Application of Antibody Against SDE3 Protein of *Candidatus Liberibacter asiaticus* in Detection of Huanglongbing of Citrus

Abstract

The present invention discloses the application of the antibody against the SDE3 protein of *Candidatus Liberibacter asiaticus* in the detection of citrus huanglongbing, belonging to the technical field of molecular biology. The SDE3 protein antigen is the protein of A1) or A2): A1) the protein with the amino acid sequence of SEQ ID No.2; A2) a soluble fusion protein obtained by fusing a tag protein at the carboxyl terminus and / or amino terminus of the protein shown in A1) and having the same activity as A1). The antibody provided by the present invention can simply and accurately detect whether a plant is infected with *Candidatus Liberibacter asiaticus*, and can provide great support for the detection of *Candidatus Liberibacter asiaticus* in plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to the application of the SDE3 protein antibody of Candidatus Liberibacter asiaticus in the detection of Huanglongbing of citrus. Background Art

[0002] Huanglongbing (HLB) of citrus is a devastating insect-borne bacterial disease that harms the global citrus industry. Candidatus Liberibacter asiaticus is a phloem-limited and difficult-to-culture Gram-negative α-proteobacterium. According to its geographical classification, it can be divided into three subspecies, namely Candidatus Liberibacter asiaticus (Las), Candidatus Liberibacter americanus (Lam), and Candidatus Liberibacter africanus (Laf). Las and Lam are transmitted by the Asian citrus psyllid (Diaphorina citri), while Laf is transmitted by the African citrus psyllid (Trioza erytreae). Among them, Las is the most widely distributed. It was first reported in Asia but has spread to Africa, the Middle East, and throughout the Americas, and the symptoms caused by it are more severe.

[0003] Previous studies have shown that Las lacks a typical type III (T3SS) secretion system but contains a complete Sec secretion system (Secretory pathway). The Sec secretion system is a conserved pathway within bacteria and is involved in the transmembrane transport process of bacterial proteins, which is very important for their survival. The chaperone protein SecB recognizes the pre-secretory protein with an N-terminal signal peptide, guides it to the inner membrane to bind to SecA, and SecA guides the protein to the SecYEG channel and provides energy for protein translocation. The pre-protein passes through the SecYEG channel through the inner membrane, the signal peptide is cleaved, and it is transferred to the periplasm. Then it is secreted extracellularly through the B-barrel protein, autotransporter, or outer membrane vesicles in the outer membrane. Currently, some progress has been made in the functional research of the excreted proteins of Candidatus Liberibacter asiaticus.

[0004] At present, there are no effective disease-resistant genes and therapeutic agents for HLB. The prevention and control still rely on three main methods: pest control, tissue culture detoxification of sterile seedlings, and timely felling of diseased trees once they are discovered. The symptoms of early-infected trees are difficult to identify. If not felled in time, they will all become the sources of further transmission of the Asian citrus psyllid. As long as one tree in a citrus orchard is diseased, the migration of the Asian citrus psyllid carrying the Huanglongbing bacterium can destroy the entire orchard within a few years. Therefore, whether it is the early detection of sterile seedlings or the early diagnosis of diseased trees, the citrus industry urgently needs a rapid, reliable and highly sensitive pathogen early detection technology to achieve the goals of early detection and early management of Huanglongbing and block the spread of Huanglongbing.

[0005] At present, the detection methods of the Huanglongbing bacterium mainly include field detection, electron microscopy detection, spectral imaging detection, PCR detection, serological method detection and other means. Since there are no obvious symptoms on the leaves of citrus infected with Huanglongbing in the initial stage, its incubation period ranges from several months to one year or even longer; moreover, even when symptoms appear, they are extremely easy to be confused with the symptoms caused by other diseases or nutrient deficiencies and are difficult to detect in the initial stage of infection. Therefore, the field detection method based on symptoms has great limitations in the early detection of Huanglongbing. Due to the influence of objective factors such as sample collection and processing, the detection rate of the electron microscopy detection method is relatively low, generally 60% - 70%, and there are a large number of missed detections. The spectral imaging detection method not only requires expensive instrument equipment but also requires professional technical personnel. PCR detection has become a common method for laboratory detection of the Huanglongbing bacterium because it can be carried out in batches and can simultaneously complete qualitative and quantitative analysis. However, due to the uneven distribution of the bacteria in the plant, a large number of samples need to be collected to exclude false negatives, which is time-consuming and laborious. In recent years, serological methods have also been applied to the detection of Huanglongbing, such as the preparation of polyclonal antibodies against the Serralysin protein of the Huanglongbing bacterium and the preparation of polyclonal antibodies against the outer membrane protein of the Huanglongbing bacterium. However, the antibodies prepared are mainly used for the detection of samples in the late stage of HLB infection, and there are few reports on their application in the detection of early pathogen infection. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for early detecting whether a sample is infected with Huanglongbing at the pathogen stage. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0007] The present invention provides an application of an antibody in detecting Huanglongbing of plants or in preparing a kit for detecting Huanglongbing of plants. The antibody is a specific antibody against the SDE3 protein antigen, and the SDE3 protein antigen is a protein of A1) or A2):

[0008] A1) A protein with an amino acid sequence of SEQ ID No.2;

[0009] A2) A soluble fusion protein with the same activity as A1), obtained by fusing a tag protein to the carboxyl terminus or / and amino terminus of the protein shown in A1).

[0010] Furthermore, the antibody is a polyclonal antibody obtained by using the SDE3 protein antigen as an immunogen.

[0011] In the above application, the antibody can be a rabbit-derived, mouse-derived, horse-derived, sheep-derived, pig-derived, rabbit-derived or guinea pig-derived antibody. Specifically, the polyclonal antibody can be a rabbit polyclonal antibody.

[0012] Furthermore, the plant is any one of the following

[0013] C1) Dicotyledonous plants;

[0014] C2) Rutaceae plants;

[0015] C3) Plants of the genus Citrus or the genus Citrus maxima;

[0016] C4) Citrus (Citrus reticulata Blanco), Guangdong Red Orange (Citrus sinensis Osbeck), Guangxi Sugar Orange (Citrus reticulata Blanco), Hainan Sanhong Pomelo (Citrus maxima (Burm.) Merr.).

[0017] Furthermore, in the above application, the test sample is from the phloem of a Rutaceae plant.

[0018] Specifically, the test sample can be the sap of the plant phloem.

[0019] The specific detection method is as follows: Cut open the stem and the main vein of the leaf of the test sample to allow the sap to be fully absorbed by the nylon membrane. Repeat this operation 6 - 8 imprints for each sample point, then place the nylon membrane in a 4°C refrigerator overnight to allow the sample to dry thoroughly. Then block the nylon membrane at room temperature for 2 - 3 h (5% skim milk powder dissolved in 1×TBST buffer), and then add the SDE3 antibody prepared in the present invention (5% skim milk powder dissolved in 1×TBST buffer) at a ratio of 1:10000 and continue to incubate at room temperature for 2 - 3 h. Then wash the nylon membrane with 1×TBST buffer for 5 min, wash a total of 3 - 4 times, and then add the secondary antibody AP-conjugated goat anti-rabbit IgG(H+L) (Beijing Hongyue Innovation Technology Co., Ltd.) at a ratio of 1:5000 and incubate at room temperature for 1 h. Then wash the nylon membrane with 1×TBST buffer for 5 min, wash a total of 3 - 4 times, and perform signal detection according to the instructions of the BCIP / NBT color development kit (PR1100 - 125 ml) of Beijing Solarbio Science & Technology Co., Ltd.

[0020] Further, the SDE3 protein antigen is prepared by a method comprising the following steps: expressing the coding gene of the SDE3 protein antigen in a prokaryotic microorganism to obtain the SDE3 protein antigen.

[0021] Further, the coding sequence of the coding strand of the coding gene of the SDE3 protein antigen is SEQ ID No.1.

[0022] Further, expressing the coding gene of the SDE3 protein antigen in a prokaryotic microorganism includes introducing the coding gene of the SDE3 protein antigen into a recipient Escherichia coli to obtain a recombinant Escherichia coli expressing the SDE3 protein antigen, and culturing the recombinant Escherichia coli to express and obtain the SDE3 protein antigen.

[0023] Further, the recombinant Escherichia coli is a recombinant microorganism expressing a protein with the amino acid sequence of SEQ ID No.2 obtained by introducing pET-28a-SDE3 into Escherichia coli BL21(DE3), and the pET-28a-SDE3 is a recombinant vector obtained by replacing the small fragment between the EcoRI and XhoI recognition sites of the vector pET-28a(+) with a DNA fragment with the nucleotide sequence of SEQ ID No.1.

[0024] Further, the kit contains the antibody.

[0025] The present invention also provides a foregoing SDE3 protein antigen or biological material, and the biological material is any one of the following:

[0026] H1) A nucleic acid molecule encoding the foregoing SDE3 protein antigen;

[0027] H2) An expression cassette containing the nucleic acid molecule of H1);

[0028] H3) A recombinant vector containing the nucleic acid molecule of H1), or a recombinant vector containing the expression cassette of H2);

[0029] H4) A recombinant microorganism containing the nucleic acid molecule of H1), or a recombinant microorganism containing the expression cassette of H2), or a recombinant microorganism containing the recombinant vector of H3);

[0030] H5) A recombinant cell line containing the nucleic acid molecule of H1), or a recombinant cell line containing the expression cassette of H2).

[0031] Among the above biological materials, the expression cassette containing a nucleic acid molecule as described in (H2) refers to DNA that can express the protein described above in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for the nucleic acid molecule to express any one of the above proteins. The regulatory sequences can guide the coding sequence to express any one of the above proteins in a suitable host cell under their compatible conditions. The regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences should include a promoter and transcription and translation termination signals. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, regulatory sequences with linkers can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence recognizable by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter can be any nucleic acid sequence with transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, that is, a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, that is, the untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of the protein and can guide the encoded protein into the cell secretion pathway. Any signal peptide coding region that can guide the expressed protein into the secretion pathway of the host cell used can be used in the present invention. It may also be necessary to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory systems are those that can respond to chemical or physical stimulants (including in the presence of regulatory compounds) to turn on or off gene expression. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequence.

[0032] The present invention also relates to a recombinant expression vector comprising a nucleic acid molecule encoding any one of the above proteins, a promoter, and transcriptional and translational termination signals of the present invention. When preparing the expression vector, the nucleic acid molecule encoding any one of the above proteins can be positioned in the vector so as to be operably linked to appropriate expression control sequences. The recombinant expression vector can be any vector that facilitates recombinant DNA manipulation and expression of the nucleic acid sequence (such as a plasmid or a virus). The choice of the vector usually depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed circular plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure existing outside the chromosome and capable of replicating independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that will integrate into the genome when introduced into the host cell and replicate together with the chromosome into which it is integrated. In addition, a single vector or plasmid, or two or more vectors or plasmids, or transposons that collectively contain all the DNA to be introduced into the host cell genome can be applied. The vector contains one or more selectable markers that facilitate the selection of transformed cells. A selectable marker is a gene whose product confers resistance to a biocide or virus, resistance to heavy metals, or confers prototrophy to auxotrophs, etc. Examples of bacterial selectable markers are the dal gene of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that enable the vector to be stably integrated into the host cell genome or ensure that the vector replicates autonomously in the cell independently of the cell genome. In the case of autonomous replication, the vector can also contain a replication origin that enables the vector to replicate autonomously in the target host cell. The replication origin can carry a mutation that makes it temperature-sensitive in the host cell (see, for example, fEhrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75: 1433). One or more copies of the nucleic acid molecule encoding any one of the above proteins of the present invention can be inserted into the host cell to increase the yield of the gene product. The increase in the copy number of the nucleic acid molecule can be achieved by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selectable marker together with the nucleic acid molecule, and by culturing the cells in the presence of a suitable selection reagent to select cells containing the amplified copy of the selectable marker gene and thus containing an additional copy of the nucleic acid molecule. The operations for ligating the above elements to construct the recombinant expression vector described in the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0033] The term "operably linked" is defined herein as a conformation in which the regulatory sequence is positioned at an appropriate location relative to the coding sequence of the DNA sequence so that the regulatory sequence directs the expression of the protein.

[0034] The application of the specific antibody against the SDE3 protein antigen provided by the present invention in detecting citrus huanglongbing in plants or in preparing a kit for detecting citrus huanglongbing in plants can quickly and accurately detect whether a plant is infected with citrus huanglongbing. The detection process is simple and accurate, without the need to extract proteins or DNA, and is suitable for rapid field detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the electrophoresis result of the nucleotide fragments of the mature exosome proteins SDE3 - SDE8 amplified with the gDNA of the midrib of diseased leaves as the template.

[0036] Figure 2 It is the electrophoresis result of the nucleotide fragments of the mature exosome proteins SDE3 - SDE8 amplified with the gDNA of the midrib of leaves as the template.

[0037] Figure 3 It is the sequence alignment result of the amplification products of the SDE3 detection primers.

[0038] Figure 4 It is the sequence alignment result of the amplification products of the SDE4 detection primers.

[0039] Figure 5 It is the sequence alignment result of the amplification products of the SDE7 detection primers.

[0040] Figure 6 It is the sequence alignment result of the nucleotide sequences of the mature SDE3 protein in different isolates of 'Candidatus Liberibacter asiaticus' (Las).

[0041] Figure 7 The SDE3 antibody can detect the SDE3 protein in the bark phloem and the midrib of leaves of diseased Hainan Sanhong pomelo.

[0042] Figure 8 It is to detect samples of Gannan navel oranges by dot blotting using the SDE3 antibody.

[0043] Figure 9 It is the electrophoresis result of the nucleotide fragments of the mature exosome proteins SDE3 - SDE8 amplified with the gDNA of the midrib of leaves as the template.

[0044] Figure 10 It is the sequence alignment result of the amplification products of the SDE3 detection primers.

[0045] Figure 11 It is the sequence alignment result of the amplification products of the SDE4 detection primers.

[0046] Figure 12 It is the sequence alignment result of the amplification products of the SDE5 detection primers.

[0047] Figure 13It is the sequence alignment result of the amplification products of the SDE7 detection primers.

[0048] Figure 14 It is the sequence alignment result of the amplification products of the SDE8 detection primers.

[0049] Figure 15 It is to detect Guangxi red orange samples by dot blot using the SDE3 antibody.

[0050] Figure 16 It is to detect Guangdong sugar orange samples by dot blot using the SDE3 antibody. Specific embodiments

[0051] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0052] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0053] Example 1 Screening of extracellular proteins of Candidatus Liberibacter asiaticus (Las)

[0054] Through the bioinformatics analysis software TMHMM v2.0 and Signal P 3.0, extracellular proteins with unknown functions in Candidatus Liberibacter asiaticus Las_psy62 without transmembrane domains, signal peptide structures, and molecular weights less than 15 kDa were selected. A total of 6 proteins were screened and named SDE3 - SDE8 proteins in sequence. The SDE3 - SDE8 proteins are also highly conserved in three different strains, Las_psy62, Las_ishi - 1, and Las_gxpsy. Specifically, as shown in Table 1.

[0055] Table 1 Information related to SDE3 - SDE8

[0056] Number (KEGG KEGG T00948) Gene Name Amplification Location CLIBASIA_02470 SDE3 Positions 669763 to 669431 CLIBASIA_04580 SDE4 Positions 1014684 to 1014406 CLIBASIA_04410 [[ID=2,6]]SDE5 Positions 979636 to 979334 CLIBASIA_01330 SDE6 Positions 277162 to 276815 CLIBASIA_00755 SDE7 Positions 147737 to 147426 CLIBASIA_00235 SDE8 Positions 47875 to 47546

[0057] Full - length primers were designed respectively with the nucleotide sequences of the mature SDE3 - SDE8 proteins without signal peptides as templates as primers for detecting Candidatus Liberibacter asiaticus. The primer information is shown in Table 2:

[0058] Table 2 Primers for amplifying the nucleotide sequences of mature SDE3 - SDE8 proteins

[0059]

[0060]

[0061] Note: F, forward primer; R, reverse primer

[0062] Using the genomic DNA (gDNA) from the midrib of citrus leaves infected with Huanglongbing as a template, PCR amplification was performed with the primers shown in Table 2. The reaction program and system are as follows:

[0063] PCR reaction system (50 μl): 2×M5 HiPer plus Taq HiFi PCR mix (enzyme, product number MF002-plus-01) purchased from Beijing Polymer Beauty Biotechnology Co., Ltd. 25 μl, forward primer F (10 μM) 1.25 μl, reverse primer R (10 μM) 1.25 μl, gDNA template 2 μl, supplemented with ddH2O to make up 50 μl. The PCR program was pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 54°C for 25 s, extension at 72°C for 15 s, 35 cycles, and flat extension at 72°C for 5 min.

[0064] The nucleotide fragments of the mature SDE3-SDE8 proteins obtained by PCR amplification were subjected to agarose gel electrophoresis, and the results are as Figure 1 shown. The first lane from the left is the DNA Maker, the second lane is the PCR product of SDE3 amplified by SDE3-F / SDE3-R, the third lane is the PCR product of SDE4 amplified by SDE4-F / SDE4-R, the fourth lane is the PCR product of SDE5 amplified by SDE5-F / SDE5-R, the fifth lane is the PCR product of SDE6 amplified by SDE6-F / SDE6-R, the sixth lane is the PCR product of SDE7 amplified by SDE7-F / SDE7-R, and the seventh lane is the PCR product of SDE8 amplified by SDE8-F / SDE8-R. It can be Figure 1 seen that the aforementioned primers can amplify the nucleotide fragments of SDE3-SDE8 respectively.

[0065] Example 2 Sensitivity detection of SDE3-SDE8 primers

[0066] To compare whether there are differences in the sensitivity between the SDE3-SDE8 primers and the rplA primers reported by predecessors when detecting Huanglongbing samples, the inventor used the midrib gDNA of the infected Sanhong pomelo leaves (collected in Hainan Province) that had been sequenced and verified (hereinafter referred to as the infected gDNA of Hainan Sanhong pomelo) as a positive control. The Gannan navel orange fruit trees with obvious field symptoms and previously confirmed as infected by other institutions were used as control diseased plants (extracting the gDNA of the midribs of their leaves, hereinafter also referred to as "Gannan navel orange control diseased plants"). The Gannan navel orange fruit trees with obvious field symptoms but never detected were named diseased plant No. 1. The gDNA of the midribs of the leaves of the healthy branches (hereinafter also referred to as "healthy branches of Gannan navel orange diseased plant No. 1") and diseased fruit branches (hereinafter also referred to as "diseased fruit branches of Gannan navel orange diseased plant No. 1") of diseased plant No. 1 were extracted as test samples, specifically as follows:

[0067] 1. Negative control group: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with concentrations all being 10 μM), and ddH2O was added to make up 50 μl. The PCR reaction procedures of each primer were as shown for each primer in Example 1.

[0068] 2. Positive control group: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with concentrations all being 10 μM), 2 μl of infected gDNA of Hainan Sanhong pomelo, and ddH2O was added to make up 50 μl. The PCR reaction procedures of each primer were as shown for each primer in Example 1.

[0069] 3. Experimental group: Control diseased plants of Gannan navel oranges, healthy branches and diseased fruit branches of diseased plant No. 1: 2×M5 HiPerplus Taq HiFi PCR mix (enzyme) 25 μl, SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each) 1.25 μl each, Gannan navel orange control diseased plant gDNA (or gDNA of healthy branches of diseased plant No. 1 of Gannan navel oranges, or gDNA of diseased fruit branches of diseased plant No. 1 of Gannan navel oranges) 2 μl, add ddH2O to make up to 50 μl. The PCR reaction procedures of each primer are as shown for each primer in Example 1.

[0070] Perform agarose gel electrophoresis on the products obtained by the above PCR amplification, and the electrophoresis results are as Figure 2 shown. Figure 2 From top to bottom in [Figure], there are the electrophoresis results of the PCR amplification products of rplA, SDE3 - SDE8. From left to right, they are Marker (lane 1), negative control (lane 2), positive control (lane 3), control diseased plant (lane 4), healthy branches of diseased plant No. 1 (lanes 5 and 6, two replicates), diseased fruit branches of diseased plant No. 1 (lanes 7 and 8, two replicates). It can be Figure 2 seen that the rplA primer only amplified the rplA target fragment (703 bp) when using the diseased gDNA of Hainan Sanhong pomelo (positive control) as the template, and no rplA target fragment was amplified in the gDNA of the control diseased plants of Gannan navel oranges with obvious field symptoms and previously detected as diseased by other institutions, as well as the healthy branches and diseased fruit branches of diseased plant No. 1 of Gannan navel oranges with obvious field symptoms but never detected. The SDE3 primer and SDE6 primer only did not amplify the SDE3 and SDE6 target bands in the negative control, and the SDE3 and SDE6 target bands were amplified in the other three experimental groups and the positive control group. The SDE4, SDE5, SDE7, and SDE8 primers can obtain amplified fragments when using the positive control as the template, and can also obtain fragments of the same size as the positive control when using the gDNA of the midrib of the leaves of the diseased fruit branches of diseased plant No. 1 as the template.

[0071] Sequence the above amplified fragments. The sequencing alignment results of SDE3, SDE4, and SDE7 are as Figure 3 , Figure 4 and Figure 5As shown, the results indicate that the amplified fragments obtained with primers SDE3, SDE4, and SDE7 (amplifying the control diseased strain, the healthy branches of diseased plant No. 1, and the diseased fruit branches of diseased plant No. 1) are all correct fragments, while the amplification products of primers SDE5, SDE6, and SDE8 failed to obtain correct sequencing results. Thus, under the same amplification conditions, the sensitivity of primers SDE3, SDE4, and SDE7 is higher than that of the reported rplA primer. Sequencing the amplification products of other samples to be tested detected by primers SDE3 and SDE4 showed that all fragments are correct sequences ( Figure 3 and Figure 4 ), indicating that among the above primers, the sensitivity of primer SDE3 for detecting Huanglongbing samples is the highest.

[0072] Example 3 Preparation of SDE3 Polyclonal Antibody

[0073] The 11 sequenced Asian species of Candidatus Liberibacter asiaticus (Las) used in this example are as follows: CP010804.2 CLas A4, CP029348.1 CLas AHCA1, CP019958.1 CLas JXGC, AP014595.1 CLas Ishi-1, CP045565.1 CLas GDHZ11D, CP060689.1 CLas Myan16, CP004005.1 CLas gxpsy, CP041385.1 CLas isolate TaiYZ2, CP054558.1 CLas isolate CoFLP, CP040636.1 CLas isolate JRPAMB1, CP001677.5 CLas psy62.

[0074] Analysis of the SDE3 nucleotide sequences of the 11 sequenced Asian species of Candidatus Liberibacter asiaticus (Las) showed that, as Figure 6 shown, SDE3 is highly conserved among the 11 Asian species of Candidatus Liberibacter asiaticus (Las).

[0075] Since PCR detection requires expensive PCR instruments, molecular biology reagents, trained experimental operators, and cumbersome gDNA extraction steps, it is not suitable for on-site and immediate detection in the field. Therefore, in this example, SDE3 polyclonal antibody will be prepared and its sensitivity will be verified in the subsequent examples. The specific steps are as follows:

[0076] 1. Recombinant vector construction

[0077] SDE3 gene nucleotide sequence (SEQ ID No.1):

[0078] ATGCTTAATTGCAACGAAACTTTAATGCAAGCCGATATGAATCAATGCACAGGAAATTCTTTTGCACTAGTAAAAGAGAAACTAGAAGCAACATATAAAAAAGTCTTAGAAAAAGTTGAAAAGCATCAAAGAGAATTATTTGAAAAATCACAAATGGCATGGGAAATATACCGAGGTTCTGAATGCGCCTTTGCTGCTTCTGGAGCAGAAGAAGGAACTGCACAATCAATGATTTATGCGAATTGTCTACAAGGACATGCCATCGAACGAAATGAGAAACTAGAATCCTACCTTACATGTCCAGAAGGCGATCTGCTCTGCCCATTTATAAATAAT

[0079] Amino acid sequence of SDE3 protein (SEQ ID No.2):

[0080] MLNCNETLMQADMNQCTGNSFALVKEKLEATYKKVLEKVEKHQRELFEKSQMAWEIYRGSECAFAASGAEEGTAQSMIYANCLQGHAIERNEKLESYLTCPEGDLLCPFINN

[0081] The SDE3 gene (a DNA molecule with nucleotide sequence at positions 1 - 336 of SEQ ID No.1, encoding a protein with amino acid sequence at positions 1 - 112 of SEQ ID No.2) was constructed into the EcoRI and XhoI recognition sites of the pET-28a(+) vector (Shenzhen Yibaisun Technology Co., Ltd.), obtaining a recombinant expression vector of the SDE3 gene, named pET-28a-SDE3. pET-28a-SDE3 is a recombinant vector in which the SDE3 gene replaces the fragment between EcoRI and XhoI on pET-28a(+) (the SDE3 gene is inserted after the T7 promoter on pET-28a(+)), and other nucleotide sequences on the pET-28a(+) vector remain unchanged. pET-28a-SDE3 expresses a protein with amino acid sequence SEQ ID No.2.

[0082] 2. Preparation of recombinant bacteria

[0083] The pET-28a-SDE3 expression vector was transformed into Escherichia coli BL21(DE3) competent cells, and then evenly spread on an LB plate containing kanamycin, and cultured at 37 °C for 16 hours. Single colonies were picked and cultured overnight with shaking, and the plasmids were extracted for sequencing. The sequencing results showed that the recombinant Escherichia coli containing pET-28a-SDE3 was named BL21(DE3) / pET-28a-SDE3.

[0084] 3. Induce protein expression

[0085] The aforementioned BL21(DE3) / pET-28a-SDE3 strain was inoculated into 10 ml of LB liquid medium containing kanamycin and cultured overnight at 37 °C. The culture solution was transferred to 1 L of LB liquid medium containing kanamycin and cultured with shaking at 28 °C and 200 rpm until the OD600nm value reached 0.6 - 0.8. Then, isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 75 μM was added, and induction was carried out overnight at 16 °C and 160 rpm. The cells were collected by centrifugation at 4 °C and 4000 rpm for 20 min. 25 mL of lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 1% Triton X-100, 2 mM PMSF, pH 8.0) was added to the cell pellet to resuspend the cells, and 5 - 10 mg of lysozyme was added simultaneously. The cells were sonicated until they were no longer viscous, and the supernatant and pellet were collected by centrifugation at 12,000 rpm for 30 min at 4 °C. After SDS electrophoresis detection, it was determined that the fusion expression protein of SDE3 was in the supernatant, and the supernatant was named BL21(DE3) / pET-28a-SDE3 protein supernatant.

[0086] 4. Purify the protein

[0087] Pre-balance 1 mL of Ni-NTA beads (GE Healthcare) with wash buffer (50 mM sodium phosphate, 300 mM NaCl) in advance. Add the collected supernatant to the pre-balanced beads and bind them at 4°C for 4 - 6 h. Centrifuge at 500 g at 4°C to collect the bound beads, load the beads into a PD-10 column, and wash the beads 3 times with wash buffer. Add 50 mM imidazole to the wash buffer to elute the miscellaneous proteins, and then increase the imidazole concentration to 200 mM to elute the bound target protein. Use SDS-PAGE gel to detect the extracted protein. If the bands are clear and the concentration is sufficient, concentrate the protein with an ultrafiltration tube and replace the final protein buffer with 1×PBS (0.14 M NaCl, 2.7 mM KCl, 10 mM NaHPO4, 1.8 mM KH2PO4), and add 5% glycerol to store the protein at -80°C for later use. It is verified that the purified protein is the SDE3 protein antigen with the amino acid sequence shown in SEQ ID No. 2.

[0088] 5. Preparation of SDE3 polyclonal antibody

[0089] The specific preparation process of the SDE3 polyclonal antibody is as follows:

[0090] Concentrate the purified protein in Step 4 and prepare the SDE3 polyclonal antibody according to the following method.

[0091] The specific method is as follows: Use a New Zealand white rabbit (2.0 kg) as the immunized animal. Collect blood from the marginal ear vein before immunization and isolate the serum as the negative serum. Use the SDE3 protein antigen prepared in Step 4 as the immunizing antigen. Emulsify the immunogen with an equal amount of adjuvant (complete Freund's adjuvant (FCA) for the primary immunization and incomplete Freund's adjuvant (FICA) for the booster immunizations). The immunization method and immunization schedule are shown in Table 3. Perform the first booster immunization by injecting into the marginal ear vein 14 days after the first immunization (the first booster), the second booster immunization by injecting into the marginal ear vein 12 days after the first booster immunization (the second booster), the third booster immunization by injecting into the marginal ear vein 10 days after the second booster immunization (the third booster), and collect blood from the carotid artery 7 days after the third booster immunization to obtain the serum containing the SDE3 protein antigen-specific antibody.

[0092] Table 3 Immunization method and immunization schedule

[0093] Immunization Times (Interval in days) Immunization Drug Immunization Dose Immunization Route Primary Immunization Recombinant SDE3 + FCA 400 μg / animal Multiple intradermal injections into the back First Booster (14 d) Recombinant SDE3 + FICA 200 μg / animal Multiple intradermal injections into the back Second Booster (12 d) Recombinant SDE3 + FICA 200 μg / animal Multiple intradermal injections into the back Third Booster (10 d) Recombinant SDE3 + FICA 200 μg / animal Multiple intradermal injections into the back

[0094] Use the ELISA method to detect the serum antibody titer. The specific operation is as follows:

[0095] 1. Reagent preparation:

[0096] Coating solution: Sodium carbonate - sodium bicarbonate buffer, pH 9.6

[0097] PBS buffer, pH 7.4

[0098] Blocking solution: 1% BSA or 1% skim milk powder (diluted with PBS)

[0099] Washing solution: PBS - T (0.05% Tween, PBS)

[0100] Chromogenic solution: 1% Solution A + 10% Solution B (Solution A: 1% TMB in DMSO; Solution B: 0.1% H2O2 citrate buffer)

[0101] Stop solution: 2M sulfuric acid

[0102] Secondary antibody: Goat anti - rabbit IgG / HRP

[0103] 2. Experimental procedures

[0104] 1) Dilute the SDE3 antigen with the coating solution to a final concentration of 2 μg / ml, 100 μl / well, 4°C, overnight; then wash twice with the washing solution.

[0105] 2) Block with the blocking solution, 200 μl / well, incubate in an incubator at 37°C for 2 h; then wash once with the washing solution.

[0106] 3) Dilute the polyclonal antiserum starting from 200 - fold with a 2 - fold gradient (diluted with PBS), the blank control (blank) is PBS, and the negative control (negative) is the negative serum diluted 200 - fold (diluted with PBS); both are 100 μl / well, incubate in an incubator at 37°C for 1 h; then wash three times with the washing solution.

[0107] 4) Add the secondary antibody diluted 20,000 times with PBS, 100 μl / well, incubate in an incubator at 37°C for 1 h; after taking out, wash three times with the washing solution.

[0108] 5) Develop color, 100 μl / well of the chromogenic solution, and the color development time is 5 - 15 min.

[0109] 6) Add 50 μl of the stop solution to each well to terminate.

[0110] 7) Measure the absorbance values at dual wavelengths (450, 630), record and save the data, and plot for analysis. The titer is the dilution factor corresponding to 1 / 2 of the maximum OD value.

[0111] The detection results are shown in Table 4, indicating that the titer of the specific antibody against the SDE3 protein antigen reaches 102,400, meeting the requirements.

[0112] Table 4 Titer information

[0113] Dilution Factor 200 400 800 1600 3200 6400 <![CDATA[OD of Anti-SDE3 450nm value]]> 1.677 1.684 1.688 1.732 1.655 1.646 Dilution Factor 12800 25600 51200 102400 Blank Negative <![CDATA[OD of Anti-SDE3 450nm value]]> 1.641 1.518 1.389 1.134 0.043 0.201

[0114] Example 4 Sensitivity Detection of SDE3 Polyclonal Antibody

[0115] 1. Detection of Huanglongbing by Western blot

[0116] Use the SED3 polyclonal antibody prepared in Example 3 to detect whether SED3 protein is contained in the bark phloem of healthy Hainan Sanhong pomelo, the midrib of healthy Hainan Sanhong pomelo leaves, the bark phloem of diseased Hainan Sanhong pomelo, and the midrib of diseased Hainan Sanhong pomelo leaves.

[0117] Collect the juices of the bark phloem of healthy Hainan Sanhong pomelo, the midrib of healthy Hainan Sanhong pomelo leaves, the bark phloem of diseased Hainan Sanhong pomelo, and the midrib of diseased Hainan Sanhong pomelo leaves as test samples, add 2×SDS respectively and then run SDS-PAGE electrophoresis, and perform Western blot detection with SDE3 antibody. The SED3 antibody is diluted at 1:20000, incubated with the primary antibody for 1 h, the secondary antibody is diluted at 1:5000, and incubated for 0.75 h. The internal reference is rbcL protein. The results are as Figure 7 shown. The expression level of SDE3 protein is high in the juice of the bark phloem of diseased Hainan Sanhong pomelo, and the expression level of SDE3 protein is relatively low in the juice of the leaves of diseased Hainan Sanhong pomelo. SED3 protein is not detected in the juices of the bark phloem of healthy Hainan Sanhong pomelo and the midrib of healthy Hainan Sanhong pomelo leaves. Thus, it can be seen that SDE3 is specifically secreted into the phloem and accumulates more in the bark phloem, and the SDE3 polyclonal antibody prepared in Example 3 can specifically detect Huanglongbing samples.

[0118] 2. Detection of Huanglongbing by Dot Blot

[0119] Using the stems and midribs of the Gannan navel orange samples in Example 2 as experimental materials, cut the tissues with a sterilized surgical blade and immediately dot them on a nylon membrane, allowing the juice in the samples to be fully absorbed by the nylon membrane. Repeat this operation 6 - 8 imprints for each sample. Then place the nylon membrane in a 4°C refrigerator overnight to allow the samples to dry thoroughly. Next, block the nylon membrane at room temperature for 2 - 3 h (5% non-fat dry milk dissolved in 1×TBST buffer), and then add the SDE3 antibody prepared in Example 3 at a ratio of 1:10000 (5% non-fat dry milk dissolved in 1×TBST buffer) and continue to incubate at room temperature for 2 - 3 h. Then wash the nylon membrane with 1×TBST buffer for 5 min, washing a total of 3 - 4 times. Then add the secondary antibody AP-conjugated goat anti-rabbit IgG(H+L) (Beijing Hongyue Innovation Technology Co., Ltd.) at a ratio of 1:5000 and incubate at room temperature for 1 h. Then wash the nylon membrane with 1×TBST buffer for 5 min, washing a total of 3 - 4 times. Signal detection is carried out according to the instructions of the BCIP / NBT color development kit (PR1100 - 125 ml) from Beijing Solarbio Science & Technology Co., Ltd.

[0120] The results are as Figure 8 shown. No obvious blue-purple imprints were seen in the stems of healthy plants, while the presence of the SDE3 protein could be detected in the stems of the control diseased plants of Gannan navel orange, the healthy branches and diseased fruit branches of Diseased Plant No. 1, that is, blue-purple imprints (the imprints shown within the circles in the figure) could be detected, indicating that these samples were samples infected with Candidatus Liberibacter asiaticus, which was consistent with the results of SDE3 primer PCR detection in Example 2. Although there were relatively many blue-purple imprints in the midribs of the leaves of the control diseased plants, the healthy branches and diseased fruit branches of Diseased Plant No. 1, there were also a few blue-purple imprints in healthy plants. Combining the results of PCR detection, it can be seen that compared with the midribs of leaves, the stem is a better experimental material for dot blot hybridization.

[0121] Dot blot hybridization for detecting Huanglongbing samples is more sensitive than PCR detection in Example 5

[0122] In this example, Guangdong red oranges and Guangxi sugar oranges with only field symptoms of Huanglongbing were used as the test samples to compare the sensitivity of PCR amplification and dot blot hybridization for detecting Huanglongbing samples.

[0123] Extract the gDNA of the midribs of the leaves of Guangdong red oranges and Guangxi sugar oranges respectively according to the method of Example 2, which are subsequently referred to as the gDNA of diseased Guangdong red oranges and the gDNA of diseased Guangxi sugar oranges. Referring to the PCR amplification conditions of the SDE3 - SDE8 primers in Example 1, set up groups as follows:

[0124] 1. Negative control group: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each), and ddH2O was added to make up 50 μl. The PCR reaction program for each primer was as shown for each primer in Example 1.

[0125] 2. Positive control group: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each), 2 μl of gDNA from diseased Hainan Sanhong pomelo, and ddH2O was added to make up 50 μl. The PCR reaction program for each primer was as shown for each primer in Example 1.

[0126] 3. Healthy plant group: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each), 2 μl of gDNA from healthy Guangxi red orange plants, and ddH2O was added to make up 50 μl. The PCR reaction program for each primer was as shown for each primer in Example 1.

[0127] 4. Experimental group:

[0128] (1) Control diseased plants: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each), 2 μl of gDNA from the control diseased Gannan navel orange plants in Example 2, and ddH2O was added to make up 50 μl. The PCR reaction program for each primer was as shown for each primer in Example 1.

[0129] (2) Guangxi red oranges: 25 μl of 2×M5 HiPer plus Taq HiFi PCR mix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each), 2 μl of gDNA from diseased Guangxi red oranges, and ddH2O was added to make up to 50 μl. The PCR reaction program for each primer was as shown for each primer in Example 1.

[0130] (3) Guangdong Satsumas: 25 μl of 2×M5 HiPerplus Taq HiFi PCRmix (enzyme), 1.25 μl each of SDE3-F and SDE3-R (or SDE4-F and SDE4-R, or SDE5-F and SDE5-R, or SDE6-F and SDE6 / R, or SDE7F and SDE7-R, or SDE8-F and SDE8-R, or rplA-F and rplA-R, with a concentration of 10 μM each), 2 μl of gDNA from diseased Guangdong Satsumas, and ddH2O was added to make up to 50 μl. The PCR reaction program for each primer was as shown for each primer in Example 1.

[0131] The electrophoresis results are as Figure 9 shown. From top to bottom are the PCR electrophoresis results of the amplification products of rplA, SDE3 to SDE8. From left to right are Marker (lane 1), negative control (lane 2), positive control (lane 3), healthy plant (lane 4), Gannan navel oranges (lane 5), Guangxi red oranges (lanes 6, 7, and 8, three replicates), and Guangdong Satsumas (lanes 9, 10, and 11, three replicates). The rplA primer could only obtain an amplification fragment of 703 bp when using the positive control as a template, and no amplification bands were observed in other tested samples. For the SDE3-SDE8 primers, in addition to obtaining amplification fragments when using the positive control as a template, only when using the gDNA from the midrib of leaf No. 9 of Guangdong Satsumas as a template could fragments of the same size as the positive control be obtained. Sequencing of the amplification fragments showed that the amplification fragments obtained by the SDE3, 4, 5, 7, and 8 primers were all correct fragments ( Figures 10 - 14 ), while the amplification product of the SDE6 primer failed to obtain a correct sequencing result. Sequencing of the amplification products of other tested samples detected by the SDE3 primer showed that all fragments were correct sequences ( Figure 10 ). Thus, it can be concluded that the SDE3 primer can sensitively detect Huanglongbing samples from different regions.

[0132] 2. Dot blot detection of Huanglongbing

[0133] Using the stems of Guangxi red oranges and Guangdong sugar oranges infected with Huanglongbing as experimental materials, cut the tissues with a sterilized surgical blade and immediately place them on a nylon membrane. Ensure that the phloem sap in the samples is fully absorbed by the nylon membrane. Repeat this operation 6-8 imprints for each sample. Then place the nylon membrane in a 4°C refrigerator overnight to allow the samples to dry thoroughly. Next, block the nylon membrane at room temperature for 2-3 hours (5% skim milk powder dissolved in 1×TBST buffer), and then add the SDE3 antibody prepared in Example 3 (5% skim milk powder dissolved in 1×TBST buffer) at a ratio of 1:10000 and continue to incubate at room temperature for 2-3 hours. Then wash the nylon membrane with 1×TBST buffer for 5 minutes, wash 3-4 times in total. Then add the secondary antibody AP-conjugated goat anti-rabbit IgG(H+L) (Beijing Hongyue Innovation Technology Co., Ltd.) at a ratio of 1:5000 and incubate at room temperature for 1 hour. Then wash the nylon membrane with 1×TBST buffer for 5 minutes, wash 3-4 times in total. Signal detection is carried out according to the instructions of the BCIP / NBT color development kit (PR1100-125ml) of Beijing Solarbio Science & Technology Co., Ltd.

[0134] The results are as Figure 15 and Figure 16 shown. No obvious blue-purple imprints were seen in the stems of healthy plants, while the presence of SDE3 protein (blue-purple imprints) could be detected in the stems of Guangxi red oranges, Guangdong sugar oranges, Hainan three-red honey pomelos, and Gannan navel oranges, indicating that these samples were infected with Huanglongbing bacteria (the same as the apparent traits of diseased branches). The detection results of Gannan navel oranges were consistent with the results of SDE3 primer PCR detection in Example 2. The detection results of all samples of Guangxi red oranges (No. 5, No. 6, and No. 7) and sample No. 9 of Guangdong sugar oranges were consistent with the results of SDE3 primer PCR detection in this Example 5. This shows that the SDE3 antibody can be used for dot blot hybridization to detect whether a plant is infected with Huanglongbing and the detection results are reliable. The results of PCR detection of samples No. 8 and No. 10 of Guangdong sugar oranges showed that samples No. 8 and No. 10 of Guangdong sugar oranges were not infected with Huanglongbing bacteria, but the dot blot hybridization detection results showed that samples No. 8 and No. 10 of Guangdong sugar oranges were both infected with Huanglongbing bacteria, indicating that dot blot hybridization detection of Huanglongbing samples is more sensitive than PCR detection. <(

[0135] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, in accordance with the principles of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art. Some basic features can be applied within the scope of the appended claims below.

Claims

1. Use of an antibody in detecting plant Huanglongbing or in preparing a kit for detecting plant Huanglongbing, wherein the antibody is a specific antibody against an SDE3 protein antigen, and the SDE3 protein antigen is a protein of A1) or A2): A1) a protein having an amino acid sequence of SEQ ID No. 2; A2) A soluble fusion protein having the same activity as A1) is obtained by fusing a tag protein to the carboxyl terminus or / and amino terminus of the protein shown in A1).

2. The use according to claim 1, characterized in that: The antibody is a polyclonal antibody obtained by using the SDE3 protein antigen as an immunogen.

3. The use according to claim 1 or 2, characterized in that: The plant is any one of the following C1) Dicotyledons; C2) Rutaceae; C3) Citrus or pomelo plants; C4) Mandarin oranges, Guangdong red oranges, Guangxi sugar oranges or Hainan three-red honey pomeloes.

4. The use according to claim 3, characterized in that: In the application, the sample to be tested is from the phloem of Rutaceae plants.

5. The use according to claim 4, characterized in that: The SDE3 protein antigen is prepared according to a method comprising the following steps: expressing the coding gene of the SDE3 protein antigen in a prokaryotic microorganism to obtain the SDE3 protein antigen.

6. The use according to claim 5, characterized in that: The coding sequence of the coding chain of the gene encoding the SDE3 protein antigen is sequence SEQ ID No.

1.

7. The use according to claim 6, characterized in that: The method of expressing the coding gene of the SDE3 protein antigen in a prokaryotic microorganism includes introducing the coding gene of the SDE3 protein antigen into a recipient Escherichia coli to obtain a recombinant Escherichia coli expressing the SDE3 protein antigen, culturing the recombinant Escherichia coli, and expressing the SDE3 protein antigen.

8. The use according to claim 7, characterized in that: The recombinant Escherichia coli is a recombinant microorganism that expresses a protein with an amino acid sequence of SEQ ID No. 2, obtained by introducing pET-28a-SDE3 into Escherichia coli BL21 (DE3). The pET-28a-SDE3 is a recombinant vector that is obtained by replacing a small fragment between the EcoRI and XhoI recognition sites of the vector pET-28a (+) with a DNA fragment with a nucleotide sequence of SEQ ID No.

1.

9. The use according to claim 8, characterized in that: The kit contains the antibody.

10. A biological material related to the SDE3 protein antigen according to claim 1, wherein the biological material is any one of the following: H1) a nucleic acid molecule encoding the SDE3 protein antigen according to claim 1; H2) an expression cassette containing the nucleic acid molecule described in H1); H3) a recombinant vector containing the nucleic acid molecule described in H1) or a recombinant vector containing the expression cassette described in H2); H4) a recombinant microorganism containing the nucleic acid molecule described in H1), or a recombinant microorganism containing the expression cassette described in H2), or a recombinant microorganism containing the recombinant vector described in H3); H5) A recombinant cell line containing the nucleic acid molecule described in H1) or a recombinant cell line containing the expression cassette described in H2).

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