Screening method for antibody detection targets of unknown pathogens and application

Abstract

The application discloses a screening method for antibody detection target of unknown pathogen, and comprises the following steps: 1) collecting and separating the unknown pathogen, and performing whole genome sequencing; 2) predicting the protein coding gene of the strain or strain of the unknown pathogen and screening potential target; 3) in vitro expression of the screened target; 4) antibody detection using the screened target; 5) the target that meets the antibody growth and decline rule in the antibody detection of step 4) can be used as the antibody detection target of the unknown pathogen. Therefore, when a new pathogen is generated or the core gene sequence / protein sequence of the pathogen is unknown, the method is used to realize rapid and efficient screening of the antibody detection target of the unknown pathogen and establishment of the antibody detection method of the unknown pathogen, thereby laying a foundation for diagnosis, treatment and prevention and control of the unknown pathogen.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method and application for screening antibody detection targets for unknown pathogens. Background Technology

[0002] When a disease with an unknown pathogen emerges, how to efficiently and quickly diagnose / detect the unknown pathogen becomes a thorny issue.

[0003] In livestock farming, duck mycoplasma (MA) disease, also known as duck infectious sinusitis, is a respiratory infectious disease of ducks caused by mycoplasma (mycoplasma). It is rarely reported internationally, and research on it is limited. Its genome has an ANI value of less than 74% with other mycoplasma species, indicating it is a completely new species. Duck mycoplasma is characterized by a relatively slow development time, long duration, and long transmission period within the duck body. Infected ducks and carrier ducks are the main sources of infection. Susceptible ducks are infected through the air via the respiratory tract, and it can also be transmitted vertically through eggs contaminated with the secretions and excrement of infected or carrier ducks. Ducks aged 2-3 weeks are susceptible, with a morbidity rate as high as 80% and a low mortality rate. Infected ducks exhibit inflammation and swelling of the infraorbital sinus, with serous, mucous, or caseous secretions. Duck mycoplasma is an opportunistic pathogen, becoming pathogenic under weakened conditions or certain stress conditions. This disease reduces the duck's resistance and often occurs concurrently with influenza A and E. coli infections, affecting production performance.

[0004] Pathogen detection methods are divided into antibody detection methods and antigen detection methods. Antigen detection methods are based on known conserved gene sequences, while antibody detection methods are based on conserved proteins with good immune responses, making them more challenging. For pathogens with few reference sequences and unclear protein functions, establishing corresponding antibody detection methods is very difficult, but both antigen and antibody detection methods are essential for studying pathogen characteristics.

[0005] Therefore, there is an urgent need to create a highly applicable method for screening antibody detection targets for unknown pathogens, so as to provide a foundation for the establishment of antibody detection methods for unknown pathogens and lay the foundation for the research, diagnosis and treatment of unknown pathogens. Summary of the Invention

[0006] The purpose of this invention is to provide a highly applicable method and application for screening antibody detection targets for unknown pathogens, in order to solve the problems of rapid and effective screening of antibody detection targets for unknown pathogens and the rapid and efficient establishment of antibody detection methods for unknown pathogens when the pathogen gene sequence or protein sequence is unknown.

[0007] According to a first aspect of the present invention, a method for screening antibody detection targets for unknown pathogens is provided, the method comprising the following steps:

[0008] 1) Collect and isolate unknown pathogens, and perform whole-genome sequencing;

[0009] 2) Prediction of protein-coding genes of unknown pathogens or strains and screening of potential targets.

[0010] 3) Express the selected targets in vitro;

[0011] 4) Use the screened targets for antibody detection;

[0012] 5) In step 4), antibodies that conform to the antibody fluctuation pattern can be used as antibody detection targets for unknown pathogens.

[0013] Therefore, when a new pathogen emerges or the core gene sequence / protein sequence of the pathogen is unknown, this method can be used to achieve rapid and effective screening of antibody detection targets for the unknown pathogen and to establish an antibody detection method for the unknown pathogen.

[0014] In some implementations, the prediction of protein-coding genes and the screening of potential targets include: using Prokka v1.13.7 software to analyze the whole genome sequence of an unknown pathogen to predict its protein-coding genes; based on the better antigenicity of membrane proteins and lipoproteins, the aim is to screen for target proteins in the unknown pathogen that simultaneously possess membrane protein and lipoprotein characteristics: firstly, Prokka v1.13.7 software is used to analyze the membrane localization of the hypothetical protein, and proteins located on the membrane are selected, thereby screening out membrane proteins; then, among the screened membrane proteins, the binding protein domains and annotated protein functions are analyzed, and proteins that simultaneously possess the lipoprotein signal peptidase SpII are screened out; finally, proteins that simultaneously meet the membrane protein and lipoprotein characteristics predicted by the software are screened out as potential targets.

[0015] In some implementations, the unknown pathogen is Mycoplasma duckii.

[0016] According to a second aspect of the present invention, a method for screening antibody detection targets for unknown pathogens is provided for use in screening antibody detection targets for unknown pathogens. This enables rapid antibody detection of an unknown pathogen upon its attack.

[0017] According to a third aspect of the present invention, a method for screening antibody detection targets for unknown pathogens is provided, and its application in screening antibody detection targets for duck mycoplasma disease is provided. This enables rapid antibody detection of this duck mycoplasma disease.

[0018] In some embodiments, the application of a method for screening antibody detection targets for unknown pathogens in screening antibody detection targets for duck mycoplasma disease includes the following steps:

[0019] 1) Collect and isolate the pathogenic strains of duck mycoplasma disease and perform whole-genome sequencing;

[0020] 2) Prediction of protein-coding genes and screening of potential targets in duck mycoplasma strains;

[0021] 3) Express the selected targets in vitro;

[0022] 4) Use the screened targets for antibody detection;

[0023] 5) In step 4), antibodies that conform to the pattern of antibody growth and decline can be used as antibody detection targets for duck mycoplasma disease.

[0024] In some embodiments, the nucleotide sequence of the antibody detection target encoding duck mycoplasma disease is shown in SEQ ID No:3, SEQ ID No:5, or SEQ ID No:7;

[0025] The amino acid sequence of the target protein for the screening of duck mycoplasma disease antibody detection is shown in SEQ ID No:4, SEQ ID No:6, or SEQ ID No:8.

[0026] According to a fourth aspect of the present invention, an antibody detection target for duck mycoplasma disease is provided, wherein the nucleotide sequence encoding the antibody detection target for duck mycoplasma disease is shown in SEQ ID No:3, SEQ ID No:5, or SEQ ID No:7. Therefore, the nucleotide sequence of this target can be used to prepare reagents for detecting duck mycoplasma disease, thereby achieving rapid and efficient antibody detection of duck mycoplasma disease.

[0027] According to a fifth aspect of the present invention, an antibody detection target for duck mycoplasma disease is provided, the amino acid sequence of which is shown in SEQ ID No:4, SEQ ID No:6, or SEQ ID No:8. Therefore, the amino acid sequence of this target protein can be used to prepare reagents for detecting duck mycoplasma disease, thereby achieving rapid and efficient antibody detection of duck mycoplasma disease.

[0028] According to a sixth aspect of the present invention, an application of an antibody detection target for duck mycoplasma disease is provided in the preparation of a reagent for detecting duck mycoplasma disease antibodies. Thus, by using this reagent, rapid and efficient antibody detection of duck mycoplasma disease can be achieved.

[0029] According to a seventh aspect of the present invention, an application of an antibody detection target for duck mycoplasma disease is provided in the preparation of a kit for detecting duck mycoplasma disease antibodies. Thus, by using this kit, rapid and efficient antibody detection of duck mycoplasma disease can be achieved.

[0030] The beneficial effects of this application are:

[0031] 1. A method for screening antibody detection targets for unknown pathogens is provided. This method enables rapid and efficient screening of antibody detection targets for unknown pathogens and rapid and efficient establishment of antibody detection methods for unknown pathogens, thereby laying the foundation for the diagnosis, treatment and prevention of unknown pathogens.

[0032] 2. A method for screening antibody detection targets for unknown pathogens is provided, and its application in screening antibody detection targets for unknown pathogens is discussed. This enables rapid antibody detection of unknown pathogens during their attack.

[0033] 3. A method for screening antibody detection targets for unknown pathogens is provided, and its application in screening antibody detection targets for duck mycoplasma disease is discussed. This enables rapid antibody detection of this duck mycoplasma disease.

[0034] 4. A nucleotide sequence of an antibody detection target for duck mycoplasma disease is provided. The nucleotide sequence of this target can be used to prepare reagents for detecting duck mycoplasma disease, thereby achieving rapid and efficient antibody detection of duck mycoplasma disease.

[0035] 5. An amino acid sequence of an antibody detection target for duck mycoplasma disease is provided. The amino acid sequence of this target can be used to prepare reagents for detecting duck mycoplasma disease, thereby achieving rapid and efficient antibody detection of duck mycoplasma disease.

[0036] 6. An application of an antibody detection target for duck mycoplasma disease in the preparation of an antibody detection reagent for duck mycoplasma disease is provided. By using this reagent, rapid and efficient antibody detection of duck mycoplasma disease can be achieved.

[0037] 7. An application of an antibody detection target for duck mycoplasma disease in the preparation of a kit with duck mycoplasma disease antibody detection function is provided. By using this kit, rapid and efficient antibody detection of duck mycoplasma disease can be achieved. Attached Figure Description

[0038] Figure 1 The genome sequences of 16 MA strains are shown: the two innermost loops represent GC content and GC skew plots, and the 16 outermost loops show paired nucleotide sequences between the complete genome of LC01 as a reference and the genome assemblies of other MA strains.

[0039] Figure 2 The results of whole-genome protein sequence analysis of Mycoplasma duckissis: Based on membrane protein and lipoprotein characteristics, proteins located on the membrane were selected. According to COG annotation classification, lipoprotein classification, and protein function, 13 proteins were selected as potential targets for subsequent vaccine design, namely: GIG_00396, GIG_01286, GIG_01885, GIG_03093, GIG_03662, GIG_00030, GIG_03894, GIG_00346, GIG_02828, GIG_03188, GIG_03969, GIG_02453, and GIG_00456;

[0040] Figure 3 SDS-PAGE analysis results of purified fusion proteins: M is the molecular weight standard of the protein, 1 is the purified 01885 fusion protein, 2 is the purified 03093-1 fusion protein, 3 is the purified 03093-2 fusion protein, 4 is the purified 03093-3 fusion protein, 5 is the purified 00396 fusion protein, 6 is the purified 03662 fusion protein, and 7 is the blank control;

[0041] Figure 4 OD of unimmunized duck serum negative samples 450nm The critical value result of the value calculation;

[0042] Figure 5 The results of antibody fluctuation analysis for inactivated vaccines targeting 03093-1 are as follows: the horizontal axis represents the antibody detection time point, and the vertical axis represents the OD (October Deficiency Rate). 450nm value

[0043] Figure 6 The results of mycoplasma antibody detection in breeding ducks of different ages from an unimmunized duck farm in Guangdong Province were obtained by using the 03093-1 target protein as the target for mycoplasma antibody detection. Detailed Implementation

[0044] The screening method for antibody detection targets of unknown pathogens includes the following steps:

[0045] 1) Collect and isolate unknown pathogens, and perform whole-genome sequencing;

[0046] 2) Prediction of protein-coding genes of unknown pathogens and screening of potential targets: Prokka v1.13.7 software was used to analyze the whole genome sequence of unknown pathogens to predict their protein-coding genes. Based on the better antigenicity of membrane proteins and lipoproteins, the aim was to screen for target proteins in unknown pathogens that simultaneously possess membrane protein and lipoprotein characteristics. First, Prokka v1.13.7 software was used to analyze the membrane localization of hypothetical proteins, and proteins located on the membrane were selected to screen for membrane proteins. Then, the structural domains of the selected membrane proteins and the annotated protein functions were analyzed to screen for proteins that simultaneously possess lipoprotein signal peptidase SpII. Finally, proteins that simultaneously meet the membrane protein and lipoprotein characteristics predicted by the software were screened as potential targets for further validation.

[0047] 3) Express the selected targets in vitro;

[0048] 4) Use the screened targets for antibody detection;

[0049] 5) In step 4), antibodies that conform to the antibody fluctuation pattern can be used as antibody detection targets for unknown pathogens.

[0050] Example: Screening antibody detection targets using Mycoplasma anatis (MA) disease of unknown pathogen in ducks as an example:

[0051] I. The complete genome sequence of Mycoplasma duckii

[0052] Since the inventors have already disclosed the pathogenic strain sequence information of duck mycoplasma in the literature (Zhou Q, Mai K, Yang D, Liu J, Yan Z, Luo C, Tan Y, Cao S, Zhou Q, Chen L, Chen F. Comparative genomic analysis of Mycoplasma anatisstrains. Genes Genomics. 2021 Nov; 43(11):1327-1337.), these sequences can be directly analyzed to draw the whole genome sequence map of duck mycoplasma. The literature collected and isolated pathogenic strains of duck mycoplasma (isolated as many strains as possible, a total of 15 strains were isolated in the literature). The 15 isolated pathogenic strains of duck mycoplasma were sequenced and comparative genomics studies were performed. The information of the 15 isolated and sequenced pathogenic strains of duck mycoplasma is shown in Table 1. In Table 1, “GenBank accession” is the NCBI accession number of the corresponding strain. The last strain in Table 1 is NCTC. 10156 is the only publicly available complete genome sequence of *Mycoplasma duckii* in other literature. The literature used PacBio Sequel and IonTorrent Personal Genome Machine (PGM) sequencers to perform draft genome sequencing and complete genome sequencing of the strain, obtaining the complete genome sequence of *Mycoplasma duckii*. Figure 1 The image shows the whole genome sequence map of duck mycoplasma: the two innermost loops represent GC content and GC skew, and the outermost 16 loops show paired nucleotide sequences between the complete genome of LC01 as a reference and the genome assemblies of other MA strains.

[0053] Table 1. Detailed information on 115 strains of Mycoplasma duckii on NCBI.

[0054]

[0055]

[0056] II. Prediction of protein-coding genes and selection of potential targets

[0057] Prediction of protein-coding genes and screening of potential targets: Prokka v1.13.7 software was used to analyze the whole genome sequence of Mycoplasma duckii to predict its protein-coding genes. Based on the better antigenicity of membrane proteins and lipoproteins, the aim was to screen for target proteins in Mycoplasma duckii that simultaneously possess membrane protein and lipoprotein characteristics. First, Prokka v1.13.7 software was used to analyze the membrane localization of the hypothetical proteins, and proteins located on the membrane were selected to screen for membrane proteins. Then, the structural domains of the selected membrane proteins and the annotated protein functions were combined to screen for proteins that simultaneously possess the lipoprotein signal peptidase SpII. Finally, proteins that simultaneously meet the membrane protein and lipoprotein characteristics predicted by the software were screened as potential targets for further validation.

[0058] The results are as follows Figure 2 As shown: Thirteen targets were screened out and denoted according to their Seq IDs (which correspond to loci on the whole genome of Mycoplasma duckii) as follows: GIG_00396, GIG_01286, GIG_01885, GIG_03093, GIG_03662, GIG_00030, GIG_03894, GIG_00346, GIG_02828, GIG_03188, GIG_03969, GIG_02453, and GIG_00456, which are abbreviated as: 00396, 01286, 01885, 03093, 03662, 00030, 03894, 00346, 02828, 03188, 03969, 02453, and 00456.

[0059] Among them, genes 01286, 03188, and 02453 failed to amplify by PCR; genes 02828, 00030, 00456, 03894, 00346, and 03969 showed low homology and also needed to be excluded; therefore, four possible candidate targets were finally obtained: 01885, 00396, 03093, and 03662. However, among the four candidate targets 01885, 00396, 03093, and 03662: 00396 and 03662 do not have signal peptides; 03093 is 2616 bp in length, and after removing the signal peptide at the front, there is still 2541 bp, which may not be conducive to prokaryotic expression, so 03093 needs to be expressed in segments, divided into anterior and posterior segments according to the antigenic epitope analysis results; 01885 needs to have its signal peptide truncated. Therefore, the four candidate targets were optimized and improved into six new target sequences: the 01885 target (with the signal peptide truncated), the 03093 target (with the complete sequence, its first and last segments expressed separately, designated as 03093-1, 03093-2, and 3093-3), the 00396 target, and the 03662 target. The nucleotide and amino acid sequences of these six improved targets are as follows (SEQ ID No: 1-12):

[0060]

[0061] Target amino acid sequence (SEQ ID No: 2):

[0062] MNKNRENNDVINISLISPWMEDENKPEQIRKSFESKLQAIIGNKVKIKVSYSSDDYLV

[0063] NLQNINKGVVDLAFVSHSSFENYISENQEKSNRIKPIIQTLTKSFKYDTDNFIYVDGTE

[0064] NDKIISNAKLQSELFNEKRYSEWDYPWNGSVYEYFYDDKLVDFQRGIIWISGTDEVR

[0065] EKIIKAWNEKDWKTFRSFGIVHGSADSGSKYLLPEKLLKKHFNKNDNAFSSLASEITN

[0066] YSNYFINGKTKDMLKNNHFHICFDNEGSYSWTQNTKDNINKYTPLNNEKMEILALTDPLQYNIGVVNTNKVNYEIQNLIVSSLIQLYTSKQDDWGKTVGFYGY.

[0067] Target nucleotide sequence of 03093-1 (SEQ ID No: 3) (2439bp): ATGGAAGAGAAGGCCAAACCGAAAGAACCAGCCGAGAACCCGAACATCCCGAGTGAGCCGGGCGATCAGCCAACCAATCCGGAAACCGGCGATACGAATAGCGGCAGCAGCAACAGTGGTAGCAACAGCGGTAGCGACAATGGCAACAGCAATGGCACGGAACAGACGAAGAAGCTCACGCTGAAGATCGTGGGCACCATTCCAGAAGCCCTCAG

[0068] CGGTCTGAAAATTGTTCTGAATAATAAAACCATTCCGGAAGTTTTCTACAATACCC

[0069] AGTTTACCATGGAAAATCTGATCCTCACGAACTACAAAAAAGACAAATTTATCGA

[0070] AAATGAGACGCTGCTCCTCACCCAGATCCAGAATAGCAAGACGAATGAGCTCAAA

[0071] GCGCAGATCAAATTCGTTGATAAGAATAATGTTCCGTACAACGTGGACATTCTCAT

[0072] CACCAACTTCGCCGAATGGCCAACGTTTGACGAAGCCGCGAGTAACGTGAGTTTC

[0073] GATAACCTCACCAAGAAGGACTATCTGGCGAAGTATACCAAAGACGACGACATCA

[0074] AATTTGTGAGCAATTATCCAAGCAGTATCATTAGTACGCAAGATCTGAAATGGCGC

[0075] ATCGATGAGGAAAGCAACCGTGTGTTCGTGACGACCAAGTTTCGCTACACGGTTA

[0076] AGCCGAGTGAGATCAGCACCAAAACCTACGAGTTTACCATCGATAACTACGGTCA

[0077] AGGCGACCGTCGCGAAATCGGTAATGTGGAGATCGGTAGCAGCAAGATCGAAGT

[0078] GGGTGACGATGGTAAGGGCAAGGTGGATACCAACGTGAAGGCCAGCTTTGGCGC

[0079] GCTGCTGCAAGAAATTGCGGAGATGAACAACAAGTTCGACGCGTGGATCTATGAC

[0080] ATCACGAACAACAGCAGCATTAACCAGAGTGGCCACTTTTGGCTCACCGGTGTTC

[0081] GCGAAAACAGCCTCAATGCGCTGCGCGAATTTCAAGAAATCTACAATAGCATCCG

[0082] CAACACGGATGACGCGAACCGCATCCGTGAGGCGGCCGAAGAAATCAAAAGCAA

[0083] AATCAACGAGATCCAGAAGAATTGGGAGAGCTACATCGCCCGTACCTTCCCGCCA

[0084] AGCCTCGAATTTGCGAGCAATCCGAACAAGGCGGAGATCAAGAAGATGATTCTGG

[0085] ACACCTTCGCGGGTCTGAAAAACAAGAGTTACGAAAATCTGATTTTTCGCGAGTA

[0086] CAAGCCAAACTACAAGCCGACGGAAGAAGATAACAAGCCGAACATCAAGATTGA

[0087] CGCGGACTACGCCTACAATGCGATGATGGAGATTCTGAAAGATGAATATTACAAGT

[0088] ATCCGTACGTGAGTCTGGTGGCGGAAAGCGAGCTGAAATTTACGACCAAAAACA

[0089] ACAGTCTGATCGGCTTCAGCGTTGGTAACAGTCTGCTCAATACGCGCGTGCAGAC

[0090] CAAAAAGGACATGGTGCAGTATGCCATCAATGCCCTCAGTCAGATCGAGAGCAGT

[0091] ATGCCGGTGCACCAGAAGGTGTATCTGCTGGGTCGTTTTGTGATGGAAAATCTGG

[0092] TGTATACCAGCGCCAGTAACAGCGCCGGCCTCAGCACCCTCGATGAAGCCTATGC

[0093] GCTGAATGAAGGCGTGTGCAAGGAGTACGTTGAGCAGCTCGCGCTGCTGCTCAGT

[0094] CTGGCGAACATCCGCTTCAAGATCTACACCGGTGAGCAACACACGTGGCTGAGTA

[0095] TCCAAGACGAGAACAATAATTGGTTTATTAGCGACCCGACCCACAGCGACTTCGG

[0096] CATCCCGGAGAAATACCATCCGGTTGGCAAGGATAGCACGTTCAACCAAGATGAG

[0097] GAAATGCTGCAGTTCATCCGTCGCGTGCACAGCGTTAGCAGCAACCAAACGCTCT

[0098] TTAGTCTGACGCCGACCAACGCGTTCAACGACAGCAGCCTCATCAGCGAGAGCTA

[0099] CCAGAGCAATCTGTTCACGACGAAGGTGATCGGTAAGATCCGCACGGAGAAAAG

[0100] CAAAAGCAGCGCGCTGAGCTTCTACAATAATAACTTCTATCTCATCGTGGACAATG

[0101] GCAGCGGTGCGAAACTGTTCAACTTTGACCCGAACGACACGAGCCACGAGTATG

[0102] TGTTCAACCAAGTGAGCAACTTTAACAACTACAGTGGTACGCTGACGACCAACGC

[0103] GGTGAATATCGGCAAGTATCTGTACTTTATGACGAACAATGGCAGCAGTAAGAGCA

[0104] TCTATAGCCTCAATCTGGAGACGAACCAGACCGAGCTGATCCGCAACAATCTGAC

[0105] CGGTGAGAATTTCTTTATCAAACAGAATATCAACACGAAAGAACTCGACTTCTATC

[0106] TGGGTGTGGACGATAGCAGCAAATACACGCTGGCCTTCAGCATTAGCCCGAAAGA

[0107] AGATAGCTATAAGCAGCTGGAAGTGAACACGATCAAACGCGTGGCCTACATGGCG

[0108] ATCGGTCTGCTGAACTTCGAGTAA

[0109] Target amino acid sequence of 03093-1 (SEQ ID No:4):

[0110] MEEKAKPKEPAENPNIPSEPGDQPTNPETGDTNSGSSNSGSNSGSDNGNSNGTEQTK

[0111] KLTLKIVGTIPEALSGLKIVLNNKTIPEVFYNTQFTMENLILTNYKKDKFIENETLLLTQ

[0112] IQNSKTNELKAQIKFVDKNNVPYNVDILITNFAEWPTFDEAASNVSFDNLTKKDYLA

[0113] KYTKDDDIKFVSNYPSSIISTQDLKWRIDEESNRVFVTTKFRYTVKPSEISTKTYEFTID

[0114] NYGQGDRREIGNVEIGSSKIEVGDDGKGKVDTNVKASFGALLQEIAEMNNKFDAWI

[0115] YDITNNSSINQSGHFWLTGVRENSLNALREFQEIYNSIRNTDDANRIREAAEEIKSKIN

[0116] EIQKNWESYIARTFPPSLEFASNPNKAEIKKMILDTFAGLKNKSYENLIFREYKPNYKP

[0117] TEEDNKPNIKIDADYAYNAMMEILKDEYYKYPYVSLVAESELKFTTKNNSLIGFSVGN

[0118] SLLNTRVQTKKDMVQYAINALSQIESSMPVHQKVYLLGRFVMENLVYTSASNSAGLS

[0119] TLDEAYALNEGVCKEYVEQLALLLSLANIRFKIYTGEQHTWLSIQDENNNWFISDPTH

[0120] SDFGIPEKYHPVGKDSTFNQDEEMLQFIRRVHSVSSNQTLFSLTPTNAFNDSSLISESY

[0121] QSNLFTTKVIGKIRTEKSKSSALSFYNNNFYLIVDNGSGAKLFNFDPNDTSHEYVFNQ

[0122] VSNFNNYSGTLTTNAVNIGKYLYFMTNNGSSKSIYSLNLETNQTELIRNNLTGENFFIKQNINTKELDFYLGVDDSSKYTLAFSISPKEDSYKQLEVNTIKRVAYMAIGLLNFE.03093-2 Target Nucleotide Sequence (SEQ ID No: 5) (900 bp): ATGAGCGCCAGTAACAGCGCCGGCCTCAGCACCCTCGATGAAGCCTATGCGCTGAATGAAGGCGTGTGCAAGGAGTACGTTGAGCAGCTCGCGCTGCTGCTCAGTCTGGCGAACATCCGCTTCAAGATCTACACCGGTGAGCAACACACGTGGCTGAGTATCCAAGACGAGAACAATAATTGGTTTATTAGCGACCCGACCCACAGCGACTTCGGCATCCCGGAGAAATACCATCCGGTTGGCAAGGATAGCACGTTCAACCAAGATGAGGAAATGCTGCAGTTCATCCGTCGCGTGCACAGCGTTAGCAGCAACCAAACGCTCTTTAGTCTGACGCCGACCAACGCGTTCAACGACAGCAGCCTCATCAGCGAGAGCTACCAGAGCAATCTGTTCACGACGAAGGTGATCGGTAAGATCCGCACGGAGAAAAGCAAAAGCAGCGCGCTGAGCTTCTACAATAATAACTTCTATCTCATCGTGGACAATGGCAGCGGTGCGAAACTGTTCAACTTTGACCCGAACGACACGAGCCACGAGTATGTGTTCAACCAAGTGAGCAACTTTAACAACTACAGTGGTACGCTGACGACCAACGCGGTGAATATCGGCAAGTATCTGTACTTTATGACGAACAATGGCAGCAGTAAGAGCATCTATAGCCTCAATCTGGAGACGAACCAGACCGAGCTGATCCGCAACAATCTGACCGGTGAGAATTTCTTTATCAAACAGAATATCAACACGAAAGAACTCGACTTCTATCTGGGTGTGGACGATAGCAGCAAATACACGCTGGCCTTCAGCATTAGCCCGAAAGAAGATAGCTATAAGCAGCTGGAAGTGAACACGATCAAACGCGTGGCCTACATGGCGATCGGTCTGCTGAACTTCGAGTAA.

[0123] 03093-2 Target Amino Acid Sequence (SEQ ID No: 6):

[0124] MSASNSAGLSTLDEAYALNEGVCKEYVEQLALLSLANIRFKIYTGEQHTWLSIQDE

[0125] NNNWFISDPTHSDFGIPEKYHPVGKDSTFNQDEEMLQFIRRVHSVSSNQTLFSLTPTN

[0126] AFNDSSLISESYQSNLFTTKVIGKIRTEKSSSALSFYNNNFYLIVDNGSGAKLFNFDP

[0127] NDTSHEYVFNQVSNFNNYSGTLTTNAVNIGKYLYFMTNNGSSKSIYSLNLETNQTEL

[0128] IRNNLTGENFFIKQNINTKELDFYLGVDDSSKYTLAFSISKEDSYKQLEVNTIKRVAYMAIGLLNFE.

[0129]

[0130] Target amino acid sequence of 03093-3 (SEQ ID No: 8):

[0131] MEEKAKPKEPAENPNIPSEPGDQPTNPETGDTNSGSSNSGSNSGSDNGNSNGTEQTK

[0132] KLTLKIVGTIPEALSGLKIVLNNKTIPEVFYNTQFTMENLILTNYKKDKFIENETLLLT

[0133] QIQNSKTNELKAQIKFVDKNNVPYNVDILITNFAEWPTFDEAASNVSFDNLTKKDYL

[0134] AKYTKDDDIKFVSNYPSSIISTQDLKWRIDEESNRVFVTTKFRYTVKPSEISTKTYEFTI

[0135] DNYGQGDRREIGNVEIGSSKIEVGDDGKGKVDTNVKASFGALLQEIAEMNNKFDAW

[0136] IYDITNNSSINQSGHFWLTGVRENSLNALREFQEIYNSIRNTDDANRIREAAEEIKSKI

[0137] NEIQKNWESYIARTFPPSLEFASNPNKAEIKKMILDTFAGLKNKSYENLIFREYKPNYKPTEEDNKPNIKID.

[0138] Target nucleotide sequence of 00396 (SEQ ID No: 9) (183 bp):

[0139] ATGAGCTTCTGGATCTATCTGATCGCGCTGATCGTTTGTCTGGTTCTGGCCATCTT

[0140] CTGTCTGAGTCTGTACCCGATCAGCATGAAGAAGATGCGCAACTACAAGCAAGC

[0141] CCAGATGATCGAGTACAAGAAGAACCACCCGAAGAGCAAGCTGACCGATTACA

[0142] ATGCCACCGGCATGTACTAA

[0143] Target amino acid sequence (SEQ ID No: 10):

[0144] MSFWIYLIALIVCLVLAIFCLSLYPISMKKMRNYKQAQMIEYKKNHPKSKLTDYNAT GMY.

[0145] Target nucleotide sequence (SEQ ID No: 11) (543 bp):

[0146] ATGAACTTTCTGATCGCCACCTTCATCTTCGCGCTGATCGTGGTGATCAGTCTGGT

[0147] GTACCGCTTTCTGATCTACAACGCGCTGATCCAGAACAGCAAGGAGCTGAACAG

[0148] CAATCTGCCGTACACCAGCGTGAAGTACGCGAAGAAGGTGCTGGACAAGAAGA

[0149] ATCAGCTGATCCAGTTCGAATTCAACCAGAACTACTTCACGTTCGAGCTGGACAA

[0150] CCGCAAGAAGATCATTCATTTTAATGAGAGCATCATCGGCGACTACAGCGTGTA

[0151] CAGTCTGACCATGAGCTATCTGCAGTGCTACAAGTTCATGCTCACCAAAAAATGG

[0152] ATGAACATCGTGCAGTACGTGATCAAGCTGCTGTTCCCGATCATCTGGATCAGCA

[0153] TTTTCGTGCTGCTGTTCTTCCAGTTCTGGATCATCAGCGCGATTCTGTTCATCAGC

[0154] ATTCTGCTGATCGCGCTGCTGAGCTTCGGCATCTTCTACAAAACGCGCCGTCTGA

[0155] TCAACGACAGCGTGATGGAACAGCTGAAGTACGTGCTCGCCGAAAATTAA

[0156] Target amino acid sequence 03662 (SEQ ID No:12):

[0157] MNFLIATFIFALIVVISLVYRFLIYNALIQNSKELNSNLPYTSVKYAKKVLDKKNQLIQ

[0158] FEFNQNYFTFELDNRKKIIHFNESIIGDYSVYSLTMSYLQCYKFMLTKKWMNIVQYVI KLLFPIIWISIFVLLFFQFWIISAILFISILLIALLSFGIFYKTRRLINDSVMEQLKYVLAEN. III. In vitro expression of target genes.

[0159] Construction of pGEX plasmids: Target sequence fragments 01885, 03093-1, 03093-2, 03093-3, 00396 and 03662 were ligated into the pGEX-4T-1 backbone vector, and transformed and purified to obtain recombinant target plasmids with correct sequencing results.

[0160] Prokaryotic expression of recombinant target protein: Target plasmids with correct sequencing results were transformed into Rosetta bacteria and aseptically plated on LB / AMP plates, incubated at 37°C for 18 h. Single clones with positive PCR results were picked and inoculated at a 1:100 ratio into 10 mL of LB / AMP liquid medium, and cultured on a shaker at 37°C and 200 rpm until OD500 was reached. 600nm When the value was approximately 0.6, IPTG was added to a final concentration of 0.4 mmol / L, and expression was induced at 25℃ and 150 rpm for 6 h. 1 mL of bacterial culture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the culture was resuspended in 50 μL of PBS. 50 μL of 2× protein loading buffer was added, and the culture was boiled at 100℃ for 5 min. The supernatant was then centrifuged again and analyzed by SDS-PAGE to select strains showing positive protein expression.

[0161] The purification method for recombinant target proteins is as follows:

[0162] After equilibrating GST-tag Purification Resin 2-3 times with an equal volume of buffer to the gel, add 4 mL of bacterial lysis supernatant to every 0.5 mL of gel (1:8 ratio), and gently shake at 4°C on a side-shaking or horizontal shaker for 60 min. Load the mixture of lysis buffer and GST-tag Purification Resin into an appropriate empty column tube, open the cap at the bottom of the purification column, and allow the liquid to flow out under gravity. Wash the column 5 times, adding 1-2 column volumes of lysis buffer each time. After the GST-tagged protein binds to the purification column and is thoroughly washed with washing buffer, elute three times with glutathione-containing elution buffer. The eluent is the target protein, and its concentration is determined using a nucleic acid protein concentration analyzer.

[0163] The results are as follows Figure 3 As shown, the recombinant proteins of four targets (01885, 03093-1, 03093-2, and 03093-3) were successfully purified, while no expression bands were observed for 00396 and 03662. Therefore, 00396 and 03662 are not suitable as antibody detection targets for duck mycoplasma disease, and the four targets (01885, 03093-1, 03093-2, and 03093-3) will continue to be screened.

[0164] IV. Further Screening of Target Proteins

[0165] Four target proteins (01885, 03093-1, 03093-2, and 03093-3) were used for coating, and eight negative and eight positive sera were randomly selected for ELISA. As shown in Table 2, the OD of the blank group without serum was significantly higher than that of the target protein 01885. 450nm The value is abnormally high, so this target is excluded.

[0166] Table 2 Preliminary screening results of target protein 01885

[0167]

[0168] V. Establishment of Antibody Detection Methods

[0169] The reaction conditions for indirect ELISA were finally determined using the checkerboard method and the single-variable method. The specific experimental steps are as follows:

[0170] (1) Coating antigen: The target proteins (i.e., the three target proteins 03093-1, 03093-2, and 03093-3 were tested separately) were added to the microplate at a concentration of 1 μg / mL using the coating solution, 100 μL / well, and coated overnight at 4℃;

[0171] (2) Washing: Discard the liquid in the well, spin dry, add PBST to wash, 250 μL / well, repeat 3 times, and pat dry.

[0172] (3) Sealing: Pat dry the residual liquid in the well, add 5% skim milk powder diluted with PBST, 300 μL / well, 37℃, 1h; wash, same as step (2);

[0173] (4) Incubate primary antibody: Pat dry any remaining liquid in the well, add primary antibody serum diluted with PBS, 1:200, 100 μL / well, 37℃, 1.5 h; wash, same as step (2);

[0174] (5) Incubate the secondary antibody: Pat dry any remaining liquid in the wells, add enzyme-labeled secondary antibody diluted with PBS, 1:1000, 100 μL / well, 37℃, 1.5 h; wash, same as step (2);

[0175] (6) Color development: Add TMB, 100 μL / well, 37℃, 5 min;

[0176] (7) Termination: Add 2M H2SO4, 50μL / well;

[0177] (8) Reading: Quickly place the plate into the microplate reader and detect the OD value at a wavelength of 450nm.

[0178]

[0179]

[0180] As shown in Table 3, the target protein 03093-1 was added to the ELISA plate at a concentration of 1 μg / mL, 100 μL / well, with primary antiserum diluted 1:200. The highest P / N value was determined to be the ELISA condition.

[0181]

[0182] As shown in Table 4, the target protein 03093-3 was added to the ELISA plate at a concentration of 1 μg / mL, 100 μL / well, with primary antibody serum diluted 1:200, and the highest P / N value was determined as the ELISA condition.

[0183] Using the same method described above, the target protein 03093-2 was added to the ELISA plate at a concentration of 1 μg / mL, 100 μL / well, with primary antibody serum diluted 1:200, and the highest P / N value was used to determine the ELISA conditions.

[0184] At this point, the three targets 03093-1, 03093-2, and 03093-3 have all shown good characteristics after ELISA verification. Considering that 03093-1 is the complete 03093 protein including 03093-2 and 03093-3, it is theoretically more specific. Therefore, 03093-1 was initially selected for further verification based on theoretical principles.

[0185] The established indirect ELISA method was used to detect the serum from 30 clinically unimmunized ducks. Each sample was tested in triplicate, and the OD of negative samples was calculated. 450nm The mean (X) and variance (SD) of the values. The critical value = X + 3SD, and the calculated critical value is 0.3657. The results are as follows: Figure 4 As shown. Therefore, the criteria for determining whether duck mycoplasma is negative or positive, namely OD... 450nm A value greater than or equal to 0.3657 is considered positive, and a value less than 0.3657 is considered negative.

[0186] V. Determination of Antibody Detection Targets

[0187] Healthy ducks aged 9 days were immunized with an inactivated duck mycoplasma vaccine. Blood samples were collected every week for four consecutive weeks. The antibody levels and decreases after immunization were detected using the methods described in Section IV, "Establishment of Antibody Detection Methods." The results are as follows: Figure 5 This indicates that the 03093-1 target protein can reflect the antibody growth pattern after immunization, ultimately confirming that the 3093-1 target protein can serve as a target for detecting duck mycoplasma diseases. VI. Application of the Antibody Target for Duck Mycoplasma Diseases (03093-1 Target)

[0188] The 03093-1 target protein was used as a target for the detection of duck mycoplasma antibodies. Duck mycoplasma antibodies were detected in breeding ducks of different ages from an unvaccinated duck farm in Guangdong. The results showed (…). Figure 6 The antibody levels of the ducklings in the brooding period were relatively low, while the antibody levels of the growing and laying ducks were high, indicating that Mycoplasma duckii was widely present in the farm and that prevention and control measures needed to be strengthened.

[0189] The routine monitoring method for duck mycoplasma disease in duck farms only requires regular blood sampling of the duck flock, and then using the method in "IV. Establishment of Antibody Detection Methods" to detect the antibody status in the blood, which can reveal the duck flock's duck mycoplasma infection status.

[0190] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0191] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0192] This invention is not limited to the above description of the embodiments. Any improvements and modifications made by those skilled in the art based on the content disclosed herein without creative effort should be within the protection scope of this invention.

Claims

1. A method for screening antibody detection targets for unknown pathogens, wherein, The method includes the following steps: 1) Collect and isolate unknown pathogens, and perform whole-genome sequencing; 2) Prediction of protein-coding genes and screening of potential targets for unknown pathogens: Prokka v1.13.7 software was used to analyze the whole genome sequence of unknown pathogens to predict their protein-coding genes. Based on the better antigenicity of membrane proteins and lipoproteins, the aim was to screen for target proteins in unknown pathogens that simultaneously possess membrane protein and lipoprotein characteristics. First, Prokka v1.13.7 software was used to analyze the membrane localization of hypothetical proteins, and proteins located on the membrane were selected to screen for membrane proteins. Then, the structural domains of the bound proteins and the annotated protein functions were analyzed among the screened membrane proteins to screen for proteins that simultaneously possess lipoprotein signal peptidase SpII. Finally, proteins that simultaneously meet the membrane protein and lipoprotein characteristics predicted by the software were screened as potential targets for further validation. 3) Express the selected targets in vitro; 4) Antibody detection using the screened targets: The antibody detection method includes determining the reaction conditions for indirect ELISA using the checkerboard method and the single variable method, and using the established indirect ELISA method to detect the antibody growth and decline patterns after immunization with inactivated vaccines; 5) In step 4), antibodies that conform to the antibody fluctuation pattern can be used as antibody detection targets for unknown pathogens.

2. The screening method according to claim 1, wherein the unknown pathogen is Mycoplasma duckii.

3. The application of the method of claim 1 in screening antibody detection targets for unknown pathogens.

4. The application of the method according to claim 1 or 2 in screening antibody detection targets for mycoplasma diseases in ducks.

5. The application according to claim 4, wherein, The applications include: 1) Collect and isolate strains of duck mycoplasma disease and perform whole-genome sequencing; 2) Prediction of protein-coding genes and screening of potential targets for Mycoplasma duckis: Prokka v1.13.7 software was used to analyze the whole genome sequence of Mycoplasma duckis to predict its protein-coding genes. Based on the better antigenicity of membrane proteins and lipoproteins, the aim was to screen for target proteins in Mycoplasma duckis that possess both membrane protein and lipoprotein characteristics. First, Prokka v1.13.7 software was used to analyze the membrane localization of the hypothetical protein and select proteins located on the membrane to screen for membrane proteins. Then, the structural domains of the selected membrane proteins and the annotated protein functions were combined to screen for proteins that also possess the lipoprotein signal peptidase SpII. Finally, proteins that simultaneously meet the membrane protein and lipoprotein characteristics predicted by the software were screened as potential targets for further validation. 3) Express the selected targets in vitro; 4) Antibody detection using the screened targets: The antibody detection method includes determining the reaction conditions for indirect ELISA using the checkerboard method and the single variable method, and using the established indirect ELISA method to detect the antibody growth and decline patterns after immunization with inactivated vaccines; 5) In step 4), antibodies that conform to the pattern of antibody fluctuation can be used as antibody detection targets for duck mycoplasma disease.

6. The application according to claim 5, wherein, The nucleotide sequences of the antibody detection targets encoding duck mycoplasma disease selected are shown in SEQ ID No:3, SEQ ID No:5, or SEQ ID No:7; The amino acid sequence of the target protein for the screening of duck mycoplasma disease antibody detection is shown in SEQ ID No:4, SEQ ID No:6, or SEQ ID No:

8.

7. An antibody detection target for duck mycoplasma disease, wherein, The nucleotide sequence encoding the antibody detection target for the aforementioned duck mycoplasma disease is shown in SEQ ID No:3, SEQ ID No:5, or SEQ ID No:

7.

8. An antibody detection target for duck mycoplasma disease, wherein, The amino acid sequence of the antibody target protein for mycoplasma infection in ducks is shown in SEQ ID No:4, SEQ ID No:6, or SEQ ID No:

8.

9. The application of the antibody detection target for duck mycoplasma disease as described in claim 7 or 8 in the preparation of a reagent for detecting duck mycoplasma disease antibodies; or its application in the preparation of a kit with the function of detecting duck mycoplasma disease antibodies.

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