Polyclonal antibody of citrus hipp7 protein and preparation method and application thereof

Abstract

This invention relates to the field of genetic engineering technology, specifically disclosing a polyclonal antibody against citrus HIPP7 protein, its preparation method, and its applications. The method includes selecting amino acid 1-246 of the HIPP7 protein sequence for prokaryotic expression, synthesizing the sequence and ligating it into the vector pET41a to construct pET41a-HIPP7, transforming it into *E. coli*, culturing it in LB liquid medium containing kanamycin, inducing expression with IPTG, isolating and purifying the protein, and preparing a polyclonal antibody against citrus HIPP7 protein by immunizing rabbits with the HIPP7 protein. The polyclonal antibody against citrus HIPP7 protein obtained by this invention can specifically detect citrus HIPP7 protein and can be used to detect the accumulation of HIPP7 protein in different citrus tissues and under different treatment conditions, as well as to detect the accumulation of HIPP7 protein in transgenic plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a polyclonal antibody against citrus HIPP7 protein, its preparation method, and its application. Background Technology

[0002] Heavy metal-associated isoprenylated plant protein (HIPP) is a type of metal chaperone protein containing a heavy metal-associated domain (HMA) and a C-terminal isoprenylated motif. Metal chaperone proteins bind tightly to intracellular heavy metal ions, preventing them from reacting with other cellular components and damaging the cell, thus playing a crucial role in the safe transport of heavy metal ions within the cell. While metal chaperone proteins are less numerous in algae, fungi, and animals, in plants they have diversified into a large family comprising two types: heavy metal-associated plant protein (HPP) and heavy metal-associated isoprenylated plant protein (HIPP), with HIPP proteins found only in vascular plants. Based on sequence conservation, the HIPP family of proteins can be divided into five distinct subfamilies. HIPP proteins have three main functions: (a) involvement in heavy metal homeostasis and detoxification mechanisms, particularly tolerance to cadmium; (b) transcriptional responses to cold and drought stress; and (c) involvement in plant-pathogen interactions.

[0003] Studies have found that HIPP family genes function as susceptibility genes in multiple plant-pathogen interaction systems. In the wheat-stripe rust interaction system, the wheat TaHIPP1 gene is induced by stripe rust, the exogenous hormone ABA, and damage stress. Silencing TaHIPP1 using VIGS enhances wheat resistance to stripe rust, indicating that TaHIPP1 is a susceptibility gene in the wheat-stripe rust interaction and plays an important role in wheat's defense response to biotic and abiotic stresses. During the infection of *Nicotiana benthamiana* by *Potato Mop-Top Virus* (PMTV), its motility protein TGB1 interacts with *N. bhikkiens* NbHIPP26, leading to the activation of the drought stress response pathway in vascular tissues, thereby promoting the long-distance transport of PMTV into the phloem. TGB1 is involved in the viral ribonucleoprotein complex and long-distance viral transport, while HIPP26 acts as a signal from the plasma membrane to the nucleus under abiotic stress. Studies have shown that NbHIPP26 is specifically expressed in vascular bundles. Silencing the NbHIPP26 gene inhibits long-distance viral transport in tobacco but does not inhibit its intercellular transport. The TGB1-HIPP26 complex is localized in microtubules and accumulates in the nucleolus, but is almost absent on the plasma membrane and plasmodesmata. These results indicate that the interaction between TGB1 and HIPP26 reverses the lipidation of NbHIPP26, releasing it from the membrane and thereby activating the drought stress response and promoting long-distance viral transport. Other studies have found that HIPP27 in Arabidopsis is a host susceptibility gene required for infection and development of beet cyst nematodes. HIPP27 belongs to the Arabidopsis IV subfamily and is strongly upregulated in Arabidopsis leaves, young roots, and nematode-induced syncytia. HIPP27 loss-of-function T-DNA insertion mutants show reduced susceptibility to nematodes, while transgenic Arabidopsis overexpressing HIPP27 shows increased susceptibility, indicating that HIPP27 is essential for nematode syncytia formation and development. Ultrastructural analysis revealed that the absence of HIPP27 leads to physiological or metabolic abnormalities, including the accumulation of large starch granules in syncytial plastids and epidermal cytoplasms adjacent to the syncytial cells. Citrus leaves infected with Huanglongbing (HLB) also exhibit a typical phenomenon of massive starch accumulation, suggesting that members of the HIPP family may function as susceptibility genes in the interaction between citrus and HLB. Therefore, the development of polyclonal antibodies specifically recognizing HIPP7 protein in citrus is of great significance for citrus breeding. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a polyclonal antibody against citrus HIPP7 protein, its preparation method, and its application. The polyclonal antibody against HIPP7 protein obtained by this invention can specifically detect citrus HIPP7 protein and can be used to detect the accumulation of HIPP7 protein in different citrus tissues and under different treatment conditions. It can also be used to detect the accumulation of HIPP7 protein in transgenic plants.

[0005] This invention provides a method for preparing a polyclonal antibody against citrus HIPP7 protein, comprising the following steps:

[0006] S1, Construction of protein purification vector: The 1-246 amino acid region of the HIPP7 protein sequence was selected for prokaryotic expression, and the synthesized sequence was ligated into the vector pET41a to construct pET41a-HIPP7;

[0007] The amino acid sequence of the HIPP7 protein is shown in SEQ ID NO.1;

[0008] S2, HIPP7 protein induction and purification: pET41a-HIPP7 obtained in S1 was transformed into Escherichia coli, cultured in LB liquid medium containing kanamycin, and HIPP7 protein was obtained after induction of expression by IPTG inducer and then isolated and purified.

[0009] S3. The HIPP7 protein isolated and purified in S2 was used to immunize rabbits. After four immunizations, the serum was separated and purified to obtain a polyclonal antibody against citrus HIPP7 protein.

[0010] Furthermore, in S2, the final concentration of the IPTG inducer is 0.6-1.2 mM.

[0011] Furthermore, in S3, the four immunization processes are as follows: the first immunization uses a complete adjuvant, the second immunization is performed two weeks after the first immunization, and the second to fourth immunizations use an incomplete adjuvant.

[0012] Furthermore, in S3, the initial immunization antigen concentration was 1 mg / mL, and 0.5 mg per rabbit.

[0013] Furthermore, in S3, the interval between each injection for the second to fourth immunizations was one week, the antigen concentration was 1 mg / mL, and the dose per rabbit was 0.25 mg.

[0014] The present invention also provides a polyclonal antibody against citrus HIPP7 protein prepared by the above method.

[0015] The present invention also provides the application of the polyclonal antibody against citrus HIPP7 protein in the detection of HIPP7 protein, wherein the polyclonal antibody against citrus HIPP7 protein is used to detect HIPP7 protein expressed in prokaryotes.

[0016] Furthermore, the polyclonal antibody against citrus HIPP7 protein is used to detect transiently expressed HIPP7 protein in plants.

[0017] Furthermore, the polyclonal antibody against citrus HIPP7 protein was used to detect the accumulation of HIPP7 protein in different citrus tissues and citrus tissues under different treatment conditions.

[0018] Furthermore, the polyclonal antibody against the citrus HIPP7 protein was used to detect the accumulation of HIPP7 protein in transgenic plants.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The polyclonal antibody against HIPP7 protein provided by this invention can be used to detect HIPP7 protein expressed in prokaryotes, and can also be used to detect HIPP7 protein transiently expressed in plants.

[0021] 2. The polyclonal antibody against HIPP7 protein provided by this invention can be used to specifically detect HIPP7 protein in citrus. It can be used to detect the accumulation of HIPP7 protein in citrus tissues under different treatment conditions, and can also be used to detect the accumulation of HIPP7 protein in transgenic plants. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a sequence analysis diagram of the citrus HIPP7 protein in this invention, including analysis of its secondary structure, hydrophilicity, antigenic index, and amino acids appearing on the protein surface.

[0024] Figure 2 This is a sequence alignment diagram of the citrus HIPP7 homologous protein in this invention.

[0025] Figure 3 This is the SDS-PAGE result of the low-level HIPP7 protein expression induction assay in this invention. Lane M: protein marker; Lane 1: total protein before induction; Lane 2: total protein after induction.

[0026] Figure 4 This is the SDS-PAGE result of HIPP7 protein affinity purification in this invention. Lane M: protein marker; Lane 1: fragmented protein; Lane 2: eluent; Lanes 3-4: HIPP7 protein elution samples.

[0027] Figure 5This is the dialysis result of HIPP7 protein in this invention. Lane M is the protein marker; lane 1 is the dialyzed HIPP7 protein sample.

[0028] Figure 6 This is the result of Western blot (WB) of the purified HIPP7 protein detected by the purified anti-HIPP7 antibody in this invention.

[0029] Figure 7 The Western blot results for detecting transiently expressed HIPP7-GFP protein in tobacco using purified anti-HIPP7 antibody in this invention;

[0030] Figure A shows the detection of transiently expressed HIPP7-GFP protein in tobacco using an anti-GFP antibody.

[0031] Figure B shows the detection of transiently expressed HIPP7-GFP protein in tobacco using anti-HIPP7 antibody;

[0032] Figure C is a gel image obtained by SDS-PAGE protein electrophoresis with Coomassie Brilliant Blue staining. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0034] Example 1

[0035] This embodiment provides a citrus HIPP7 protein antibody, its preparation method, and its application.

[0036] I. Construction of Protein Purification Vectors

[0037] First, the HIPP7 protein sequence (amino acid sequence as shown in SEQ ID NO.1) was analyzed to predict its secondary structure, including α-helices, β-sheets, turns, and random coils. In particular, its hydrophilicity, antigenic index, and possible amino acid regions on the protein surface were predicted. Figure 1 ).

[0038] Based on protein sequence analysis, the full-length HIPP7 sequence can be used for prokaryotic protein expression. Sequence alignment analysis of members of the HIPP protein family revealed that the C-terminal 15 amino acids are relatively conserved. Figure 2To avoid non-specific binding of the antibody, the C-terminus of the protein was removed during prokaryotic expression. Therefore, the 1-246 amino acid region (underlined) was ultimately selected for prokaryotic expression. The synthesized sequence was directly ligated into the vector pET41a to construct pET41a-HIPP7, which was then transformed into E. coli strain BL21.

[0039] SEQ ID NO.1:

[0040] MGEENKEEQKKEEAKKEKKEEEKKEEETPEIVLKVDMHCEACARKVARALKGFEGVDDITADSKASKVV VKGKTADPIKVCERLQKKSGRKVELISPLPKPPPPDADDQEKKEQQKVEKKEEPPAAITVVLNVRMHCEACAQGLRK RIRKIQGVECVETNLASGQVIVKGVVDPVKLVNDVNKKTRKQASIVKDEEKKQEEKKEGEKKDGGEEAKVDEEKNKQ QLDFNINRSEYWATKNYSEFAYA PQIFSDENPNACFVM

[0041] II. HIPP7 protein induction and purification

[0042] 1. Low-level induction of HIPP7 protein expression

[0043] Transformed *E. coli* BL21 (pET41a-HIPP7) was inoculated into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C with shaking at 200 rpm. The induction group was treated with 1 mM IPTG, while the control group received no IPTG. Induction was performed at 37°C for 4 h. Cells were collected by centrifugation at 12000 rpm for 10 min, the supernatant was discarded, and 80 μL of *E. coli* lysis buffer was added to lyse the cells. 20 μL of 5× protein loading buffer was added, and the cells were incubated at 95°C for 10 min. 10 μL of the protein sample was then analyzed by SDS-PAGE electrophoresis. Figure 3 ).

[0044] 2. Large-scale expression and purification of HIPP7 protein

[0045] (1) Inoculate 3 ml of BL21 (pET41a-HIPP7) bacterial culture that has been cultured overnight into 200 ml of LB medium containing 50 μg / mL kanamycin. The induction method is the same as in 2.1. Collect the bacterial cells by centrifugation at 12000 rpm for 10 min, and resuspend them in 10 ml of 0.01 M PBS (pH 7.4) buffer. Under low temperature conditions, sonicate at 100 W for 16 min. Centrifuge at 4℃ at 12000 rpm for 10 min and retain the supernatant.

[0046] (2) Purification of HIPP7 protein using Ni-NTA resin

[0047] First, 1.5 mL of Ni-NTA resin was added to an empty affinity chromatography column. The Ni-NTA resin was then washed with 5 mL of deionized water, followed by equilibration with 5 mL of non-denaturing wash buffer. The protein lysis supernatant was added to the Ni-NTA resin column at a flow rate of approximately 1 mL / min. The flow-through buffer was repeated once, and 20 μL of the flow-through buffer was collected for electrophoresis. The column was washed with 5 mL of PBS buffer, and 20 μL of the wash buffer was collected for electrophoresis. Protein was eluted with 5 mL of non-denaturing wash buffer, and 20 μL of elution buffer (elution 1) was collected for electrophoresis. Protein was eluted with 5 mL of 60 mM imidazole elution buffer, and 20 μL of elution buffer (elution 2) was collected for electrophoresis. The HIPP7 prokaryotic expression protein has a GST tag fused to its N-terminus and a HIS tag fused to its C-terminus, with a predicted size of 61.5 kDa. SDS-PAGE electrophoresis results showed that the purified HIPP7 protein exhibited a specific band at the target size location, which can be used for further dialysis purification. Figure 4 ).

[0048] (3) HIPP7 prokaryotic expression protein dialysis and concentration

[0049] The protein eluent obtained from Ni-NTA chromatography was placed in a dialysis bag and dialyzed with PBS buffer to remove salts. After dialysis, the protein solution was added to a centrifuge tube and concentrated to 2 ml at 3000 rpm. The concentrated protein sample was then analyzed by SDS-PAGE electrophoresis. Figure 5 Store in an ultra-low temperature freezer at -80℃.

[0050] III. HIPP7 protein immunity

[0051] 1. Immunize rabbits with HIPP7 protein

[0052] The concentrated protein sample was dissolved on ice and used as an antigen to immunize New Zealand white rabbits. For the first immunization, a complete adjuvant was used, with an antigen concentration of 1 mg / ml and 0.5 mg per rabbit. For the second to fourth immunizations, an incomplete adjuvant was used, with the antigen concentration halved. The rabbits were immunized via multiple subcutaneous injections, with 0.2 ml injected at each injection site. A second immunization was administered two weeks after the first, and the interval between each injection for the second to fourth immunizations was one week. One week after the third immunization, serum was collected from the middle auricular artery for ELISA testing. If the test was satisfactory, a fourth immunization was administered, with whole blood collected one week later.

[0053] 2. ELISA testing

[0054] (1) Plate coating: Dilute the purified HIPP7 protein antigen to 1 μg / mL with coating buffer, add 50 μL of antigen to the reaction wells of a polystyrene plate, react overnight at 4°C, remove the solution from the reaction wells, and wash the reaction wells once with 180 μL of 1xTBST buffer.

[0055] (2) Blocking: Add 150 μL of 1% BSA to the reaction well, incubate at 37°C for 1 hour, and remove the blocking solution.

[0056] (3) Primary antibody incubation: Add 50 μL of serially diluted serum or purified antibody to the reaction wells, and add 1% BSA to the negative control reaction wells. Incubate at 37℃ for 30 min, then remove the primary antibody and wash twice with 180 μL of 1xTBST buffer.

[0057] (4) Enzyme-labeled antibody incubation: Add freshly diluted goat anti-rabbit-HRP (1:20000) to the wells of the enzyme-labeled plate, 50 μL per well, incubate at 37℃ for 45 min, then remove the enzyme-labeled antibody and wash 3 times with 180 μL of 1xTBST buffer.

[0058] (5) Color development: Add 100 μL of freshly prepared 3,3,5,5-tetramethylbenzidine substrate solution to the reaction well and react at 37°C for 10 min.

[0059] (6) Termination of reaction: Add 90 μL of 2M sulfuric acid to the reaction well to terminate the reaction.

[0060] (7) Reading the plate: Place the microplate in the microplate reader at a wavelength of 450 nm and read the data as shown in Table 1.

[0061] Table 1. Results of serum ELISA detection

[0062]

[0063]

[0064] 3. Antibody purification

[0065] The affinity chromatography column was washed with deionized water and PBS buffer, respectively. 10 mL of serum was filtered through a 0.45 μm microporous membrane and then added to the affinity chromatography column. The flow rate was approximately 1 mL / min. The flow-through buffer was repeated once. The column was washed with 5 mL of PBS buffer, and 5 mL of non-denaturing washing buffer was added to elute proteins. The antibody solution was collected. The antibody solution was concentrated using an ultrafiltration concentrator and then dialyzed against PBS (pH 7.4) buffer at room temperature. The buffer was changed every 3 hours, for a total of 3 dialyzing cycles. The dialyzed antibody solution was filtered through a 0.22 μm microporous membrane to a concentration of 11.2 mg / mL. The antibody titer was then determined using an ELISA assay.

[0066] 4. ELISA detection of antibody titer

[0067] The ELISA detection method for anti-HIPP7 protein antibody is the same as that for 4 ELISA detection, and the results are shown in Table 2.

[0068] Table 2. ELISA detection results of antibodies

[0069]

[0070] IV. Using anti-HIPP7 antibodies for Western blotting

[0071] 1. Western blot detection of purified HIPP7 protein

[0072] Prepare a 10% SDS-PAGE gel. Add 50 ng of purified HIPP7 protein to loading buffer, boil, and load the gel. Electrophoresis is performed at 80V for 30 min, then at 110V for 2 h. After electrophoresis, transfer the protein to a nitrocellulose membrane using a wet transfer method: 250 mA for 1 h 45 min. Block with 10% milk powder for 2 h. Dilute the anti-HIPP7 antibody (1:1000) with 5% milk powder and incubate at room temperature for 1.5 h. Wash the membrane three times with TBST for 10 min each time. Add goat anti-rabbit secondary antibody (1:2000) and incubate for 1.5 h. Wash the membrane twice with TBST, once with TBS, and expose with chromogenic buffer. The prepared anti-HIPP7 antibody can detect the purified protein, and the electrophoresis results are consistent with those of the purified protein, showing multiple bands, indicating that the prepared antibody is specific for the purified HIPP7 protein. Figure 6 ).

[0073] 2. Detection of HIPP7 protein expressed in plants

[0074] The HIPP7 gene was constructed into the plant expression vector pCambia2300GFP, fusing the C-terminus of HIPP7 with GFP. The constructed plasmid was transformed into Agrobacterium GV3101, and the HIPP7-GFP protein (57.2 kDa) was expressed in *Nicotiana benthamiana* leaves using Agrobacterium-mediated transient expression. First, *Agrobacterium* was cultured overnight in LB broth at 28°C. The cells were collected by centrifugation at 5000g for 5 min, resuspended in MES solution, and the OD of the culture was adjusted. 600 The value was 0.4. The bacterial suspension was injected into *Nicotiana benthamiana* leaves using a 1 mL syringe and left to stand overnight in the dark with moisture. Three days after injection, the leaves were harvested, and total protein was extracted using RIPA strong reagent. After boiling in loading buffer, the extract was spotted into SDS-PAGE gel wells for protein electrophoresis, with non-protein-expressing tobacco leaves serving as a control. Following the same method, Western blots were performed using anti-HIPP7 and anti-GFP antibodies. The results showed that both anti-HIPP7 and anti-GFP antibodies detected the target band at approximately 57.2 kDa (the size of HIPP7-GFP), and both also detected a specific band at approximately 70 kDa. Figure 7A, B). To further confirm that the band detected by the anti-HIPP7 antibody is HIPP7-GFP expressed by the plant, we performed gel extraction and recovery at positions H1 and H2 of the band (A, B). Figure 7 C), and perform protein spectrum detection.

[0075] Table 3. Results of protein spectroscopy detection

[0076]

[0077] Comparative analysis of the protein proteomic analysis results revealed the detection of HIPP7-GFP protein-specific peptide fragments in both samples H1 and H2. Sample H1 showed 21 HIPP7-GFP-specific peptides with a coverage rate of 41.73%, while sample H2 showed 23 HIPP7-GFP-specific peptides with a coverage rate of 48.03% (Table 3). These results indicate the successful preparation of a polyclonal antibody against citrus HIPP7 protein. This antibody can be used for the detection of citrus HIPP7 protein by ELISA, Western blotting, or other serological methods.

[0078] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0079] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a polyclonal antibody against citrus HIPP7 protein, characterized in that, Includes the following steps: S1, Construction of protein purification vector: The 1-246 amino acid region of the HIPP7 protein sequence was selected for prokaryotic expression, and the synthesized sequence was ligated into the vector pET41a to construct pET41a-HIPP7; The amino acid sequence of the HIPP7 protein is shown in SEQ ID NO.1; S2, HIPP7 protein induction and purification: pET41a-HIPP7 obtained in S1 was transformed into Escherichia coli and cultured in LB liquid medium containing kanamycin. After expression was induced by IPTG inducer, HIPP7 protein was isolated and purified. S3. The HIPP7 protein isolated and purified in S2 was used to immunize rabbits. After four immunizations, the serum was separated and purified to obtain a polyclonal antibody against citrus HIPP7 protein.

2. The method for preparing a polyclonal antibody against citrus HIPP7 protein according to claim 1, characterized in that, In S2, the final concentration of the IPTG inducer is 0.6-1.2 mM.

3. The method for preparing a polyclonal antibody against citrus HIPP7 protein according to claim 1, characterized in that, In S3, the four immunization processes are as follows: the first immunization uses a complete adjuvant, the second immunization is performed two weeks after the first immunization, and the second to fourth immunizations use an incomplete adjuvant.

4. The method for preparing a polyclonal antibody against citrus HIPP7 protein according to claim 3, characterized in that, In S3, the initial immunization antigen concentration was 1 mg / mL, and 0.5 mg per rabbit.

5. The method for preparing a polyclonal antibody against citrus HIPP7 protein according to claim 4, characterized in that, In S3, the interval between each injection from the second to the fourth immunization was one week, the antigen concentration was 1 mg / mL, and the dose per rabbit was 0.25 mg.

6. A polyclonal antibody against citrus HIPP7 protein prepared by the method of any one of claims 1-5.

7. The application of the polyclonal antibody against citrus HIPP7 protein as described in claim 6 in the detection of HIPP7 protein, characterized in that, The polyclonal antibody against citrus HIPP7 protein is used to detect HIPP7 protein expressed in prokaryotes or transiently expressed HIPP7 protein in plants.

8. The application according to claim 7, characterized in that, The polyclonal antibody against citrus HIPP7 protein was used to detect the accumulation of HIPP7 protein in transgenic plants.

9. The application according to claim 8, characterized in that, The polyclonal antibody against citrus HIPP7 protein was used to detect the accumulation of HIPP7 protein in different citrus tissues and under different treatment conditions.

Images