Cell strain capable of generating antibodies for detecting Pseudomonas amygdali and diseases caused by Pseudomonas amygdali and application of cell strain

Abstract

The invention discloses a cell strain capable of generating antibodies for detecting Pseudomonas amygdali and diseases caused by the Pseudomonas amygdali and application of the cell strain. The preservation number of the cell strain is CCTCC (China Center For Type Culture Collection) NO: C2024169 and / or CCTCC NO: C2024170. The monoclonal antibody generated by the monoclonal antibody hybridoma cell strain can be prepared into handheld colloidal gold rapid test paper, and the pseudomonas amygdali in a sample can be rapidly and sensitively detected on site.

Description

Technical Field

[0001] The present application relates to the technical field of plant pathogenic bacteria detection, and in particular to a cell strain capable of producing antibodies for detecting Pseudomonas amygdalus and its induced diseases and its application. Background Art

[0002] The Pseudomonas syringae species complex is an important plant pathogenic bacteria, including Pseudomonas amygdali (including P. meliae, P, ficuserectae and P. savastanoi), P. asturiensis, P. avellanae, P. cannabina, P. caspiana, P. caricapapayae, P. cerasi, P. cichorii, P. congelans, P. syringae, P. tremae, P. viridiflava, P. alliivorans, P. capsici, P. floridensis, P. folii, P. lijiangensis, P. quasicaspiana, P. triticifolii, P. ovata and other Pseudomonas (for details, see Mulet, M.; Gomila, M.; Busquets, A. et al. Genome-based taxonomy of species in the Pseudomonas syringae and Pseudomonas lutea phylogenetic groups and proposal of Pseudomonasmaioricensis sp.nov.,isolated from agricultural soil.Microorganisms 2024,12,460.).

[0003] Tobacco wildfire and angular leaf spot are both caused by infection with Pseudomonas amygdali pv. tabaci (formerly known as Pseudomonas syringae pv. tabaci) (see Knoche KK, Parke JL, and Durbin RD. Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco. 1994, 158(1): 91-97.; Matsui H, Nishimura T, Asai S, et al. 2021. Complete genome sequence of Pseudomonas amygdali pv. tabaci strain 6605, a causal agent of tobacco wildfire disease. Microbiol Resour Announc 10:10.1128 / mra.00405-21.). The pathogens of the two diseases are similar in many aspects, and differ only in whether they can produce tabacion. The pathogen of wildfire can synthesize tabacion normally, and is of tox+ type. After infection, large circular lesions are formed on the leaves, and the lesions are surrounded by large halos; while the pathogen of angular leaf spot cannot synthesize tabacion normally, and is of tox- type. After infection, small and irregular spots are formed (Mapuranga NA new race of Pseudomonas syringae pv.tabacion tobacco in Zimbabwe. Plant Disease, 1998, 82(12):1404-1404.). Wildfire and angular leaf spot are both important bacterial leaf spot diseases of tobacco, and occur all over the world.Although the above diseases have occurred in tobacco-growing areas in northern and southwestern my country for many years, the current detection methods for the above two viruses are mainly based on indoor detection technology based on PCR amplification, such as the method for rapid detection and identification of tobacco wildfire and angular spot bacteria disclosed in CN200610128446. According to the complete sequence of HRPZ of tobacco wildfire and angular spot bacteria Chinese strains, angular spot bacteria HRPZ SEQ ID 002, the designed sequence-specific oligonucleotide PCR amplification primer pair, and the specific oligonucleotide PCR amplification primer pair of the essential gene for wildfire toxin biosynthesis, i.e., SEQ ID 005, SEQ ID 006, etc.; using the above primer pair and TAQ DNA polymerase, PCR amplification is performed, and tobacco wildfire and angular spot bacteria can be quickly identified within 2 to 5 hours according to the electrophoresis results. Compared with traditional bacterial identification methods, it is not only convenient and fast, but also accurate, reliable and highly specific. However, this method requires the collection of samples and transportation to the laboratory for detection, and the detection efficiency is low. No rapid field detection method / product for tobacco wildfire / angular spot disease has been found.

[0004] Pseudomonas amygdali (P. amygdali) species, in addition to tobacco pathogenic variants (Pv. tabaci), also include at least 13 pathogenic variants such as Pv. morsprunorum, Pv. lachrymans, Pv. mori, Pv. sesami, and cause diseases on important agricultural and economic plants such as Prunus sp. plants, sweet cherry, cucumber, and tobacco (for details, see Jung H, Kim HS, Han G, et al. Comparative analyses of four complete genomes in Pseudomonas amygdal revealed differential adaptation to hostile environments and secretion systems. Plant Pathol J. 2022Apr; 38(2): 167-174.). Although Pseudomonas amygdali (P. amygdali) is very harmful in agricultural production, the existing technology lacks effective, fast, convenient, and efficient detection means and methods for this species.

[0005] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] In response to the above technical problems, the present application provides a cell strain for detecting Pseudomonas amygdali and its diseases and its application. The antibodies secreted by the cell strain can be used to produce handheld colloidal gold rapid test paper, which can quickly, on-site and sensitively detect Pseudomonas amygdali infection in samples.

[0007] The present application provides a cell strain capable of producing antibodies for detecting Pseudomonas amygdali and its disease-inducing antibodies, and the deposit number is CCTCC NO: C2024169 and / or CCTCC NO: C2024170.

[0008] Preferably, the plant is infected with Pseudomonas amygdali to cause wildfire disease or angular leaf spot;

[0009] Preferably, the plant is tobacco.

[0010] Preferably, the antibody is a monoclonal antibody, which is composed of a capture antibody and a detection antibody; the capture antibody is secreted by a cell line with a deposit number of CCTCC NO: C2024170; and the detection antibody is CCTCC NO: C2024169.

[0011] The antigens subsequently produced by this cell line can be made into test cards to achieve rapid indicative detection of tobacco leaf sample extracts with high detection efficiency.

[0012] Another aspect of the present application also provides a method for preparing the above-mentioned cell line, wherein BALB / c mice are immunized with Pseudomonas amygdali, the pathogen of tobacco wildfire disease, and a monoclonal cell line is obtained through fusion, screening, and cloning.

[0013] Another aspect of the present application also provides a method for detecting Pseudomonas amygdaloids

[0014] (Pseudomonas amygdali) and its disease-inducing products, characterized by comprising: antibodies secreted by the above-mentioned cell lines.

[0015] Another aspect of the present application also provides a method for detecting Pseudomonas amygdaloids

[0016] (Pseudomonas amygdali) and antibodies causing diseases thereof are prepared by using the above-mentioned cell lines and collecting mouse ascites to obtain antibodies through purification.

[0017] Another aspect of the present application also provides an immunoassay card for detecting Pseudomonas amygdali and diseases caused by it, comprising: a gold label pad, a sample pad, a nitrocellulose membrane and a water-absorbing pad; a detection line is provided on the nitrocellulose membrane, and the detection line is coated with a capture antibody secreted by the cell line as described above; the gold label pad is coated with a detection antibody secreted by the cell line as described above and labeled with colloidal gold.

[0018] Preferably, a quality control line spaced apart from the detection line is further provided on the nitrocellulose membrane, and the quality control line is coated with an anti-mouse IgG secondary antibody.

[0019] Preferably, the sample processed by the immunoassay card is obtained by crushing and extracting the tobacco leaf sample to be tested;

[0020] Preferably, the sample is tobacco leaves to be tested, which are added to a PBS solution, ground, and then diluted at least 10 times with a PBS solution to obtain a solution to be tested.

[0021] Preferably, the method of using the immunoassay card is as follows: drop the extract of the tobacco leaf sample to be tested onto the sample pad, let it stand for 10 minutes, and observe whether the C line or the and / or T line appears in the display area. If only the C line appears, it is a negative result; if both the C line and the T line appear, it is a positive result; if only the T line appears, re-prepare the sample for testing.

[0022] Preferably, the immunoassay card is positive for detection of Pseudomonas amygdali or diseases caused by infection of plants by Pseudomonas amygdali.

[0023] Preferably, the immunoassay card is negative for detection of Pseudomonas lijiangensis, Pseudomonas viridiflava, Pseudomonas capsici, Pseudomonas cichorii, and Ralstonia nicotianae.

[0024] Preferably, the immunoassay card is negative for Achromobacter marplatensis, Agrobacterium deltaense, Bacillus velezensis, Escherichia coli, Kosakonia sp., Pantoea vagans, and Pectobacterium sp.

[0025] Preferably, the immunoassay card is negative for tobacco bacterial black spot, frog eye disease, black shank disease, brown spot disease, gray mold, target spot disease, common mosaic virus disease, cucumber mosaic virus disease, and potato virus Y disease.

[0026] The beneficial effects of this application include:

[0027] 1) The present application provides a cell line capable of producing antibodies for detecting Pseudomonas amygdalus and diseases caused by it and its application. The obtained antibodies can achieve a specific reaction to Pseudomonas amygdalus, and the prepared detection card can realize accurate and timely detection of tobacco leaves caused by Pseudomonas amygdalus, such as tobacco wildfire disease / angular leaf spot.

[0028] 2) The cell strains and applications thereof provided in the present application that can produce antibodies for detecting Pseudomonas amygdalus and the diseases it causes, the detection card made of the antibodies can be directly inserted into the liquid sample for detection, and the detection results can be intuitively read from the test paper, which effectively improves the detection efficiency and reduces the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Comparison photos of the experimental group and the blank control group in Example 2 of the present application;

[0030] Figure 2 Provided for this application is a graph showing the specific recognition results of the immunodetection card for Pseudomonas amygdalus in Example 3;

[0031] Figure 3 The present application provides a graph showing the specific recognition results of the immunodetection card in Example 3 for the pathogen of tobacco bacterial black spot;

[0032] Figure 4 Provided for this application is a graph showing the specific recognition results of common bacteria by the immune detection card in Example 3;

[0033] Figure 5 The test card result diagram of the positive samples 23DLYP02, 23DLYP03, 23DLYP04, and 23DLYP05 tobacco leaves in Example 4 provided in this application;

[0034] Figure 6 This is the test card result diagram of the positive sample 2309BSLY04 tobacco leaf in Example 4 provided in this application;

[0035] Figure 7 This is a graph showing the evaluation results of the effects of the test card on other tobacco diseases in Example 5 provided in this application;

[0036] Figure 8 This is a graph showing the evaluation results of the effects of the test card on other tobacco diseases in Example 5 provided in this application;

[0037] Fig. 9 This is a graph showing the evaluation results of the effects of the test card on other tobacco diseases in Example 5 provided in this application;

[0038] Fig.10 This is a graph showing the effect evaluation results of the test card in Example 5 provided in this application for common mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY);

[0039] Fig.11 Schematic diagram of the test paper of the test card obtained in Example 2 provided in the present application; wherein 1, bottom plate; 2, NC membrane; 3, absorbent paper; 4, gold label pad; 5, sample pad; 6, test line (T line); 7, quality control line (C line). The preservation information is as follows: The depository is China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, zip code: 430072, telephone: (027) 68752319, fax: (027) 68754833. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0041] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The technical means that are not described in detail in this application and are not used to solve the technical problems of this application are all set according to the common knowledge in the field, and can be implemented in a variety of common knowledge settings.

[0043] Example

[0044] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0045] Pseudomonas amygdali strain 59#1 (pathogen of tobacco wildfire), 22KMSL09 (pathogen of tobacco wildfire), 22KMSL01 (pathogen of tobacco angular spot), 22DLJC-10 (pathogen of tobacco angular spot), 22LJNL03 (pathogen of tobacco angular spot), and 22LJYL08 (pathogen of tobacco angular spot) are all Knoche KK, Parke JL, and Durbin RD. Relationship of Pseudomonas syringae pv.tabaci races to therhizosphere of Wisconsin-grown tobacco. 1994, 158(1): 91-97.; Matsui H, Nishimura T, Asai S, et al. 2021. Complete genome sequence of Pseudomonas amygdali pv.tabaci strain 6605, a causal Agent of tobacco wildfire disease. Microbiol Resour Announc 10: 10.1128 / mra.00405-21. The above bacteria are strains preserved in the applicant's laboratory. The applicant promises to distribute them to the public free of charge for verification of the effects of the present invention within 20 years from the date of application of the present invention. The strains can be obtained by contacting Lu Canhua of Yunnan Tobacco Agricultural Science Research Institute.

[0046] Pseudomonas lijiangensis LJ2 T , Pseudomonas cichorii (P. cichorii) DSM 50259 T LJ-2 recorded in the article "Lu Can-Hua, Han Tian-Hua, Jiang Ning, et al. Pseudomonas lijiangensis sp.nov., anovel phytopathogenic bacterium isolated from black spots of tobacco in Yunnan, China [J]. International Journal of Systematic and Evolutionary Microbiology, 2022, 72:005591." T 、DSM50259 TThe public can obtain the strain by contacting Lu Canhua of Yunnan Tobacco Agricultural Science Research Institute within 20 years from the filing date of this application.

[0047] The pathogen of tobacco bacterial black spot, Pseudomonas lijiangensis (P.lijiangensis) strains 21LCFQ011, 22DLYP12, 22LXQNDG-6, and Pseudomonas capsici (P.capsici) 20WSXC14, Pseudomonas (Pseudomonassp.) 21LCFQ02, Pseudomonas viridiflava (P.viridiflava) 22KMSL03 and 22DLJC11-5 are all strains preserved in the applicant's laboratory. The applicant promises to distribute them to the public free of charge for verification of the effects of the present invention within 20 years from the date of application of the present invention. The strains can be obtained by contacting Lu Canhua of Yunnan Tobacco Agricultural Science Research Institute.

[0048] Tobacco bacterial wilt pathogen Ralstonia nicotianae RS T For "Liu Jun-Ying, Zhang Jian-Feng, Wu Han-Lian, et al. Proposal to classify Ralstonia solanacearumphylotype I strains as Ralstonia nicotianae sp.nov., and agenomic comparison between members of the genus Ralstonia.Frontiers in Microbiology, 2023,14:1135872.doi:10.3389 / fmicb.2023.1135872." RS recorded in the article T The bacteria was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 14, 2022, with the collection number GDMCC 1.3533.

[0049] The strains of Achromobacter marplatensis 22KMSM04, Kosakonia sp. 22KMSL02-1, Pantoea vagans 22KMSM11-2, and Pectobacterium sp. 21LCBS03 are all strains preserved in the applicant's laboratory. The applicant promises to distribute them to the public free of charge for 20 years from the date of application of the present invention for the purpose of verifying the effects of the present invention. The strains can be obtained by contacting Lu Canhua of Yunnan Tobacco Agricultural Science Research Institute.

[0050] Agrobacterium deltaense 18A6 is the Agrobacterium deltaense 18A6 described in “Lu Canhua, Gai Xiaotong, Ma Junhong, et al. A strain of Agrobacterium 18A6 and its application, product and method [P]. Yunnan Province: CN202211444108.0, 2023-06-23.” The bacterium is a patented strain of the applicant’s unit, and the bacterium is deposited in: China Center for Type Culture Collection (CCTCC), deposit date: January 28, 2020, deposit number: CCTCC NO: M2020041.

[0051] Bacillus velezensis Z002 is the Bacillus velezensis Z002 described in “(Lu Canhua, Xia Zhenyuan, Ma Junhong, et al. A strain of Bacillus velezensis Z002, acquisition method and application thereof [P]. Yunnan Province: CN202011255055.9, 2021-02-05.)”. The bacterium is a patented strain of the applicant’s unit, and the bacterium is deposited in: China Center for Type Culture Collection (CCTCC), deposit date: October 5, 2020, deposit number: CCTCC No: M 2020567.

[0052] Escherichia coli DH5α is the Escherichia coli DH5α recorded in the article “Lu Can-Hua, Nakayasu Ernesto S., Zhang Li-Qun, et al. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation [J]. Science Signaling, 2016, 9 (412): ra11.” The public can obtain this strain by contacting Lu Canhua of Yunnan Tobacco Agricultural Science Research Institute within 20 years from the date of filing this application.

[0053] Tobacco wildfire has been recorded in the public literature (Wang Fenglong, Zhou Yihe, Ren Guangwei, eds. Illustrated Handbook of Tobacco Diseases in China. Beijing: China Agriculture Press, 2019.7). Typical diseased leaves of tobacco wildfire 23DLYP02, 23DLYP03, 23DLYP04, and 23DLYP05 were collected from diseased tobacco plants of the flue-cured tobacco variety 'K326' grown in Yongping County, Dali Prefecture, Yunnan Province in September 2023.

[0054] Tobacco bacterial black spot has been recorded in the known literature (Lu Can-Hua, Han Tian-Hua, Jiang Ning, et al. Pseudomonas lijiangensis sp.nov., a novel phytopathogenic bacterium isolated from black spots of tobacco in Yunnan, China [J]. International Journal of Systematic and Evolutionary Microbiology, 2022, 72: 005591.). Tobacco bacterial black spot disease samples 2309LCQN01 and 2309LCYQ01-1 were collected from diseased tobacco plants in Linxiang District, Lincang City in September 2023, disease samples 2209BSSD08-2 were collected from Shidian County, Baoshan City in September 2023, and disease samples 2309DLYP09 and 23DLYP10 were collected from diseased tobacco plants of the flue-cured tobacco variety 'K326' grown in Yongping County, Dali Prefecture, Yunnan Province in September 2023. The above samples were confirmed to be tobacco bacterial black spot samples through pathogen isolation and 16S rDNA sequence analysis.

[0055] Tobacco black shank, frog eye disease, red star disease, target spot disease, gray mold, mosaic virus disease (TMV), cucumber mosaic virus disease (CMV), and potato virus Y disease (PVY) have been recorded in the public literature (Wang Fenglong, Zhou Yihe, Ren Guangwei, eds. Chinese Tobacco Disease Atlas. Beijing: China Agriculture Press, 2019.7). Black shank disease leaves 2309BSLY08 and 2309BSLY09-1 were collected from Longyang District, Baoshan City in September 2023, frog eye disease samples 2309LCGM01-1 and 2309LCGM02-2 were collected by the applicant from tobacco frog eye disease fields in Gengma County, Lincang City in September 2023, and red star disease samples 2309BSSD07 and 2309BSSD03 were collected by the applicant in 2023. The samples were collected from tobacco fields in Shidian County, Baoshan City in September; target spot diseased leaves 2309DLYP06 and 2309DLYP07 were collected by the applicant in September 2023 from diseased tobacco plants of the flue-cured tobacco variety 'K326' planted in Yongping County, Dali Prefecture; gray mold disease samples 2309LCCY02-1 and 2309LCQN02 were collected by the applicant in September 2023 from diseased tobacco plants in tobacco fields in Cangyuan County and Linxiang District, Lincang City, respectively.

[0056] The PVY virus used is Potato virus Y (PVY), and the TMV virus used is Tobacco Mosaic Virus (TMV), both of which are recorded in the public literature (Guo Y, Wu W, Zhang X, et al. Triplex immunostrip assay for rapid diagnosis of Tobacco Mosaic Virus, Tobacco Vein Banding Mosaic Virus, and Potato Virus Y. Plant Dis. 2022 Dec; 106 (12): 3033-3039.).

[0057] The CMV virus used is Cucumber mosaic virus (CMV), which is recorded in the public literature (Shen Z, Yang X, Sun Y, et al. Integrated transmission electron microscopy and proteomic analyses reveal the cytoarchitectural response to cucumbermosaic virus infection in tobacco. Int J Biol Macromol. 2024Mar;262(Pt 2):130100.doi:10.1016 / j.ijbiomac.2024.130100.).

[0058] Reagents and materials: bovine serum albumin was purchased from BSA Merck; TMB was purchased from Sigma; goat anti-rabbit enzyme-labeled secondary antibody was purchased from KPL; Freund's complete adjuvant was purchased from Sigma; Freund's incomplete adjuvant was purchased from Sigma; New Zealand white rabbits were purchased from the Experimental Animal Factory of Jiangnan University; bovine serum albumin was purchased from BSA Merck; calf serum was purchased from Sijiqing; TMB was purchased from Sigma; DMEM medium was purchased from Hyclone; fetal bovine serum was purchased from Sijiqing; Freund's complete adjuvant was purchased from Sigma; Freund's incomplete adjuvant was purchased from Sigma; PEG4000 was purchased from Sigma; HT was purchased from Sigma; HAT was purchased from Sigma; myeloma cells were stored in the laboratory of Shanghai Hongjing Technology Co., Ltd., 2nd Floor, Building A, No. 189, Xinjun Ring Road, Minhang District, Shanghai; BALB / c mice were purchased from Yangzhou University; EDC and NHS were purchased from Sigma; bovine serum albumin BSA was purchased from Merck; egg albumin OVA was purchased from Sinopharm; calf serum was purchased from Sijiqing; TMB was purchased from Sigma; goat anti-mouse enzyme-labeled secondary antibody and goat anti-rabbit enzyme-labeled secondary antibody were purchased from KPL;

[0059] Example 1 Antibody Preparation

[0060] BALB / c mice were immunized with the Pseudomonas amygdali strain 59#1, the pathogen of tobacco wildfire disease preserved in our laboratory, and a monoclonal cell line targeting tobacco wildfire disease was finally obtained through fusion, screening, and cloning. The specific process of monoclonal antibody preparation is as follows:

[0061] 1. Animal Immunization

[0062] 1.1 Animal selection

[0063] Healthy animals that meet the standards for experimental animals were selected and rested for one week before immunization. A 2-month-old female New Zealand white rabbit was used for immunization.

[0064] 1.2 Preparation of immune antigens

[0065] The antigen was dissolved in sterile PBS (PH7.2) at a volume of 500ug / rabbit.

[0066] Add an equal amount of Freund's complete adjuvant for emulsification (except for the first use of Freund's complete adjuvant, all subsequent use of Freund's incomplete adjuvant).

[0067] 1.3 Animal immunization

[0068] Use 75% alcohol cotton balls to wipe and disinfect, lift the skin of the immunization site on the back, insert the needle at a 15-degree angle 1-2 cm below the skin, and stay for about 10 seconds after the injection to prevent the antigen from leaking out. The injection should be done at multiple points on the inner thigh.

[0069] Table 1 Antigen information

[0070]

[0071] Table 2 Preparation of polyclonal antibodies Immunization scheme

[0072]

[0073] 2. Potency detection

[0074] 2.1 Antibody titer detection method (ELISA endpoint method)

[0075] 2.1.1 Coating: 1ug / ml 0.05M CB of the corresponding antigen was coated on the ELISA plate, 100ul / well, and kept in a refrigerator at 4℃ overnight;

[0076] 2.1.2 Blocking: Blocking solution (2% BSA) 150ul / well, 37°C for 1 hour.

[0077] 2.1.3 Primary antibody: Serum was diluted three times starting from 1:10,000, 100 ul / well, incubated at 37°C for 1 hour.

[0078] 2.1.4 Secondary antibody: Wash three times with TBST, add goat anti-rabbit HRP 1:3000, 100ul / well, 37℃, 1 hour.

[0079] 2.1.5 Color development: Remove TBST and wash three times, then add TMB substrate color development solution, 100ul / well.

[0080] 2.1.6 Reading: Develop at room temperature for 5-10 minutes, add 50ul / well of stop solution, and read using dual wavelength (OD450 / 630) microplate reader.

[0081] 3. Antibody Purification

[0082] 3.1 Ammonium sulfate precipitation

[0083] The serum was centrifuged for 15 min (4000 rpm, room temperature), the supernatant was taken, saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded;

[0084] The precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4); saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded;

[0085] The precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4), dialyzed overnight at 4°C, and the antibody was collected.

[0086] 3.2 Column purification

[0087] 3.2.1 Column equilibration

[0088] After ammonium sulfate precipitation, continue to use Protein A column for purification. First, pass 5 ml of ultrapure water through the new column, and then use 5 ml of 0.4 M PB buffer (pH 7.0) to balance the purification column.

[0089] 3.2.2 Antibody Column

[0090] When the antibody passes through the column, it is required to pass through the column slowly so that the antibody protein can better bind to the binding site.

[0091] 3.2.3 Cleaning

[0092] Continue to equilibrate the purification column with 10 ml of 0.4 M PB buffer (pH 7.0)

[0093] 3.2.4 Antibody elution

[0094] The antibodies on the binding sites were eluted with 5 ml of 0.1 M glycine-HCl buffer (pH 2.7), and glycine was neutralized by adding 1 M Tris-HCl (pH 8.0) to keep the pH at a neutral value suitable for antibody storage.

[0095] 3.3 BCA method to detect antibody concentration

[0096] The purified antibody was dialyzed into PBS, and the concentration of the dialyzed antibody was detected by the BCA method.

[0097] 4. Results

[0098] 1. Potency detection

[0099] The ELISA method was used to detect the titer of the antiserum, the detection source: tobacco wildfire pathogen, the coating concentration: 1ug / ml, and the results are as follows (Table 4).

[0100] Table 4 Antiserum titer determination

[0101]

[0102] It can be seen from the above table that the titer of the obtained rabbit antiserum reached 1:243000.

[0103] 2. Monoclonal antibody preparation

[0104] 1. Animal immunization

[0105] Four 6-week-old female BALB / c mice were immunized with Pseudomonas amygdalus strain 59#1 for a total of three immunizations, with multiple subcutaneous injections. The antigen immunization dosage for each mouse was 25ug each time, and the first immunization was emulsified with an equal amount of Freund's complete adjuvant, the second immunization was emulsified with an equal amount of Freund's incomplete adjuvant, and the third immunization was intraperitoneally injected with an equal amount of Freund's incomplete adjuvant. The specific immunization procedure is shown in Table 5.

[0106] Table 5 Immunization scheme for preparation of monoclonal antibodies

[0107] Immunization times Immunization date Interval days Adjuvant type Immunization site and method Immunization dose first 2022 / 3 / 28 28 Freund's complete adjuvant Multiple subcutaneous injections 25ug Second time 2022 / 4 / 26 28 Freund's incomplete adjuvant Multiple subcutaneous injections 25ug The third time 2022 / 5 / 12 14 Freund's incomplete adjuvant Intraperitoneal injection 25ug

[0108] Blood was collected from the tail vein 10 days after the second immunization, and the antibody titer was measured by an indirect method to determine whether antibodies against the antigen were produced. The titers of the four mice were compared, and finally the mice with higher antibody titers were selected for cell fusion (mouse numbers: M220053, M220054). Three days before fusion, the antigen was used for direct booster immunization once, with the same dose as the previous one.

[0109] 2. Establishment of monoclonal antibody hybridoma cell lines

[0110] 2.1 Recovery of myeloma cells

[0111] Take out the frozen cells from liquid nitrogen, immediately put them in a 37℃ water bath to thaw, centrifuge, discard the supernatant, use a small amount of complete culture medium to break up the cell clumps, add complete culture medium to the cryopreservation tube, blow and beat evenly with a pipette, aspirate all and transfer to a cell culture bottle, place in a 37℃ carbon dioxide incubator for culture. After the cells have grown to the bottom of the bottle, transfer them to another cell bottle for culture, observe the cell status under an inverted microscope, and prepare the cells in good condition for fusion.

[0112] 2.2 Preparation of immune splenocytes

[0113] Take the mouse with the highest titer and boost immunization, remove the eyeball and collect blood, separate the serum as the positive control serum for detection. Kill the mouse by cervical dislocation, remove the spleen after disinfection in 75% alcohol, remove the connective tissue, prepare the spleen cell suspension, transfer to a 50ml centrifuge tube, add basal culture medium to 30ml, centrifuge at 1000rpm for 10 minutes, discard the supernatant, and add basal culture medium to 30ml.

[0114] 2.4 Cell fusion

[0115] The separated spleen cells were fused with the pre-recovered myeloma cells using PEG, centrifuged, the supernatant was discarded, and the cells were washed once by centrifugation with cell culture medium. The cell culture medium containing HAT was added and blown into a single cell suspension, which was then plated into a 96-well cell culture plate.

[0116] 2.5 Screening of positive hybridoma cells

[0117] On the 7th day after cell fusion, clones visible to the naked eye grew on the bottom of the culture plate wells. All cell wells were initially screened using the ELISA indirect method, and the cell wells showing strong positive results were recorded.

[0118] Each cell well that is positive in the initial screening should be expanded and cloned in a 24-well cell culture plate in a timely manner.

[0119] 2.6 Cloning of positive hybridoma cells

[0120] The cell suspension is diluted continuously by limiting dilution until each well contains only a single cell, and then transferred to a 96-well plate for culture. After several cloning operations, when the positive rate of all cloned cell wells is 100%, it can be determined that a hybridoma cell line secreting monoclonal antibodies has been obtained. The obtained positive hybridoma cells are expanded and cultured in a cell culture bottle, and then frozen and ascites is prepared.

[0121] 3. Large-scale preparation and purification of monoclonal antibodies

[0122] 3.1 Preparation of mouse ascites

[0123] Six-week-old healthy female mice were sensitized by intraperitoneal injection of 0.5 ml of liquid paraffin. One to two weeks later, each mouse was intraperitoneally injected with 1-2X10 6 hybridoma cells, observe the mouse status for 7-10 days, and collect ascites when the mouse abdominal cavity is obviously swollen. The supernatant from centrifugation is the ascites, which is packaged, labeled, and set aside.

[0124] 3.2 Purification of monoclonal antibodies

[0125] 3.2.1 Ammonium sulfate precipitation

[0126] The ascites was centrifuged for 15 min (4000 rpm, room temperature), the supernatant was taken, saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded; the precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4), saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded;

[0127] The precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4), dialyzed overnight at 4°C, and frozen at -20°C for later use.

[0128] 3.2.2 Column purification

[0129] 3.2.2.1 Column balance

[0130] Protein G column was used for purification. 5 ml ultrapure water was first passed through the new column, and then 5 ml 0.4 M PB buffer (pH 7.0) was used to balance the purification column.

[0131] 3.2.2.2 Antibody column

[0132] When the antibody passes through the column, it is required to pass through the column slowly so that the antibody protein can better bind to the binding site.

[0133] 3.2.2.3 Cleaning

[0134] The purification column was equilibrated with 10 ml of 0.4 M PB buffer (pH 7.0) to wash away impurity proteins.

[0135] 3.2.2.4 Antibody elution

[0136] The antibodies on the binding sites were eluted with 5 ml of 0.1 M glycine-HCl buffer (pH 2.7), and glycine was neutralized by adding 1 M Tris-HCl (pH 8.0) to keep the pH at a neutral value suitable for antibody storage.

[0137] 3.2.3 BCA method to detect antibody concentration

[0138] The purified antibody was dialyzed into PBS, and the concentration of the dialyzed antibody was detected by the BCA method.

[0139] 4. Monoclonal antibody characterization:

[0140] The type and subtype of monoclonal antibodies were determined using the mouse antibody subtype identification kit, and the titer of monoclonal antibodies was determined using the indirect ELISA method.

[0141] 5. Results and Analysis

[0142] Immunogen and Detection Information

[0143] Table 6 Information on immunogens and detection agents

[0144]

[0145] 2. Mouse serum titer determination

[0146] Table VII Mouse titer determination

[0147]

[0148] 3. Hybridoma cell line subtype detection

[0149] A total of 6 positive monoclonal hybridoma cell lines were screened and established, and the secreted antibody subtypes were confirmed as shown in Table 4.

[0150] Table 8 Cell line subtype detection data

[0151]

[0152] 4. Monoclonal Antibody Titer Determination

[0153] The hybridoma cells were collected, mouse ascites was prepared and collected, and after purification, the antibody titer was determined by the ELISA indirect method. The titer data are shown in Table 9.

[0154] Table 9 Monoclonal antibody titer test data

[0155]

[0156] 5. Conclusion

[0157] 1. Obtained 6 positive monoclonal hybridoma cell lines. 2. Prepared monoclonal antibody ascites. A total of 6 ascites were collected and purified. 6 were IgG1. 3. The titer of the ascites prepared for testing reached 1:256000 or above for 5 strains.

[0158] After sample screening, six monoclonal antibodies were screened in pairs using the double antibody sandwich gold label method, and an antibody pair that can be paired for Pseudomonas amygdalus detection was obtained, FL5392-1 and FL5392-6, which had the best recognition effect on Pseudomonas amygdalus, among which FL5392-1 was used as the gold label antibody (detection antibody) and FL5392-6 was used as the capture antibody at the detection line.

[0159] 6. Cell line preservation

[0160] The hybridoma cell lines secreting FL5392-1 and FL5392-6 were deposited in China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072. The deposit number of FL5392-1 monoclonal antibody hybridoma cell line is CCTCC NO: C2024169, and the classification is named as hybridoma cell line FL5392-1Hybridoma cell line FL5392-1; the deposit number of FL5392-6 monoclonal antibody hybridoma cell line is CCTCC NO: C2024170, and the classification is named as hybridoma cell line FL5392-6 Hybridoma cell line FL5392-6.

[0161] (2) Ascites preparation and purification

[0162] 1. Cell recovery: Take the monoclonal antibody hybridoma cell lines with the deposit numbers of CCTCC NO: C2024169 and CCTCC NO: C2024170, and expand the culture in a cell culture bottle;

[0163] 2. Preparation of ascites: Take 6-8 week old BALB / c mice and inject liquid paraffin into the peritoneum, 0.2 mL per mouse. Ten days later, a certain number of monoclonal antibody hybridoma cells were inoculated into the peritoneum. The abdomen of the mouse was obviously swollen, and the ascites was collected with an elbow dropper;

[0164] 3. Monoclonal antibody purification: centrifuge the ascites for 15 min (4000 rpm, room temperature), take the supernatant, add saturated ammonium sulfate drop by drop slowly to half saturation under stirring at 4°C, continue stirring for 30 min, centrifuge for 30 min (13000 rpm, 4°C), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4); add saturated ammonium sulfate drop by drop slowly to 33% under stirring at 4°C, continue stirring for 30 min, centrifuge for 30 min (13000 rpm, 4°C), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4), dialyze at 4°C overnight, determine the antibody content, and freeze at -20°C to obtain the antibody for use.

[0165] After ammonium sulfate precipitation, continue to use Protein G column for purification. First, use 5 ml ultrapure water to pass through the new column, and then use 5 ml 0.4 M PB buffer (pH 7.0) to equilibrate the purification column; the antibody is passed through the column slowly during the process so that the antibody protein can better bind to the binding site; continue to equilibrate the purification column with 10 ml 0.4 M PB buffer (pH 7.0); 5 ml 0.1 M glycine-hydrochloric acid buffer (pH 2.7) is used to elute the antibody on the binding site, and 1 M Tris-HCl (pH 8.0) is added to neutralize glycine to keep the pH neutral for antibody storage, thereby obtaining the purified antibody.

[0166] Example 2 Preparation of Pseudomonas amygdaloids Immunity Detection Card

[0167] See also Fig.11 , the sample pad and the gold label pad fixed with the above-mentioned specific monoclonal antibody labeled with colloidal gold, the cellulose acetate membrane and the absorption pad are pasted side by side on the bottom plate. 40ng of colloidal gold-labeled mouse anti-pseudomonas amygdalus monoclonal antibody (the antibody produced by the FL5392-1 cell line is used as the detection antibody) is fixed on the gold label pad, 0.8μg of mouse anti-pseudomonas amygdalus monoclonal antibody (the antibody produced by the FL5392-6 cell line is used as the capture antibody) is fixed in the detection area on the cellulose acetate membrane, and the anti-mouse IgG secondary antibody (Hangzhou Longji, PS00901) is fixed in the control area. Then place it in a vacuum dryer at 37°C for 2h to obtain an immunodetection card. The preparation operations of the immunodetection card not described in detail are all carried out according to the existing ones.

[0168] Evaluation of the effect of the Pseudomonas amygdaloids immune detection card obtained in Example 2 on detecting the actual standard product:

[0169] Blank control groups were set up separately: 200 μL of blank sample solution was added to the sample cup. The blank sample solution was pH 7.4, 0.2 mol / L PBS: 8 g sodium chloride, 3.35 g disodium hydrogen phosphate dodecahydrate, 0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, and dissolved in double distilled water to a volume of 1 L.

[0170] Experimental group: The difference from the blank control group is that 100 μL of 0.1 μg / ml Pseudomonas amygdalus strain 59#1 standard was added to the PBS solution; the solution of the treatment group was added to the sample cup;

[0171] Experimental process: Insert the rapid test paper into the sample cup, react at room temperature for 10 minutes, and then observe the color development results in the result observation area.

[0172] The results show that: in the blank control group, the T line in the observation area of ​​the test paper does not show color, but the C line shows color. In the observation area of ​​the experimental group, the C line and the T line show color at the same time. Figure 1 .

[0173] From the above, it can be seen that the test paper can realize accurate and real-time indicative detection of Pseudomonas amygdalus contained in liquid samples through C and T lines.

[0174] Example 3 Specificity of Pseudomonas amygdaloids Immunoassay Card Detection

[0175] The bacteria of each test treatment were cultured (48 h) in LB liquid culture medium (the total mass fraction of the culture medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride and 0.5% sucrose, the balance being water, natural pH) at 28°C under a shaking incubator. 100 μL 10^8 CFU / mL of each test bacterium was taken in a sample cup, and the same operating steps as the blank sample in Example 2 were followed.

[0176] The results show that the Pseudomonas amygdali immunoassay card obtained in Example 2 can specifically identify Pseudomonas amygdali (P. amygdali) strains 59#1 (wildfire pathogen), 22KMSL01 (angular spot pathogen), 22KMSL09 (wildfire pathogen), 22DLJC-10 (angular spot pathogen), 22LJNL03 (angular spot pathogen), and has a weak ability to identify 22LJYL08 (angular spot pathogen); see the results for details. Figure 2 .

[0177] The test card does not identify the tobacco bacterial disease pathogen Pseudomonas lijiangensis LJ2 T, 21LCFQ011, 22DLYP12, 22LXQNDG-6, Pseudomonas capsici (P. capsici) 20WSXC14, other Pseudomonas (Pseudomonas sp. 21LCFQ02, Pseudomonas viridiflava (P. viridiflava) 22KMSL03, 22DLJC11-5, Pseudomonas cichorii (P. cichorii) DSM 50259), Ralstonia nicotianae (R. nicotianae) RS T .

[0178] The test card does not identify other common bacteria: Achromobacter marplatensis 22KMSM04, Agrobacterium deltaense 18A6, Bacillus velezensis Z002, Escherichia coli DH5α, Kosakonia sp. 22KMSL02-1, Pantoea vagans 22KMSM11-2, Pectobacterium sp.

[0179] 21LCBS03( Figures 3 to 5 ).

[0180] The above research results show that the detection card obtained in Example 2 has strong specificity for Pseudomonas amygdalis and can specifically detect wildfire disease and angular leaf spot disease caused by multiple strains of Pseudomonas amygdalis (P. amygdali).

[0181] Example 4 Evaluation of the effectiveness of the Pseudomonas amygdaloids immunoassay card in detecting tobacco wildfire and angular leaf spot diseased leaves

[0182] Take 0.1 g of leaf sample, add 1 mL of PBS (0.01 M, pH 7.4) and grind thoroughly. Dilute the ground liquid 10 times with PBS buffer or use it directly as an extract.

[0183] 200 μL of healthy tobacco leaves were added to the sample cup as the sample extract of negative leaves, and the same experimental process as the blank control standard in Example 2 was followed. The results showed that only the control line C line was colored in the observation area, and the detection line T line was not colored, which was the CK group.

[0184] Four typical diseased leaves infected with tobacco wildfire were collected from Yongping County, Dali Prefecture as positive samples 23DLYP02, 23DLYP03, 23DLYP04, and 23DLYP05. 200 μL of the positive leaf sample extract (the extract preparation method is as described above) was added to the sample cup, and the test was performed in the same manner as the negative sample.

[0185] The results show that: after testing the above 5 samples with the test card, both the control line C and the test line T in the observation area are colored ( Figure 4 ).

[0186] In addition, angular leaf spot diseased leaf No. 2309BSLY04 was collected from Longyang District, Baoshan City, and the extract of the leaf was tested with the test card obtained in Example 2, and the result was positive ( Figure 6 ). The above research results show that the test card can effectively detect tobacco leaf disease samples infected with tobacco wildfire disease and angular leaf spot in natural environment.

[0187] Example 5 Evaluation of the effectiveness of the Pseudomonas amygdaloids immune test card in detecting other tobacco diseases

[0188] The detection card in Example 2 and the detection method in Example 3 were used to detect tobacco leaves infected with tobacco bacterial black spot (2309LCQN01, 2309LCYQ01-1, 2209BSSD08-2, 2309DLYP09, 23DLYP10), frog eye disease (2309LCGM01-1, 2309LCGM02-2), black shank disease (2309BSLY08, 2309BSLY09-1), brown spot disease (2309BSSD07, 2309BSSD03), target spot disease (2309DLYP06, 2309DLYP07), gray mold (2309LCCY02-1, 2309LCQN02), common mosaic virus disease (TMV), cucumber mosaic virus disease (CMV), and potato virus Y (PVY). The tobacco leaves were prepared into an extract according to the method in Example 4 and then tested.

[0189] CK is the same as in Example 3.

[0190] Results Figures 7-10 , indicating that the Pseudomonas amygdaloids immune detection card obtained in Example 2 cannot identify the above-mentioned common tobacco diseases, and the detection card can specifically detect tobacco wildfire disease / angular spot disease caused by Pseudomonas amygdaloids.

[0191] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cell strain capable of producing antibodies for detecting Pseudomonas amygdali and its disease-inducing antibodies, characterized in that: The deposit number is CCTCC NO:C2024169 and / or CCTCC NO:C2024170.

2. The cell line according to claim 1, characterized in that When plants are infected with Pseudomonas amygdali, they can cause wildfire or angular leaf spot; Preferably, the antibody is a monoclonal antibody, which is composed of a capture antibody and a detection antibody; the capture antibody is secreted by a cell line with a deposit number of CCTCC NO: C2024170; the detection antibody is CCTCC NO: C2024169; Preferably, the plant is tobacco.

3. The method for preparing a cell line according to claim 1 or 2, characterized in that: BALB / c mice were immunized with Pseudomonas amygdali, and the monoclonal cell line as claimed in claim 1 was obtained by fusion, screening and cloning.

4. A product for detecting Pseudomonas amygdali and diseases caused by it, characterized in that: include: The antibody secreted by the cell line according to claim 1 or 2.

5. An immune detection card for detecting Pseudomonas amygdali and diseases caused by it, characterized in that: include: Gold label pads, sample pads, nitrocellulose membranes and absorbent pads; A detection line is arranged on the nitrocellulose membrane, and the detection line is coated with the capture antibody secreted by the cell line according to claim 1 or 2; and the gold label pad is coated with the detection antibody secreted by the cell line according to claim 1 or 2 labeled with colloidal gold.

6. The immune detection card according to claim 5, characterized in that: A quality control line spaced apart from the detection line is also arranged on the nitrocellulose membrane, and an anti-mouse IgG secondary antibody is coated on the quality control line.

7. The immune detection card according to claim 5, characterized in that: The samples processed by the immunoassay card are obtained by crushing and extracting the plant samples to be tested; Preferably, the sample is tobacco leaves to be tested, which are added to a PBS solution, ground, and then diluted at least 10 times with a PBS solution to obtain a solution to be tested.

8. The immune detection card according to claim 5, characterized in that: How to use the immunoassay card: Add the extract of the tobacco leaf sample to be tested onto the sample pad, let it stand for 10 minutes, and then observe whether the C line or the T line appears in the display area. If only the C line appears, it is a negative result; if both the C line and the T line appear, it is a positive result; if only the T line appears, re-prepare the sample for testing.

9. The immune detection card according to claim 5, characterized in that: The immunoassay card is positive for Pseudomonas amygdali or diseases caused by plants infected by Pseudomonas amygdali.

10. The immune detection card according to claim 5, characterized in that: The immunoassay card is negative for Pseudomonas lijiangensis, Pseudomonas capsici, Pseudomonas viridiflava, Pseudomonas cichorii, Ralstonia nicotianae or diseases caused by the above bacteria; Preferably, the immunoassay card is negative for Achromobacter marplatensis, Agrobacterium deltaense, Bacillus velezensis, Escherichia coli, Kosakonia sp., Pantoea vagans, and Pectobacterium sp.; Preferably, the immunoassay card is negative for tobacco bacterial black spot, frog eye disease, black shank disease, brown spot disease, gray mold, target spot disease, common mosaic virus disease, cucumber mosaic virus disease, and potato virus Y disease.

Images