CN115678858B - Hybridoma cell strain secreting monoclonal antibody against potato broomcorn top virus and application of monoclonal antibody - Google Patents
Hybridoma cell strain secreting monoclonal antibody against potato broomcorn top virus and application of monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting a monoclonal antibody against Potato broom-top virus (PMTV) and application of the monoclonal antibody. The prokaryotic expression purified PMTV coat protein is used as antigen to immunize BALB/c mice, and 1 hybridoma cell strain 4D2 capable of secreting anti-PMTV monoclonal antibody is obtained through hybridoma technology, and the preservation number is CGMCC No.45330. The indirect ELISA titer of the ascites of the monoclonal antibody secreted by the cell strain reaches 10 ‑7 The antibody types and subclasses are IgG1 and kappa light chains, and the monoclonal antibody has specific immune reaction with PMTV, but does not react with potato virus Y, potato leaf curl, potato virus S, potato virus M and potato virus A, and does not react with tobacco mosaic virus and healthy plant tissues. Wherein the sensitivity of detecting PMTV disease leaves by using the ACP-ELISA and dot-ELISA methods established by the 4D2 monoclonal antibody reaches 1:10240 and 1:1280 times dilution (w/v, g/mL) respectively. The establishment of the hybridoma cell strain and the virus serology detection method provides support for detection diagnosis, epidemiological investigation and scientific prevention and control of PMTV.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a hybridoma cell strain secreting a monoclonal antibody against potato broomcorn top virus and application of the monoclonal antibody.
Background
Potato top virus (PMTV) is a representative species of the family of plant baculoviruses (Virgaviridae), the genus Potato top virus (Pomovirus), and is a plus-sense single-stranded RNA virus (ssRNA) possessing a trisomy genome, RNA1 being about 6.0kb in length, RNA2 being about 3.5kb in length, and RNA3 being about 2.5 to 3.0kb in length. The virus particles are in the form of non-enveloped rigid rods with the diameter of 18-20 nm, and the three typical lengths are 65-80 nm, 150-160 nm and 290-310 nm respectively. RNA1 of PMTV contains an Open Reading Frame (ORF) encoding a 148kDa protein and a 206kDa read-through protein which may be involved in replication, wherein the 148kDa protein has a methyltransferase (Mtr) domain at the N-terminus and a helicase (Hel) domain at the C-terminus. The C-terminus of the 206kDa through protein has an RNA dependent RNA polymerase (RdRp) domain. RNA2 is RNA-CP, which encodes an ORF of Capsid Protein (CP) terminated with UAG, and encodes a CP reading protein of 61-79 kDa. RNA3 encodes a Triple Gene Block (TGB) consisting of three partially overlapping ORFs, namely TGBp1, TGBp2 and TGBp3, and an 8K protein or cysteine-rich protein (CRP). TGB is also present in other positive strand RNA viruses such as Allexivirus, benyvirus, carlavvirus, fovevirus, hordeivirus, pecluvirus and Potexvirus. Current studies demonstrate that the systemic movement of PMTV is independent of capsid protein expression and viral particle assembly, whereas these TGB and 8K proteins are involved in the intercellular movement and systemic transport of viruses through interactions.
post-PMTV infection symptoms depend on potato variety and environmental conditions. At lower temperatures, symptoms may be more pronounced, but as the temperature increases, the symptoms diminish. Leaf symptoms include bright yellow spots on the lower leaf, bright yellow V-shape, arc or ring pattern, leaf deformation, and internode shortening; symptoms on tubers often include necrosis and internal discoloration, including rust brown arcs, rings or spots, sometimes PMTV-infected tubers may not show symptoms; whereas symptoms of PMTV outside tubers are unusual, showing a surface annular bulge.
The united nations grain and agricultural organization (FAO) data shows that in 2005, the potato yield in developing countries exceeded developed countries for the first time, but china is currently the largest potato producing country, with chinese potato planting area and potato yield accounting for over 20% of the world. PMTV can result in significant loss of potato yield. A study of seed potatoes in Scotland showed that PMTV produced yields reduced as much as 67%. This virus also causes distortion and necrosis of tuber meats, which create morphological defects, producing unsightly potatoes, facing the commercial rejections of processors and packagers. At present, research on potato broomcorn virus is relatively mature abroad, but related research is less in China, but PMTV is reported in many places in China in recent years, and inspection and quarantine of the PMTV is required to be paid attention as the influence of a plurality of factors such as climate condition change, potato seed and tissue culture seedling introduction and the like.
The current PMTV risk is mainly represented by: 1) Recognition and detection are difficult: after infection, symptoms are affected by varieties and climate, when the symptoms are slight, yellow leaf symptoms caused by other viruses are easy to be confused, tuber symptoms are similar to TRV and PVY, and the method is not beneficial to field diagnosis, epidemic situation mastering and targeted removal of disease plants; 2) Easy propagation: PMTV is mainly transmitted through zoospores of eschar aleyrodis, and dormant spores releasing zoospores can still transmit viruses after being kept for more than 2 years under the condition of drying, so that even if seed potatoes are not toxic, the possibility that PMTV is transmitted along with the seed potatoes cannot be eliminated as long as toxic dormant spores are attached to the seed potatoes; 3) Once the intrusion is difficult to eradicate: dormant spores of eschar are present in the form of sporocysts that are resistant to adverse conditions and their hosts are very broad, so toxic solanum lycopersicum are difficult to eradicate once they are introduced. At present, PMTV virus-transmitting mediator potato eschar is found in inner Mongolia, jilin, gansu, jiangxi, fujian, guangdong, guizhou, yunnan and other places in China, and the domestic and international seed potato transportation and germplasm resource exchange are frequent, so that the risk of virus spreading and epidemic is high. At present, the main strategy for preventing the virus diseases in China is to strengthen the inspection and quarantine of the viruses and plant non-toxic potato seedlings.
In order to strengthen the inspection and quarantine technology of PMTV in China, it is extremely necessary to establish a practical detection technology for detecting PMTV with simplicity, rapidness, economy, effectiveness and high flux. The serological method has the advantages of simplicity, economy, accuracy, high flux detection and the like compared with the conventional methods of plant inoculation indication, electron microscope observation, molecular detection and the like, and is one of the most common and practical methods for plant virus detection and diagnosis. While serological methods are established depending on high quality viral antibodies. The invention prepares 1 hybridoma cell strain secreting PMTV specific monoclonal antibody by taking prokaryotic expression PMTV Capsid Protein (CP) as antigen through hybridoma technology, establishes a high-flux dot-ELISA serological method and a kit for detecting PMTV by taking the secreted monoclonal antibody as a core, and provides reagents and technical support for detection diagnosis of the virus in potato in China, port inspection and quarantine, epidemiological investigation, virus genome function analysis, resistance breeding, scientific prevention and control establishment and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a hybridoma cell strain secreting anti-potato broomcorn virus monoclonal antibody and application of the monoclonal antibody.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a hybridoma cell strain 4D2 for secreting the anti-potato broomcorn virus monoclonal antibody, which can secrete the anti-potato broomcorn virus specific monoclonal antibody, wherein the hybridoma cell strain 4D2 is preserved in China general microbiological culture collection center (CGMCC) No.45330 at 11-7 of 2022.
In a second aspect, the present invention provides an anti-potato broomcorn virus monoclonal antibody secreted by the hybridoma cell according to the first aspect. The indirect ELISA titer of the monoclonal antibody ascites reaches 10 -7 The antibody types and subclasses are IgG1 and kappa light chains, and the monoclonal antibody has specific immune reaction with the 19.7kDa coat protein of the potato broomcorn top virus, and the sensitivity of detecting the crude extract of the infected PMTV leaf tissue by using the monoclonal antibody to establish an ACP-ELISA and a dot-ELISA method respectively reaches 1:10240 and 1:1280 times dilution (w/v, g/mL).
The monoclonal antibody against the potato broomcorn top virus can specifically react with potato broomcorn top virus (PMTV) without reacting with Potato Virus Y (PVY), potato leaf curl virus (PLRV), potato Virus S (PVS), potato Virus M (PVM), potato Virus A (PVA) and Tobacco Mosaic Virus (TMV) which invade potatoes or with healthy potato plant tissues.
In a third aspect, the present invention provides an application of the monoclonal antibody against potato broom top virus in the above second aspect, which is various immunological detection methods and immunological detection kits established with monoclonal antibody as a core.
Compared with the prior art, the invention has the beneficial effects that: 1) The hybridoma cell strain provided secretes a large amount of anti-PMTV specific monoclonal antibodies, and the detection methods such as ACP-ELISA, dot-ELISA serology and the like established by taking the monoclonal antibodies as cores can rapidly, specifically, sensitively and accurately detect PMTV; 2) The monoclonal antibody prepared by the invention is used for detecting the PMTV virus, has low technical requirements on operators, and does not need expensive equipment such as an electron microscope, a PCR instrument and the like; 3) The monoclonal antibody prepared by the invention can be effectively used for detection and diagnosis of field PMTV and port inspection and quarantine, and can also be used for epidemiological investigation of the virus diseases, functional analysis of virus genome, resistance breeding, scientific prevention and control and the like.
Drawings
FIG. 1RT-PCR amplification of PMTV CP gene results;
FIG. 2 shows the result of SDS-PAGE analysis of purified PMTV CP recombinant protein;
FIG. 3 results of a specific assay for detecting PMTV by dot-ELISA;
FIG. 4 is a plot of the sensitivity analysis results of the dot-ELISA method for detecting PMTV;
FIG. 5ACP-ELISA (A), RT-PCR (B), dot-ELISA (C) test samples for PMTV results.
Preservation of organisms
Hybridoma cell line 4D2 secreting anti-potato broomcorn virus monoclonal antibody was deposited at China general microbiological culture Collection center, address: beijing, chaoyang area, north Chenxi way No. 1, no. 3, post code: 100101 and the preservation number is CGMCC No.45330.
Detailed Description
Hybridoma cell strain 4D2 for secreting potato-resistant broomcorn top virus is preserved in China general microbiological culture Collection center (CGMCC) of China academy of sciences microbiological culture Collection center (CGMCC) No.45330 in 11-7-2022, and can secrete potato-resistant broomcorn top virus.
An anti-potato broomcorn virus monoclonal antibody secreted by the hybridoma cell strain 4D2, and the anti-potato broomcorn virus monoclonal antibody ascites indirect ELISA effectThe price reaches 10 -7 The antibody types and subclasses are IgG1 and kappa light chains, and the monoclonal antibody has specific immune reaction with the 19.7kDa coat protein of the potato broomcorn top virus, and the sensitivity of detecting the crude extract of the infected PMTV leaf tissue by using the monoclonal antibody to establish an ACP-ELISA and a dot-ELISA method respectively reaches 1:10240 and 1:1280 times dilution (w/v, g/mL).
The monoclonal antibody against potato broomcorn top virus can specifically react with potato broomcorn top virus (PMTV) without reacting with Potato Virus Y (PVY), potato leaf curl virus (PLRV), potato Virus S (PVS), potato Virus M (PVM), potato Virus A (PVA) and Tobacco Mosaic Virus (TMV) or with healthy plant tissues.
The application of the monoclonal antibody against potato top virus in the virus detection is various immunological detection methods and immunological detection kits which are established by taking monoclonal antibody as a core.
The hybridoma cell strain provided by the invention can secrete a large amount of anti-potato broom top virus monoclonal antibodies, and the monoclonal antibodies secreted by the hybridoma cell strain are strong in specificity, high in potency and good in stability. The serological method for detecting PMTV with high flux by taking the monoclonal antibody as a core can be applied to detection and diagnosis of PMTV in field plant samples and port inspection and quarantine, thereby providing reagents and technical support for scientific prevention and control of PMTV in China.
The invention is further described below with reference to examples and figures.
1. Hybridoma cell preparation and monoclonal antibody preparation thereof
1. Preparation of immunogens
Prokaryotic expression of the PTMV CP gene and purification of recombinant proteins were performed by the following procedure:
1) Total RNA of PMTV-infected potato plants was extracted using Trizol.
2) Specific primers for the PMTV CP gene were designed based on the complete PMTV gene sequence (Access No. KM822700) already reported in NCBI GenBank: PMTV-CP-F (5'-gccatggctgatatcggatccATGGCTGAAAACAGAGGTGAGC-3') and PMTV-CP-R (5'-tgcggccgcaagcttgtcgacCTATGCACCAGCCCAGCG-3'), the lower case sequences are BamHI and SalI restriction endonuclease sites, respectively, homologous to the vector. The PMTV CP gene was amplified from total RNA extracted from PMTV-infected potatoes by RT-PCR (FIG. 1) and verified by DNA sequencing and nucleic acid blast comparison.
3) Homologous recombination: amplified PCR products were cut with Bam HI and Sal I after recovery and cloned into His-tagged prokaryotic expression vector pET-32a (Novagen, darmstadt, germany) by homologous recombination. And (3) performing heat shock conversion on the recombinant product pET-32a-PMTV-CP into escherichia coli DH5 alpha, plating for culture, selecting single colony for PCR detection, and screening pET-32a-PMTV-CP positive clones to send to a company for sequencing.
4) After sequencing, sequence comparison is carried out, after confirming that the PMTV CP sequence is error-free and the reading frame is accurate, the recombinant plasmid pET-32a-PMTV-CP which is successfully constructed is transformed into a E.coli BL21 (DE 3) strain (GE Healthcare, bucks, UK) through heat shock, after recovery culture, plating and colony PCR verification are carried out, and the recombinant positive expression strain is obtained.
5) pET-32a-PMTV-CP expression strain is inoculated into 2mL LB culture medium containing ampicillin (Amp) for culturing for 12h at 37 ℃, bacterial liquid is transferred into the LB culture medium containing Amp in a ratio of 1:100 times dilution, and shake culture is carried out for 2-3h to OD at 37 DEG C 600 The value is 0.6-0.8, IPTG is added to the final concentration of 0.5mM, and the recombinant protein expression is induced by overnight shake culture at 16 ℃.
6) Purifying PMTV-CP recombinant protein by imidazole method: after centrifugation at 6000rpm for 10min, the supernatant was removed, the bacterial pellet was resuspended in 50mL PBS, sonicated until the solution was clear, after centrifugation the supernatant was mixed with 500. Mu.L of High Affinity nickel ion resin High Affinity ni-charge resin FF (GenScript, USA) and incubated for 2h with slow rolling at 4 ℃.
7) Pouring the mixed solution into a protein purification column, pouring the effluent column passing solution back into the column again, washing the column with imidazole washing liquid with the concentration of 20mM and 50mM after the liquid is completely discharged, eluting with imidazole eluent with the concentration of 250mM and 300mM, and collecting the eluent to obtain the purified PMTV CP recombinant protein.
8) 20 mu L of eluent is respectively taken for SDS-PAGE electrophoresis analysis, the electrophoresis result of the recombinant protein is shown in figure 2, and the recombinant protein is successfully purified.
9) PMTV CP recombinant protein dialysis: preparing 2L of 0.01M PBS in advance, pre-cooling in a refrigerator at 4 ℃, adding the purified PMTV CP recombinant protein into a clean dialysis bag, clamping by a clamp, putting into the pre-cooled PBS, dialyzing for 24 hours, changing the PBS every 2 hours during the dialysis, and performing SDS-PAGE electrophoresis after the dialysis.
2. Immunization of animals
8 week old BALB/c female mice were immunized with purified PMTV CP protein: 50 mug of PMTV CP protein is mixed with equal volume Freund complete adjuvant, after full emulsification, subcutaneous injection is carried out through abdominal cavity, 3 weeks are spaced, after full emulsification with a non-equal amount of antigen and equal volume Freund incomplete adjuvant is carried out, the intraperitoneal injection is carried out for secondary avoidance, after 3 weeks, the intraperitoneal injection is carried out by mixing the antigen with doubling dose with equal volume of physiological saline, and after 3 days, splenocytes are taken for cell fusion.
3. Cell fusion
Mixing the above immunized mouse spleen cells with mouse myeloma cells (Sp 2/0) at a ratio of 9:1 in serum-free RPMI 1640 (Gibco) medium, centrifuging at 1500rpm for 5min, removing supernatant medium, adding 1mL 50% PEG (molecular weight 1500) fusion agent into a centrifuge tube containing cells, placing in a water bath at 37deg.C for 2min, stopping fusion with serum-free RPMI 1640 medium, centrifuging at 1500rpm for 5min, absorbing supernatant, suspending precipitate with RPMI 1640 medium containing HAT, fetal bovine serum and green chain mycin, packaging into 96-well cell plate holes, and packaging at 37deg.C for 5% CO 2 Is cultured and hybridoma cells are selected in a cell culture box.
4. Hybridoma cell, positive hole screening and cell cloning
After 6-7d culture in cell incubator, the positive wells were screened by indirect ELISA method using purified PMTV CP protein and/or crude extract of potato leaf infected with PMTV as coating antigen to obtain 360 positive wells when the fused cells proliferated to above 15% of the bottom of the covered wells (about 12-13 d) with medium containing HT, fetal bovine serum, and green streptomycin. And carrying out specificity analysis on 360 holes, screening out 20 cell holes with good specificity, and carrying out cloning by a limiting dilution method to finally obtain 1 hybridoma cell strain 4D2 capable of secreting PMTV highly specific and sensitive monoclonal antibody, namely the hybridoma cell with the preservation number of CGMCC No.45330. After in vitro passage for more than 3 months and repeated cryopreservation and recovery, the cell strain can well grow and stably secrete antibodies. After the enlarged culture, the product is used for ascites preparation and liquid nitrogen preservation.
5. Monoclonal antibody ascites preparation and purification
Taking BALB/c male mice of about 12 weeks old, injecting 300 μl pristane into abdominal cavity, and injecting about 7×10 into abdominal cavity after 7-10d 5 The hybridoma cells of 4D2 can obviously expand the abdomen of the mouse after 6-10D of injection, ascites is taken by a needle head, centrifugation is carried out for 3min at 8000rpm, and the supernatant is collected to obtain the monoclonal antibody ascites.
Taking 1-time volume of ascites, adding 2-time volume of physiological saline to dilute, dropwise adding saturated ammonium sulfate solution (pH 7.2) while stirring at room temperature, standing at 4 ℃ for 10min, centrifuging at 12000rpm for 10min, pouring out supernatant, suspending and precipitating with 2mL of PBS, dialyzing in 4 ℃ precooled PBS for 24h (changing PBS every 4 h), and storing in a refrigerator at-80 ℃.
6. Subclass identification and ascites titer determination of monoclonal antibodies
Purified monoclonal antibody ascites was combined with standard anti-BALB/c mouse IgG from Sigma Co 1 、IgG 2a 、IgG 2b 、IgG 3 DAS-ELISA experiments are carried out on IgM, lambda and kappa antibodies according to the instruction book of the kit, and the results show that the 4D2 monoclonal antibodies are of IgG1 and kappa light chains. The purified PMTV CP protein is taken as an antigen, the monoclonal antibody ascites titer is detected by an indirect ELISA method, and the analysis result shows that the monoclonal antibody ascites titer reaches 10 -7 。
7. Specific detection of monoclonal antibodies
The ELISA plate is coated with crude extracts of leaf diseases infected with Potato Virus Y (PVY), potato leaf curl virus (PLRV), potato Virus S (PVS), potato Virus M (PVM), potato Virus A (PVA) and Tobacco Mosaic Virus (TMV), the crude extracts of healthy leaf of potato are used as negative control, the crude extracts of leaf diseases infected with PMTV are used as positive control, and the specificity of the monoclonal antibody is analyzed by an ACP-ELISA method. The steps of the ACP-ELISA method are as follows: the virus-infected disease leaves and healthy leaves are respectively ground in a mortar, ELISA coating liquid is added according to the proportion of 1:20 (w/v, g/mL) for homogenizing, and supernatant is 100 mu L after centrifugation at 5000rpm for 3minHole coating ELISA plate, overnight at 4 ℃ or for 4 hours at 37 ℃; sealing with 3% skimmed milk powder for 30-40min after PBST washing 3 times; adding 100 mu L/hole of monoclonal antibody diluted 1:5000 times, and incubating for 1h at 37 ℃; after 3 times of PBST washing, 100. Mu.L/well of an Alkaline Phosphatase (AP) -labeled rabbit anti-mouse IgG secondary antibody (Sigma Co.) diluted 1:8 times was added, and incubated at 37℃for 1 hour; after PBST is washed for 4 times, PNPP substrate is used for developing for 20-30min,2mol/L sodium hydroxide is used for stopping the reaction, and an enzyme-labeled instrument is used for reading OD 405 Samples with a ratio of negative OD value greater than 3.0 were positive. As a result, it was found that 4D2 mab was strongly positive for PMTV without any immune response to both diseased leaves infected with PVY, PLRV, PVS, PVM, PVA, TMV and crude extract of healthy potato plant tissue.
2. Establishment of serological method for detecting PMTV
1ACP-ELISA detection method for detecting PMTV
1.1 steps of ACP-ELISA method:
1) Grinding and homogenizing the leaves with ELISA coating liquid according to the ratio of 1:20 (w/v, g/mL) until no obvious solid exists, centrifuging at 5000rpm for 3min, adding 100 mu L/hole of supernatant into an ELISA plate, taking PMTV infected potato leaf tissues as positive control, taking healthy potato leaf tissues as negative control, and coating at 4 ℃ overnight or 37 ℃ for 4h;
2) Washing ELISA plate with PBST for 3 times each for 2min, adding 250 μl PBS containing 3% skimmed milk powder into each well, and sealing at 37deg.C for 30-40min;
3) Pouring off the blocking solution in the ELISA plate in the previous step, washing with PBST for 3 times, adding 100 mu L/hole of monoclonal antibody ascites properly diluted by the blocking solution, and incubating for 1h at 37 ℃;
4) Pouring out the monoclonal antibody diluent in the ELISA plate, washing with PBST for 3 times, adding 100 mu L/hole of AP-labeled goat anti-mouse IgG secondary antibody (Sigma) properly diluted by the blocking solution, and reacting for 1h at 37 ℃;
5) PBST was washed 3 times followed by PBS washing 1 time for 3min each. Adding PNPP substrate, developing at room temperature for 20-30min, and visually observing that the yellow hole of the substrate is positive, or detecting OD with Bio-Rad 680 enzyme marker after stopping reaction with 2mol/L sodium hydroxide 405 In P/N>3.0 as a positive judgment criterion.
1.2 establishment of ACP-ELISA method for detecting PMTV
Determining the optimal working concentration of the antibody in the ACP-ELISA method by adopting a square matrix test, namely taking a (w/v, g/mL) PMTV disease leaf crude extract diluted by 1:20 as an antigen coating ELISA plate; ELISA plate transversely adding PMTV monoclonal antibody diluted in 1:1000-1:512000 times ratio for 1h respectively, adding AP marked goat anti-mouse IgG secondary antibody diluted in 1:1000-1:512000 times ratio for 1h respectively in each row of ELISA plate longitudinally after PBST washing, taking the diluted crude extract of healthy potato plant as negative control, repeating 3 times for each treatment, and determining the optimal working concentration of antibody in ACP-ELISA. The results show that 4D2 monoclonal antibody is diluted 1:8000 times and AP-labeled goat anti-mouse IgG secondary antibody is diluted 1:10000 times to be the optimal working concentration, and an ACP-ELISA method for detecting PMTV is established according to the optimal working concentration.
1.3ACP-ELISA method and specificity and sensitivity of PMTV monoclonal antibody
Taking potato disease leaves infected with PMTV and crude extract of healthy potato leaf tissue as positive control and negative control respectively, taking disease leaves infected with PVY, PLRV, PVS, PVM, PVA and TMV as detection samples, adding the detection samples into an ELISA plate, repeating the experiment for three times, and analyzing the specificity of PMTV monoclonal antibody and the established ACP-ELISA method. Under the working concentration of the optimal antibody, the method detects that the disease leaves infected with PMTV have strong positive reactions, and detects that the crude extract of the disease leaves infected with PVY, PLRV, PVS, PVM, PVA, TMV and healthy potato leaves has negative reactions, and has extremely obvious difference with the OD value of a positive sample, which indicates that the monoclonal antibody and the ACP-ELISA method established by the monoclonal antibody can specifically detect PMTV.
The PMTV-infected potato leaf and healthy potato leaf were sequentially added to ELISA plates with PBS buffer from 1:10 to 1:81920 (w/v, g/mL) fold diluted crude extract, respectively, and the sensitivity of the ACP-ELISA method to detect PMTV-infected leaf was analyzed and the experiment repeated three times. The results show that the sensitivity of the ACP-ELISA detection of the disease leaves reaches 1:10240 times dilution (w/v, g/mL), which shows that the prepared monoclonal antibody and the established method have good sensitivity.
Establishment of 2dot-ELISA detection method
2.1 detection step of dot-ELISA method
1) Grinding potato plant tissue in a mortar, adding 0.01M PBS buffer solution according to the proportion of 1:20 (w/v, g/mL) for homogenizing, centrifuging at 5000rpm for 3min, and obtaining a supernatant as a plant tissue crude extract;
2) Spotting: taking 2 mu L of crude extract solution and spotting the crude extract solution on a Nitrocellulose (NC) membrane, and simultaneously setting healthy and PMTV-infected potato leaf crude extract solutions as negative and positive controls respectively, and drying the potato leaf crude extract solution in a 37 ℃ incubator for 10min;
3) NC membrane is immersed in PBST (0.01M PBS containing 0.05% Tween-20) blocking solution containing 5% skimmed milk powder, and blocked at room temperature for 30min; then, the NC membrane is put into a moderately diluted monoclonal antibody for incubation for 1h at room temperature;
5) Washing the film: washing the membrane with PBST for 3 times and 3min each time;
6) Placing NC membrane into moderately diluted AP enzyme marked goat anti-mouse IgG secondary antibody, incubating for 1h at room temperature, washing the membrane 3 times by PBST, 3min each time, and washing the membrane 1 time by PBS;
7) Preparing a color development liquid: mu.L of NBT and 33. Mu.L of BCIP substrate (Promega) were added to 10mL of substrate buffer (0.1M Tris Cl, 0.1M NaCl, 0.025M MgCl, pH 9.5) and mixed well.
8) The membrane is dried by absorbing water with absorbent paper and then is put into substrate liquid for reaction, and the membrane is developed for 15-20min at the room temperature. Visual inspection, when the positive control showed a purple spot, and the negative control showed no color change, the membrane was rinsed in tap water to terminate the reaction, photographed and the result was recorded.
2.2 establishment of the method for detecting PMTV dot-ELISA
And (3) respectively performing double dilution on PMTV monoclonal antibody and AP marked goat anti-mouse IgG secondary antibody from 1:1000 times, and determining the optimal working concentration of the monoclonal antibody and the enzyme-labeled secondary antibody in the dot-ELISA method by using a square matrix experiment. Experimental results show that the optimal working concentrations of the monoclonal antibody and the enzyme-labeled secondary antibody are respectively 1:5000 and 1:8000 times of dilution, and a dot-ELISA method for detecting PMTV in plants is established according to the optimal working concentrations of the antibodies.
2.3 specificity and sensitivity of detection of PMTV by dot-ELISA method
The specificity analysis of the dot-ELISA method was performed with PMTV-infected potato leaf and healthy potato leaf tissue crude extracts as positive and negative controls, respectively, and with infected PVY, PLRV, PVS, PVM, PVA, TMV-infected leaf as the detection sample. The method detects that the crude extract of the plant disease leaves infected with PMTV has strong positive reaction, and detects that the crude extract of the plant disease leaves infected with PVY, PLRV, PVS, PVM, PVA, TMV and the crude extract of the healthy potato leaves have negative reaction (figure 3), which shows that the method and the monoclonal antibody have excellent specificity.
The PMTV-infected leaf was homogenized in a mortar and diluted from 1:10 to 1:2560 fold with 0.01M PBS (w/v, g/mL), and the same treatment was performed on healthy potato leaf crude extract. Sensitivity of infected PMTV potato leaves was detected by dot-ELISA. Sensitivity analysis showed that when PMTV leaf crude extract was diluted 1:1280 fold (w/v, g/mL), positive spots still appeared purple with established dot-ELISA detection, i.e. the sensitivity of detection of the leaf reached 1:1280 fold dilution (FIG. 4).
3. Detection application of established serological method
PMTV was performed on 24 samples using established ACP-ELISA, dot-ELISA methods, and 7 samples were found to produce positive reactions (FIG. 5). The samples were analyzed simultaneously by RT-PCR, which indicated that all serological tests were positive for the PMTV-specific gene fragments, but none of the serological tests were negative for the specific gene fragments (fig. 5), and sequencing and sequence comparison of the PCR products indicated that the RT-PCR positive samples did infect PMTV, indicating that the detection results of the 2 serological tests were completely consistent with the RT-PCR. It was demonstrated that the ACP-ELISA and dot-ELISA methods can be used accurately and reliably for the detection of PMTV in plants (FIG. 5).
5. Potato broomcorn virus dot-ELISA detection kit
1) The main components of the kit are as follows:
the above reagents are all stored at 4deg.C
Nitrocellulose membrane (NC) 10 sheets
30g of skimmed milk powder
10X PBST 1 bottle 100mL
Substrate buffer 1 bottle 100mL
2) The operation steps of detecting the plant sample are as follows:
a. potato plant tissue is weighed, ground, homogenized by adding 0.01M PBS (pH 7.4) in a ratio of 1:20-50 (w/v, g/mL);
b. centrifuging the homogenate at 5000rpm for 3min;
c. taking 2 mu L of supernatant sample on NC membrane, setting healthy and PMTV infected plant tissue as negative and positive control respectively, and drying at room temperature for 5-10min;
immersing NC film into PBST sealing liquid containing 5% of skimmed milk powder for 30min at room temperature;
e, placing NC membrane into monoclonal antibody diluted 1:5000 times for incubation for 1h at room temperature;
f. washing the membrane with PBST for 3-4 times and 3min each time; placing NC membrane into AP enzyme marked goat anti-mouse IgG secondary antibody with dilution of 1:8000, and incubating for 1h at room temperature;
PBST membrane washing is carried out for 4 times, each time for 3min;
66. Mu.L of NBT and 33. Mu.L of BCIP substrate are added into 10mL of substrate buffer solution and mixed uniformly, the membrane is placed into the buffer solution for developing for 15-20min, when positive control shows obvious purple and negative control does not show any color, tap water is used for rinsing the membrane to stop the reaction, and the result is recorded by photographing.
3) Preservation and validity period:
storing at 2-8deg.C in dark place, and has a shelf life of 12 months.
4) 0.01M phosphate buffer (PBS pH 7.4) formulation:
adding 950mL of distilled water for dissolution, adjusting the pH to 7.4, and fixing the volume to 1000mL.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (3)
1. The hybridoma cell strain 4D2 capable of secreting the anti-potato broomcorn virus monoclonal antibody is characterized in that the hybridoma cell strain 4D2 can be secreted to be preserved in China general microbiological culture collection center (CGMCC) No.45330 on the 11 th month 7 of 2022.
2. An anti-potato broomcorn monoclonal antibody secreted by the hybridoma cell line 4D2 of claim 1.
3. The use of the anti-potato top virus monoclonal antibody according to claim 2 for detecting the virus, wherein the use is various immunological detection methods and immunological detection kits which are established by taking the anti-potato top virus monoclonal antibody as a core.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105525037A (en) * | 2015-07-24 | 2016-04-27 | 湖南农业大学 | Nested RT-PCR method and primer combination for detecting potato mop-top virus |
| CN112877480A (en) * | 2021-04-14 | 2021-06-01 | 黑龙江省农业科学院马铃薯研究所 | Primer probe set, kit and detection method for PCR detection of potatosis virus |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105525037A (en) * | 2015-07-24 | 2016-04-27 | 湖南农业大学 | Nested RT-PCR method and primer combination for detecting potato mop-top virus |
| CN112877480A (en) * | 2021-04-14 | 2021-06-01 | 黑龙江省农业科学院马铃薯研究所 | Primer probe set, kit and detection method for PCR detection of potatosis virus |
Non-Patent Citations (1)
| Title |
|---|
| Potato mop-top virus isolate Yunnan segment RNA2, complete sequence;Hu，X.，等;Genbank登录号：KM822700.1;参见全文 * |
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