule capable of encoding the eA33-1 polypeptide of the first aspect hereinbefore;
(B2) An expression cassette, which is expression cassette 1 or expression cassette 2; the expression cassette 1 is an expression cassette containing the nucleic acid molecule 1 of (B1); the expression cassette 2 is an expression cassette containing the nucleic acid molecule 2 of (B1);
(B3) The recombinant vector is a recombinant vector 1 or a recombinant vector 2; the recombinant vector 1 is a recombinant vector containing the nucleic acid molecule 1 in (B1); the recombinant vector 2 is a recombinant vector containing the nucleic acid molecule 2 in (B1);
(B4) The recombinant bacterium is a recombinant bacterium 1 or a recombinant bacterium 2; the recombinant bacterium 1 is a recombinant bacterium containing the nucleic acid molecule 1 in (B1); the recombinant bacterium 2 is a recombinant bacterium containing the nucleic acid molecule 2 in (B1);
(B5) A transgenic cell line, which is a transgenic cell line 1 or a transgenic cell line 2; the transgenic cell line 1 is a transgenic cell line containing the nucleic acid molecule 1 in (B1); the transgenic cell line 2 is a transgenic cell line containing the nucleic acid molecule 2 in (B1);
(B6) A set of nucleic acid molecules consisting of said nucleic acid molecule 1 and said nucleic acid molecule 2 of (B1);
(B7) A set of expression cassettes consisting of said expression cassette 1 and said expression cassette 2 in (B2);
(B8) A set of recombinant vectors consisting of said recombinant vector 1 and said recombinant vector 2 in (B3);
(B9) The recombinant bacteria set consists of the recombinant bacteria 1 and the recombinant bacteria 2 in the step (B4);
(B10) A set of transgenic cell lines consisting of said transgenic cell line 1 and said transgenic cell line 2 of (B5).
In a sixth aspect, the invention claims the use of a biomaterial as described in the fifth aspect hereinbefore in any one of:
(C1) Preparing a polypeptide or a polypeptide set as hereinbefore defined in the first aspect or a conjugate set as hereinbefore defined in the second aspect or a kit as hereinbefore defined in the third aspect;
(A1) Preparing a reagent or a kit for detecting the bovine nodular skin disease virus antibody;
(A2) Preparing the bovine nodular skin disease virus diagnostic antigen.
In a seventh aspect, the invention claims the use of a polypeptide or a polypeptide set as hereinbefore described in the first aspect or a conjugate set as hereinbefore described in the second aspect as an immunogen in the preparation of bovine sarcoidosis virus antibodies.
Experiments prove that a conjugate obtained by coupling the full-length A27 recombinant protein of the bovine sarcoidosis or the full-length A33 recombinant protein of the bovine sarcoidosis serving as a hapten and a carrier protein serves as a coating antigen, so that an antibody of the bovine sarcoidosis and an antibody of the foot-and-mouth disease virus cannot be effectively distinguished. In order to improve the specificity of the antibody detection of the bovine sarcoidosis, the invention selects dominant antigen epitopes eA27 and eA33 from the full-length A27 of the bovine sarcoidosis and the full-length A33 of the bovine sarcoidosis respectively, and takes a conjugate (BSA-eA 27-1 and/or BSA-eA 33-1) obtained by coupling with a carrier protein as a coating antigen, thereby effectively distinguishing the bovine sarcoidosis antibody from various virus antibodies such as a foot-and-mouth disease and the like and improving the specificity of the antibody detection of the bovine sarcoidosis. The kit for detecting the bovine nodular skin disease antibody in the serum, which is prepared by respectively using BSA-eA27-1 and/or BSA-eA33-1 as coating antigens, can accurately distinguish the bovine nodular skin disease antibody positive serum from the bovine foot-and-mouth disease virus antibody positive serum, the bovine viral diarrhea virus antibody positive serum and the bovine infectious rhinotracheitis virus antibody positive serum, has the accuracy of detecting the bovine nodular skin disease antibody positive serum consistent with the virus neutralization test result of the traditional gold standard method, and has higher sensitivity than the virus neutralization test.
The antibody detection kit prepared by taking BSA-eA27-1 and/or BSA-eA33-1 as a coating antigen has the advantages of strong specificity, high sensitivity, high accuracy, simple and quick operation, and is suitable for quick and large-scale screening and detection of bovine sarcoidosis infection serum antibodies by various veterinary departments and entry and exit inspection and quarantine bureaus in the basic level.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
pET32a (+) in the following examples is a product of Huada Gene Co. Luminol chemiluminescent substrate, available from the company luoyang research institute for modern biotechnology, ltd; chemiluminescent enzyme-labeled plates, available from Thermo corporation, usa; 10% fetal bovine serum, cell culture media and related cell culture reagents were purchased from Gibco, USA; the bovine sarcoidosis virus antibody negative and positive sample serum with the background of serum neutralization titer is preserved by a Chinese animal epidemic disease prevention control center (veterinary diagnostic center in agricultural rural areas) (the negative serum is collected from healthy cattle, the positive serum is collected from cattle clinically ill in the field, and the virus neutralization test is used for carrying out antibody titer identification by using a traditional gold standard method). Chemiluminescence immunoassay analyzer, model HEXU, purchased from Luoyang modern biotechnology, inc.
The neutralization test method of bovine sarcoidosis virus in the following examples is as follows:
(1) Treatment of serum to be tested
The serum was inactivated at 58 ℃ for 30 minutes and diluted with serum-free MEM medium in a gradient format for use.
(2) Control sera
The anti-bovine sarcoidosis virus standard negative serum and the anti-bovine sarcoidosis virus standard positive serum which have the background of serum neutralization titer are used and stored by a Chinese animal epidemic disease prevention and control center (veterinary diagnostic center of rural agricultural department).
(3) Neutralization of viruses
The concentration is 2 x 10 5 Vero cell suspension of one/ml was seeded on 96-well cell plates at 100. Mu.l/well. 5% CO at 37 ℃ 2 Incubate in incubator for 1-2 days until 70% -80% of the cells form a monolayer. 50 mul of diluted serum to be detected is mixed with equal volume of suspension of Xinjiang isolate LSDV/Xinjiang/2019 (described in the article "first LSDV isolate P32, GPCR and RPO30 gene clone in China, genetic evolution relationship and protein structure analysis, lanzhou university, academic paper, DOI:10.27204/d. Cnki. Glzhu.2020.001024", publicly available from applicant, only available for experiments of duplicate invention, not available for others) containing 100TCID50/50 mul of bovine nodular skin disease, and the suspension is mixed at 37 ℃ with equal volume of 5% CO 2 The reaction was carried out in an incubator for 1 hour.
(4) Culturing
After 1 hour of neutralization of serum and virus, 100. Mu.l of a mixed suspension of virus and serum was added to each well of the cell culture plate at 37 ℃ with 5% CO 2 The incubation was continued in an incubator.
(5) Determination of results
And (4) performing initial judgment on results 72 hours after inoculation, discarding the holes with specific lesions, replacing culture solutions of other holes with maintenance solutions, and performing rotary culture for 7 days to perform final judgment.
And (4) judging standard: those which inhibited the cytopathic effect (CPE) by 50% or more than 50% were judged to be positive. The maximum neutralizing dilution of serum was calculated according to this standard.
In the following examples, the bovine sarcoidosis virus antibody-positive sera were tested as positive sera by the virus neutralization test, and the bovine sarcoidosis virus antibody-negative sera were tested as negative sera by the virus neutralization test.
Example 1 detection of bovine sarcoidosis infection serum antibodies by chemiluminescence immunoassay
The inventor utilizes pET32a (+) in the development process to express the full-length A27 protein of bovine sarcoidosis virus and the full-length A33 recombinant protein of bovine sarcoidosis virus in Escherichia coli BL21 (DE 3) in a soluble manner. Experimental results show that the chemiluminescence immunoassay method established by respectively using the full-length A27 protein of the bovine sarcoidosis virus and the full-length A33 recombinant protein of the bovine sarcoidosis virus as envelope antigens can not effectively distinguish bovine sarcoidosis antibodies from foot-and-mouth disease virus antibodies, and has poor specificity. The invention further selects 4 dominant antigen epitope polypeptides (eA 27-1, eA27-2, eA27-3 and eA 27-4) from the full-length A27 protein of the bovine sarcoidosis virus, selects 4 dominant antigen epitope polypeptides (eA 33-1, eA33-2, eA33-3 and eA 33-4) from the full-length A33 protein of the bovine sarcoidosis virus, and is respectively coupled with BSA to serve as a chemiluminescence immunoassay method established by a coating antigen, so that the specificity is obviously improved, the bovine sarcoidosis antibody and the foot-and-mouth disease virus antibody can be effectively distinguished, but the sensitivity difference is larger. The conjugate of eA27-1 with BSA (BSA-eA 27-1) and the conjugate of eA33-1 with BSA (BSA-eA 33-1) were mixed in the following ratio of 1:1, the specificity is obviously improved, the bovine nodular skin disease antibody and the foot-and-mouth disease virus antibody can be effectively distinguished, and the sensitivity is also obviously improved. The specific experimental method is as follows:
1. involving the preparation of antigens
The following 11 coating antigens were prepared in this example:
(1)BSA-eA27-1+BSA-eA33-1，
(2) BSA-eA27-1 (eA 27-1 conjugate),
(3) BSA-eA33-1 (eA 33-1 conjugate),
(4) BSA-eA27-2 (eA 27-2 conjugate),
(5) BSA-eA33-2 (eA 33-2 conjugate),
(6) BSA-eA27-3 (eA 27-3 conjugate),
(7) BSA-eA33-3 (eA 33-2 conjugate),
(8) BSA-eA27-4 (eA 27-4 conjugate),
(9) BSA-eA33-4 (eA 33-4 conjugate),
(10) The full-length A27 protein of the bovine sarcoidosis virus,
(11) The full-length A33 recombinant protein of bovine sarcoidosis virus.
1. Dominant epitope polypeptides
Preferred epitope polypeptides were selected from the A27 protein and the A33 protein of bovine sarcoidosis virus, and polypeptides eA27-1, eA27-2, eA27-3, eA27-4, eA33-1, eA33-2, eA33-3, and eA33-4 (Table 1) having cysteine linked to the C-terminus or N-terminus were synthesized by Beijing Liuhe Huada Gene technology Co., ltd. Purity is higher than 95%, freeze-drying and storing.
TABLE 1 Polypeptides
Note: c in the sequence is cysteine residue, and is a connecting arm added at the amino terminal of the epitope polypeptide of A27 protein or A33 protein of the bovine sarcoidosis virus for connecting with the carrier protein; the other amino acid residues are derived from the A27 protein or the A33 protein of the bovine sarcoidosis virus.
2. Preparation of 11 coating antigens
10 polypeptides of eA27-1, eA27-2, eA27-3, eA27-4, eA33-1, eA33-2, eA33-3, eA33-4, eA 27-full length and eA 33-full length are coupled with BSA to obtain 10 coating antigens: (1) BSA-eA27-1 (conjugate of eA27-1 to BSA), (2) BSA-eA33-1 (conjugate of eA33-1 to BSA), (3) BSA-eA27-2 (conjugate of eA27-2 to BSA), (4) BSA-eA33-2 (conjugate of eA33-2 to BSA), (5) BSA-eA27-3 (conjugate of eA27-3 to BSA), (6) BSA-eA33-3 (conjugate of eA33-3 to BSA), (7) BSA-eA27-4 (conjugate of eA27-3 to BSA), (8) BSA-eA33-4 (conjugate of eA33-4 to BSA), (9) BSA-eA 27-full-length (conjugate of eA 27-to BSA), (10) BSA-eA 33-BSA 33-full-length BSA (conjugate of eA 33-to BSA).
BSA-eA27-1 (eA 27-1 conjugate to BSA) and BSA-eA33-1 (eA 33-1 conjugate to BSA) were mixed in the following ratio 1:1 to obtain the coating antigen (11) BSA-eA27-1+ BSA-eA33-1.
The specific preparation methods of the coating antigens (1) - (8) and (11) are as follows: BSA tag coupling kit (Readilink) manufactured by KPL corporation, USA was used TM BSA Conjugation Kit) cat No.: 5501, batch number: 148045 and prepared as envelope antigen by conjugation of the synthetic polypeptide fragments as required by the instructions.
The specific preparation methods of the coating antigens (9) and (10) are as follows:
the expressed full-length a27 protein of bovine sarcoidosis virus and the full-length a33 recombinant protein of bovine sarcoidosis virus were used as coating antigens.
The first step is as follows: construction of full-length A27 protein gene and full-length A33 protein gene recombinant expression vector
The fragment between the BamH I and XhoI recognition sites of pET32a (+) at position 1-388 (GenBank Accession No. MN901872.1) (Update Date is 28-OCT-2020) was substituted with another sequence of pET32a (+) while keeping the other sequence of pET32a (+) unchanged, to obtain a full-length recombinant expression vector of the A27 protein gene of bovine sarcoidosis virus, which was designated as pET32a-flA27.pET32a-flA27 can express fusion protein containing full-length A27 protein (the amino acid sequence is GenBank Accession No. MN901872.1, and the Update Date is No. 1-129 of 28-OCT-2020, namely SEQ ID No. 9) of bovine sarcoidosis virus.
A fragment between BamH I recognition sites and XhoI recognition sites of pET32a (+) (a small fragment between the BamH I recognition site and the XhoI recognition site) was replaced with a fragment between 113441 th to 114031 th sites (Update Date is 20-DEC-2020) having a nucleotide sequence of GenBank Accession No. NC-003027.1, and the other sequence of pET32a (+) was kept unchanged, to obtain a full-length A33 protein gene recombinant expression vector of bovine sarcoidosis virus, which was designated as pET32a-flA33.pET32a-flA33 can express fusion protein containing full-length A33 protein (the amino acid sequence is GenBank Accession No. NP-150556.1, the amino acid Date is 1-196 of 20-DEC-2020, namely SEQ ID No. 10) of bovine sarcoidosis virus.
The second step is that: construction of recombinant strains
Separately transforming two expression vectors of pET32a-flA27 and pET32a-flA33 constructed in the first step into competent cells of Escherichia coli BL21 (DE 3) respectively. This was spread evenly on LB plates containing ampicillin (50. Mu.g/mL) and cultured at 37 ℃ for 16 hours. And performing shaking culture on the single colony overnight, extracting a plasmid for sequencing, and naming the recombinant Escherichia coli containing pET32a-flA27 as BL21 (DE 3)/pET 32a-flA27 according to the sequencing result, and naming the recombinant Escherichia coli containing pET32a-flA33 as BL21 (DE 3)/pET 32a-flA33 according to the sequencing result.
The third step: soluble expression and purification of full-length A27 protein of bovine sarcoidosis virus or full-length A33 recombinant protein of bovine sarcoidosis patella
The two strains BL21 (DE 3)/pET 32a-flA27 and BL21 (DE 3)/pET 32a-flA33 were separately inoculated into LB liquid medium containing 50. Mu.g/ml ampicillin (medium obtained by adding ampicillin to LB liquid medium to 50. Mu.g/ml), and cultured at 37 ℃ to 0D with shaking using a Thermo Max Q6000 type full temperature shaker at 200rpm 600 When the value (blank in LB liquid medium containing 50. Mu.g/ml ampicillin) reached 0.6, IPTG was added for induction of expression. The inducible expression was induced with 0.75mM IPTG for 13h at 16 ℃. Collecting the bacterial sediment from the fermentation liquor after IPTG induced expression for 13h. Adding PBS to resuspend the precipitate, centrifuging at 8000rpm/min for 5min, and discarding the supernatant. Adding PBS into the washed thallus precipitate, crushing thallus under high pressure, cracking until the thallus is not viscous, centrifuging at 16000rpm/min in a centrifuge at 4 ℃ for 30min, collecting supernatant, and discarding the precipitate. The supernatant was filtered through a 0.22 μm filter and applied to a previously applied solution 1 (solutes and concentrations thereof: 20mM Tris, 150mM NaCl, dissolved in water)The agent is water, ph8.0 solution) is well balanced. The nickel column was loaded onto an AKTA machine, the impurity proteins in the nickel column were washed with 10 column volumes of solution 1 and 10 column volumes of solution 2 (solute and concentration: 20mM Tris, 150mM NaCl, 50mM imidazole, solvent: water, pH 8.0), respectively, and the protein peak was monitored on the AKTA machine. The target protein on the nickel column was washed with solution 3 (solute and concentration: 20mM Tris, 150mM NaCl, 300mM imidazole, solvent water, pH 8.0), and the eluted sample on which the target protein peak appeared was collected using AKTA, and further purified by molecular sieves using Superdex200 gel column manufactured by GE to obtain molecular sieve-purified full-length A27 protein of bovine sarcoidosis virus and full-length A33 recombinant protein of bovine sarcoidosis virus, respectively.
2. Chemiluminescence immune analysis by using chemiluminescence immune analysis reagent kit for diagnosing bovine nodular skin disease virus or chemiluminescence immune analysis reagent kit for detecting bovine nodular skin disease virus antibody
This example provides 11 kinds of chemiluminescence immunoassay kits for diagnosing bovine sarcoidosis virus or detecting bovine sarcoidosis virus antibody. These 11 kits each included a coating antigen, a horseradish peroxidase (HRP) -labeled bovine secondary antibody (cat # HM390, xu (Zheng) Biotechnology Co., ltd.), a coating buffer (cat # HC210, xu (Zheng) Biotechnology Co., ltd.), a washing solution (cat # HX221, xu (Zheng) Biotechnology Co., ltd.), and a secondary antibody diluent (cat # HM391, xu (Zheng) Biotechnology Co., ltd.). These 11 kits differ only in the coating antigen, and the other components are identical.
The 11 kits are respectively a chemiluminescence immunoassay kit for diagnosing bovine sarcoidosis virus of BSA-eA27-1+ BSA-eA33-1 with the coating antigen as the first step or a chemiluminescence immunoassay kit for detecting bovine sarcoidosis virus antibody, which is hereinafter referred to as the kit 1 of the invention; a chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or detecting the bovine nodular skin disease virus antibody, wherein the coating antigen is BSA-eA27-1 in the first step, and is hereinafter referred to as a kit 2 of the invention; the kit for chemiluminescence immunoassay of diagnosing bovine nodular skin disease virus or detecting bovine nodular skin disease virus antibody, which is coated with the antigen as the first step, of BSA-eA33-1 is hereinafter referred to as the kit 3 of the invention. A chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or a chemiluminescence immunoassay kit for detecting the bovine nodular skin disease virus antibody, which is coated with the antigen BSA-eA27-2 in the first step, is hereinafter referred to as a control kit 1; a chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or a chemiluminescence immunoassay kit for detecting the bovine nodular skin disease virus antibody, which is coated with the antigen BSA-eA33-2 in the first step, is hereinafter referred to as a control kit 2; a chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or a chemiluminescence immunoassay kit for detecting the bovine nodular skin disease virus antibody, which is coated with the antigen BSA-eA27-3 in the first step, is hereinafter referred to as a control kit 3; a chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or a chemiluminescence immunoassay kit for detecting the bovine nodular skin disease virus antibody, which is coated with the antigen BSA-eA33-3 in the first step, is hereinafter referred to as a control kit 4; a chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or a chemiluminescence immunoassay kit for detecting the bovine nodular skin disease virus antibody, which is coated with the antigen BSA-eA27-4 in the first step, is hereinafter referred to as a control kit 5; a chemiluminescence immunoassay kit for diagnosing the bovine nodular skin disease virus or a chemiluminescence immunoassay kit for detecting the bovine nodular skin disease virus antibody, which is coated with the antigen BSA-eA33-4 in the first step, is hereinafter referred to as a control kit 6; a chemiluminescence immunoassay kit for diagnosing the bovine sarcoidosis virus or a chemiluminescence immunoassay kit for detecting an antibody of the bovine sarcoidosis virus, which is coated with the full-length A27 protein of the bovine sarcoidosis virus with the antigen as the first step, is hereinafter referred to as a control kit 7; a chemiluminescence immunoassay kit for diagnosing the bovine sarcoidosis virus or a chemiluminescence immunoassay kit for detecting an antibody of the bovine sarcoidosis virus, which takes the full-length A33 protein of the bovine sarcoidosis virus with the antigen as the first step, and is hereinafter referred to as a control kit 8.
Firstly, a chemiluminescence immunoassay method (hereinafter referred to as CLIA method 1 of the invention) using BSA-eA27-1+ BSA-eA33-1 as a coating antigen is established by using the kit 1 of the invention through optimization experiments, and the coating antigen, a horseradish peroxidase (HRP) labeled secondary antibody and a secondary antibody diluent.
1. Procedure for the preparation of the
(1) Coating: the concentration of BSA-eA27-1+ BSA-eA33-1 in step one (total concentration by mass of BSA-eA27-1 and BSA-eA 33-1) is 1.0. Mu.g/ml, resulting in a coating antigen solution, the assay wells are coated with the coating buffer, 100. Mu.L/well is added to the ELISA plate, and incubation is carried out for 16h at 4 ℃.
(2) Washing: pouring out the original coating solution in the holes, and washing for 3min for 5 times by using a washing solution; patting to dry.
(3) And (3) sealing: 1% BSA blocking solution was added, 350. Mu.L/well, and incubated at 37 ℃ for 2h.
(4) Washing: the same as the step (2).
(5) Sample adding: diluting the sample to be tested at a ratio of 1: 20 with a sample diluent (1 XPBST) in a serum dilution plate; the coated plate is taken out, and a series of 6 holes of the calibrator are required to be arranged in each experiment. Firstly, 100 mu L of each of the calibrators 1-6 is sequentially added on a serum dilution plate, 100 mu L of each of the diluted samples to be detected is sequentially added in the other holes, and the samples to be detected are placed in a constant temperature incubator at 37 ℃ for incubation for 30min (+ -1 min).
(6) Washing: the same as the step (2).
(7) And (3) adding 100 mu L of horseradish peroxidase (HRP) labeled secondary antibody diluted by secondary antibody diluent into each hole, and uniformly mixing by shaking. Sucking 100 mu L of each hole and transferring the sample into the corresponding hole of the coated plate; incubate at 37 ℃ for 30min (+ -1 min).
(8) Washing: the same as the step (2).
(9) Adding luminol chemiluminescence substrate: adding 100 mu L luminol chemiluminescence substrate into each hole, and oscillating and mixing uniformly; standing in a dark place at 15-25 deg.c for 5min, and reading the light value with a chemiluminescence instrument within 15min after standing.
2. Result calculation method
The NCU (National clinical unit) is a unit established by a National clinical center, the unit is cited in the method to assign 20000NCU/ml to standard positive serum (the neutralizing antibody titer is 1: 512), a four-parameter method calibration curve is established by the standard positive serum with different dilution times, and the luminous value of a detection sample and the antibody dosage value (NCU/ml) of the calibration curve are converted, so that semi-quantitative detection is realized.
The bovine sarcoidosis virus antibody-positive serum having a background of serum neutralization titer was used to set calibrators 1 to 6 and corresponding antibody dose values (0 NCU/mL, 10NCU/mL, 20NCU/mL, 50NCU/mL, 100NCU/mL, 200 NCU/mL) according to the virus neutralization test results. And drawing a four-parameter fitting curve of the calibrator by using ELISACalc software by taking the luminous values of the calibrators 1 to 6 as vertical coordinates and the corresponding antibody dose values as horizontal coordinates, wherein the four-parameter mode is Y = (a-b)/[ 1+ (x/c) × b ] + d. Wherein a is an asymptote estimate on the curve; b is the slope of the curve; c is the dose corresponding to half of the maximum binding; d is the estimate of the asymptote under the curve.
Substituting the luminous value of the sample into the calibration curve for calculation to obtain the content of the bovine nodular skin disease virus antibody in the sample.
3. Test establishment condition and result judgment
Performing four-parameter fitting on the luminous values of the 6 calibrators and the dosage values of the corresponding antibodies, R 2 And the test is established, wherein the test is more than or equal to 0.99. Otherwise, the test does not hold.
4. Determination of negative and positive cut-off values
The CLIA test was performed using 200 bovine sarcoidosis virus antibody-negative sera by the above method, and the average value (X) and Standard Deviation (SD) of the dose values of the 200 bovine sarcoidosis virus antibody-negative sera were calculated. The average dose value X of 200 bovine nodular skin disease antibody negative sera is 28.59, SD is 3.64, so the negative and positive critical dose values
It was 39.51NCU/mL. Namely, the dosage value is more than or equal to 39.51NCU/mL, and the test result is positive; dosage value < 39.51NCU/mL was judged negative.
(II) by utilizing the kit 2 of the invention, a chemiluminescence immunoassay method (hereinafter, referred to as CLIA method 2 of the invention) with BSA-eA27-1 as a coating antigen is established through an optimization experiment, and the steps are the same as those in the step (I).
(III) by utilizing the kit 3 of the invention, a chemiluminescence immunoassay method (hereinafter, referred to as CLIA method 3 of the invention) with BSA-eA33-1 as a coating antigen is established through an optimization experiment, and the steps are the same as those in the step (I).
And (IV) a chemiluminescence immunoassay method (hereinafter referred to as control CLIA method 1) using BSA-eA27-2 as a coating antigen is established by using the control kit 1 of the invention through an optimization experiment, and the steps are the same as those in the step (I).
(V) by using the control kit 2 of the invention, a chemiluminescence immunoassay method (hereinafter referred to as control CLIA method 2) with BSA-eA33-2 as a coating antigen is established through an optimization experiment in the same step.
And (VI) establishing a chemiluminescence immunoassay method (hereinafter referred to as control CLIA method 3) by using the control kit 3 of the invention and using BSA-eA27-3 as a coating antigen through an optimization experiment, wherein the steps are the same as those of the step (I).
And (seventhly), a chemiluminescence immunoassay method (hereinafter referred to as control CLIA method 4) using BSA-eA33-3 as a coating antigen is established by using the control kit 4 of the invention through an optimization experiment in the same step.
(VIII) by using the control kit 5 of the invention, a chemiluminescence immunoassay method (hereinafter referred to as control CLIA method 5) with BSA-eA27-4 as a coating antigen is established through an optimization experiment in the same step.
(ninth), the control kit 6 of the invention is used to establish a chemiluminescence immunoassay method (hereinafter referred to as control CLIA method 6) with BSA-eA33-4 as a coating antigen through an optimization experiment, and the steps are the same as the steps in the step (I).
(tenth) by using the control kit 7 of the present invention, a chemiluminescence immunoassay method (hereinafter, referred to as control CLIA method 7) using the full-length a27 protein as a coating antigen was established through an optimization experiment in the same procedure as (i).
(eleventh) the same procedure as in (one) was established for a chemiluminescence immunoassay method (hereinafter, referred to as control CLIA method 8) using the full-length A33 protein as a coating antigen by using the control kit 8 of the present invention through an optimization experiment.
3. Specificity test
Comparing the CLIA method 1-3 and the CLIA comparison method 1-8 in the step two with bovine foot-and-mouth disease virus antibody positive serum, bovine viral diarrhea virus antibody positive serum and bovine infectious rhinotracheitis virus antibody positive serum (all positive sera are collected from the serum of clinically diseased animals), and observing whether cross reaction exists between the serum and other congeneric diseases. In addition, detection was performed simultaneously by virus neutralization assay (see above for details).
The results show that: the CLIA methods 1 to 3 of the present invention and the CLIA methods 1 to 8 of the present invention are each judged to be negative if the antibody dose value is less than 40 NCU/ml. The specificity was 100%. The neutralization indexes of the virus neutralization tests are all less than 1:5 the results are all negative according to the national standard. The results are shown in tables 1 and 2.
TABLE 1 test results of specificity of the methods 1 to 3 of the present invention and the Virus neutralization test
TABLE 2 results of specificity tests in control methods 1-8
4. Sensitivity test
The positive serum of the bovine nodular skin disease virus antibody is subjected to multiple dilution, and the CLIA method 1-3 and a serum sample with known serum neutralization titer background are respectively adopted for comparison detection to obtain the maximum dilution at the positive critical value. The results show that the highest dilution times of the positive sera detected by the CLIA methods 1-3 of the invention are all 1-1024 times. Results of virus neutralization assay the highest dilution of positive sera was 1. See table 3.
TABLE 3 CLIA methods 1-3 of the invention and neutralization assay sensitivity results
5. Repeatability test
The method 1-3 of the invention is adopted to respectively detect 10 bovine nodular skin disease virus antibody positive serums on the same batch of plates and different batches of plates, the detection is carried out for 5 times in parallel, and the variation Coefficient (CV) between batches is calculated. The results show that the CLIA method of the invention has a coefficient of variation of repeat within 8% within batches and a coefficient of variation of repeat within batches of less than 10% (tables 4 to 6). The result shows that the positive serum of the bovine sarcoidosis virus antibody has good repeatability.
TABLE 4 CLIA METHOD 1 repeatability tests of the invention
TABLE 5 CLIA METHOD 2 repeatability tests of the invention
TABLE 6 CLIA METHOD 3 repeatability tests of the invention
6. Test of compliance
80 parts of bovine serum with positive serum neutralizing titer background of bovine nodular skin disease virus antibodies and 80 parts of bovine serum with negative serum neutralizing titer background of bovine nodular skin disease virus antibodies are selected by using bovine serum stored by a Chinese animal epidemic prevention control center (veterinary diagnosis center in rural areas of agriculture). The 160 parts of bovine serum are respectively detected by a CLIA method 1-3 and a CLIA control method 1-8 of the invention, and the coincidence rate of the 160 parts of bovine serum and a neutralization test method is calculated.
The results show that the total coincidence rate of the 160 parts of bovine serum, the CLIA method 1 and the neutralization test method is 98.75% (the positive coincidence rate is 100%, and the negative coincidence rate is 97.5%), the CLIA method 2 and the bovine nodular skin disease virus neutralization test method is 95.625% (the positive coincidence rate is 93.75%, and the negative coincidence rate is 97.5%), and the CLIA method 3 and the bovine nodular skin disease virus neutralization test method are 96.25% (the positive coincidence rate is 92.5%, and the negative coincidence rate is 100%). The total coincidence rate of the detection result of the control CLIA method 1 and the neutralization test method for the bovine sarcoidosis virus is 85.625% (the positive coincidence rate is 85% and the negative coincidence rate is 86.25%), the total coincidence rate of the detection result of the control CLIA method 2 and the neutralization test method for the bovine sarcoidosis virus is 78.125% (the positive coincidence rate is 81.25% and the negative coincidence rate is 75%), the total coincidence rate of the detection result of the control CLIA method 3 and the neutralization test method for the bovine sarcoidosis virus is 75.625% (the positive coincidence rate is 80% and the negative coincidence rate is 71.25%), the total coincidence rate of the detection result of the control CLIA method 4 and the neutralization test method for the bovine sarcoidosis virus is 78.125% (the positive coincidence rate is 100% and the negative coincidence rate is 56.25%), the total coincidence rate of the detection result of the control CLIA method 5 and the neutralization test method for the bovine sarcoidosis virus is 79.375% (the positive coincidence rate is 80% and the negative coincidence rate is 100%), the control CLIA method for the negative coincidence rate is 6.25%), and the total coincidence rate of the negative test method for the negative CLIA virus is 75% (the negative coincidence rate is 75.85% and the negative coincidence test method for the negative sarcoidosis virus is 75.75%). See in particular tables 7 to 16.
The above results show that the total coincidence rate of the chemiluminescence immunoassay kit for antibody of bovine sarcoidosis virus prepared by respectively using BSA-eA27-1+ BSA-eA33-1, BSA-eA27-1 and BSA-eA33-1 as coating antigens or the chemiluminescence immunoassay kit for detecting antibody of bovine sarcoidosis virus and the neutralization test method is significantly higher than that of the chemiluminescence immunoassay kit for antibody of bovine sarcoidosis virus prepared by respectively using BSA-eA27-2, BSA-eA27-3, BSA-eA27-4, BSA-eA33-2, BSA-eA33-3, BSA-eA33-4, full-length A27 protein of bovine sarcoidosis virus and full-length A33 protein of bovine sarcoidosis virus as coating antigens or the chemiluminescence immunoassay kit for detecting antibody of bovine sarcoidosis virus.
TABLE 7 results of the measurement of bovine serum samples according to CLIA method 1 of the present invention
TABLE 8 results of the measurement of bovine serum samples according to CLIA method 2 of the present invention
TABLE 9 results of bovine serum samples tested by CLIA method 3 of the present invention
TABLE 10 bovine serum sample test results in control CLIA method 1
TABLE 11 results of bovine serum samples tested by CLIA control method 2
TABLE 12 results of bovine serum samples tested by CLIA method 3
TABLE 13 bovine serum sample test results by CLIA method 4 control
TABLE 14 bovine serum sample test results in comparison with CLIA method 5
TABLE 15 bovine serum sample test results in comparison with CLIA method 6
TABLE 16 results of bovine serum samples tested by CLIA control method 7
TABLE 17 bovine serum sample test results by CLIA control method 8
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A polypeptide or a polypeptide set, characterized in that: the polypeptide is an eA27-1 polypeptide; the polypeptide set consists of the eA27-1 polypeptide and the eA33-1 polypeptide;
the eA27-1 polypeptide is shown as the following P11, P12 or P13:
p11 and polypeptide with the amino acid sequence of SEQ ID No. 1;
p12, polypeptide with the amino acid sequence of 2-28 of SEQ ID No. 1;
p13, polypeptide which is obtained by connecting amino acid residues at the amino terminal or the carboxyl terminal of the polypeptide shown in P12 and can be coupled with carrier protein;
the eA33-1 polypeptide is shown as the following P21, P22 or P23:
p21 and polypeptide with the amino acid sequence of SEQ ID No. 5;
p22 and polypeptide with the amino acid sequence of 2-33 of SEQ ID No. 5;
p23, and a polypeptide which is capable of coupling with a carrier protein and is obtained by connecting an amino acid residue to the amino terminus or the carboxyl terminus of the polypeptide represented by P22.
2. A conjugate or set of conjugates characterized in that: the conjugate is an eA27-1 conjugate; the set of conjugates consists of the eA27-1 conjugate and the eA33-1 conjugate;
the eA27-1 conjugate is a complete antigen obtained by coupling the eA27-1 polypeptide and a carrier protein in claim 1; the eA33-1 conjugate is a complete antigen obtained by coupling the eA33-1 polypeptide and a carrier protein in claim 1.
3. The conjugate or conjugate set according to claim 2, wherein: in the set of conjugates, the mass ratio of the eA27-1 conjugate to the eA33-1 conjugate is 1:1.
4. use of a polypeptide or polypeptide set according to claim 1 or a conjugate or conjugate set according to claim 2 or 3 in any of:
(A1) Preparing a reagent or a kit for detecting the bovine sarcoidosis virus antibody;
(A2) Preparing the bovine nodular skin disease virus diagnostic antigen.
5. A kit comprising a polypeptide or a polypeptide set according to claim 1 or a conjugate or conjugate set according to claim 2 or 3.
6. The kit of claim 5, wherein: the kit is used for detecting the bovine nodular skin disease virus antibody; in said kit, a polypeptide or polypeptide set according to claim 1 or a conjugate or conjugate set according to claim 2 or 3 as a coating antigen.
7. The kit according to claim 5 or 6, characterized in that: the kit is a chemiluminescence immunoassay kit; the kit also contains horseradish peroxidase labeled secondary antibodies, coating buffer solution, washing solution, secondary antibody diluent and/or calibrator; the secondary antibody is an antibody capable of resisting the bovine sarcoidosis virus antibody.
8. Any one of the following biomaterials:
(B1) A nucleic acid molecule, which is a nucleic acid molecule 1 or a nucleic acid molecule 2; the nucleic acid molecule 1 is a nucleic acid molecule capable of encoding the eA27-1 polypeptide of claim 1; the nucleic acid molecule 2 is a nucleic acid molecule capable of encoding the eA33-1 polypeptide of claim 1;
(B2) An expression cassette, which is expression cassette 1 or expression cassette 2; the expression cassette 1 is an expression cassette containing the nucleic acid molecule 1 of (B1); the expression cassette 2 is an expression cassette containing the nucleic acid molecule 2 of (B1);
(B3) The recombinant vector is a recombinant vector 1 or a recombinant vector 2; the recombinant vector 1 is a recombinant vector containing the nucleic acid molecule 1 in (B1); the recombinant vector 2 is a recombinant vector containing the nucleic acid molecule 2 in (B1);
(B4) The recombinant bacterium is a recombinant bacterium 1 or a recombinant bacterium 2; the recombinant bacterium 1 is a recombinant bacterium containing the nucleic acid molecule 1 in (B1); the recombinant bacterium 2 is a recombinant bacterium containing the nucleic acid molecule 2 in (B1);
(B5) A transgenic cell line, which is a transgenic cell line 1 or a transgenic cell line 2; the transgenic cell line 1 is a transgenic cell line containing the nucleic acid molecule 1 in (B1); the transgenic cell line 2 is a transgenic cell line containing the nucleic acid molecule 2 in (B1);
(B6) A set of nucleic acid molecules consisting of said nucleic acid molecule 1 and said nucleic acid molecule 2 of (B1);
(B7) A set of expression cassettes consisting of said expression cassette 1 and said expression cassette 2 in (B2);
(B8) A set of recombinant vectors consisting of said recombinant vector 1 and said recombinant vector 2 in (B3);
(B9) The recombinant bacteria set consists of the recombinant bacteria 1 and the recombinant bacteria 2 in the step (B4);
(B10) A set of transgenic cell lines consisting of said transgenic cell line 1 and said transgenic cell line 2 of (B5).
9. Use of the biomaterial of claim 8 in any one of the following:
(C1) Preparing a polypeptide or a polypeptide set according to claim 1 or a conjugate set according to claim 2 or 3 or a kit according to any one of claims 4 to 7;
(A1) Preparing a reagent or a kit for detecting the bovine sarcoidosis virus antibody;
(A2) Preparing the bovine nodular skin disease virus diagnostic antigen.
10. Use of a polypeptide or polypeptide set according to claim 1 or a conjugate or conjugate set according to claim 2 or 3 as an immunogen in the preparation of antibodies to bovine sarcoidosis virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211265005.8A CN115873077A (en) | 2022-10-17 | 2022-10-17 | Reagent for detecting bovine nodular skin disease virus antibody and polypeptide used by reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211265005.8A CN115873077A (en) | 2022-10-17 | 2022-10-17 | Reagent for detecting bovine nodular skin disease virus antibody and polypeptide used by reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115873077A true CN115873077A (en) | 2023-03-31 |
Family
ID=85770438
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211265005.8A Pending CN115873077A (en) | 2022-10-17 | 2022-10-17 | Reagent for detecting bovine nodular skin disease virus antibody and polypeptide used by reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115873077A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116478280A (en) * | 2023-06-16 | 2023-07-25 | 金宇保灵生物药品有限公司 | Niu Jiejie skin disease virus positive serum and preparation method and application thereof |
-
2022
- 2022-10-17 CN CN202211265005.8A patent/CN115873077A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116478280A (en) * | 2023-06-16 | 2023-07-25 | 金宇保灵生物药品有限公司 | Niu Jiejie skin disease virus positive serum and preparation method and application thereof |
| CN116478280B (en) * | 2023-06-16 | 2023-09-12 | 金宇保灵生物药品有限公司 | Niu Jiejie skin disease virus positive serum and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110642925B (en) | African swine fever virus synthetic peptide ELISA antibody detection kit | |
| CN110618279A (en) | African swine fever virus epitope antigen polypeptide and application thereof | |
| CN103760365B (en) | A kind of BAS-ELISA kit detecting CSFV Erns and E2 antigen | |
| CN110746495A (en) | Recombinant protein E2 and application thereof | |
| CN110873792A (en) | African swine fever virus antibody detection kit | |
| CN110221065A (en) | A kind of fowl Mycoplasma synoviae indirect ELISA testing kit | |
| CN115873077A (en) | Reagent for detecting bovine nodular skin disease virus antibody and polypeptide used by reagent | |
| CN101982777B (en) | Duck plague virus antigen capturing ELISA method based on anti-recombination UL51 albumen antibody | |
| CN111978411B (en) | Porcine reproductive and respiratory syndrome subunit vaccine and preparation method and application thereof | |
| CN104297493B (en) | Soluble Type I DHV 3D albumen is in the application prepared in ELISA reagent and ELISA kit thereof | |
| CN109053896B (en) | Porcine circovirus bivalent gene engineering vaccine | |
| CN102183658B (en) | Recombinant UL55 protein-based duck plague virus antibody detection method | |
| CN112225780B (en) | Polypeptide for detecting Seneca virus antibody and application thereof | |
| CN110196325B (en) | In-plug virus diagnosis kit and test paper | |
| CN102360013B (en) | ELISA kit for detecting duck plague virus antibody, and antibody detection method thereof | |
| CN104293823A (en) | Preparation method and application of soluble I-type DHV (Duck Hepatitis Virus) 3D protein | |
| CN115873078A (en) | Polypeptide for detecting bovine nodular skin disease virus antibody | |
| CN114487397A (en) | Indirect ELISA detection kit for detecting porcine delta coronavirus | |
| CN110133290B (en) | ELISA kit for diagnosing heartworm disease | |
| CN113698474A (en) | African swine fever polyclonal antibody and African swine fever antigen detection test strip | |
| CN102012432B (en) | Colloidal gold immunochromatographic test paper based on recombinant UL51 protein as well as preparation method and application thereof | |
| CN111537736A (en) | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit and detection method for mycoplasma gallisepticum antibody | |
| CN102174087B (en) | UL55 recombinant prokaryotic expression protein for detecting DPV antibody and preparation method thereof | |
| CN114409806B (en) | CA-TM fusion protein and application thereof in detecting Meidi-Weiston virus | |
| CN114751963B (en) | Protein for detecting foot-and-mouth disease virus antibody and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |