CN112301044A - Raw tobacco NbAPX3Gene polyclonal antibody and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Bunsen cigarette NbAPX₃A gene polyclonal antibody, its preparing process and application are disclosed, which includes (1) constructing the recombinant expression carrier containing the nucleotide sequence shown by SEQ ID No.1, transforming the recombinant expression carrier to colibacillus competent cell to obtain NbAPX₃A protein; (2) with the NbAPX₃Immunizing animal with protein as antigen, separating and purifying to obtain NbAPX₃Protein polyclonal antibody, protein polyclonal antibody and protein polyclonal antibodyThe polyclonal antibody prepared by the method can specifically recognize the NbAPX of the Bunsen₃In the Bunsen cigarette NbAPX₃The protein detection and function identification and research have wide application. NbAPX₃Preparation of protein antibody for detecting Ningpo NbAPX₃Expression condition of gene in virus infection process and research of NbAPX₃The function of the protein is of great significance.

Description

Raw tobacco NbAPX3Gene polyclonal antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biotechnology products for detecting plant viruses, and particularly relates to a Bunsen cigarette NbAPX₃A gene polyclonal antibody and a preparation method and application thereof.

Background

The Nicotiana benthamiana is native to Australia, belongs to Solanaceae (Solanaceae) like hot pepper, tomato, potato and cultivated tobacco, and is an allotetraploid plant containing 19 chromosomes, and complete genome data is not annotated at present. The tobacco is one of important model plants, and is widely applied to the aspects of plant and microorganism interaction, protein interaction identification, subcellular localization and the like. Therefore, the research on the functional genes has important significance for the development of molecular biology.

The tobacco-producing L-ascorbic acid peroxidase (Nicotiana benthamiana L-ascorbate peroxidase 3) is abbreviated as NbAPX₃。NbAPX₃Not only is one of the important antioxidant enzymes in the metabolism of active oxygen of plants, but also the elimination of H in chloroplasts₂O₂The key enzyme of (2). H₂O₂Is a natural product of photosynthetic electron transfer chain and some enzyme reaction in plant chlorophyll body, and is active oxygen with toxic action. The ascorbate peroxidase is widely applied to genetic engineering, and the related resistance of plants can be improved by over-expressing the gene, while NbAPX is related to₃Antibodies to the protein have not been reported. Thus, NbAPX was developed₃The rapid detection and identification technology is in the forefront.

Disclosure of Invention

The purpose is as follows: in order to overcome the defects in the prior art, the invention provides a Bunsen NbAPX₃A gene polyclonal antibody and a preparation method and application thereof.

The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:

in a first aspect, a Bunsen NbAPX is provided₃A gene having a nucleotide sequence such as SED ID NO: 1 is shown.

In a second aspect, a Bunsen NbAPX is provided₃Recombinant protein, the amino acid sequence of which is as defined in SED ID NO: 2, respectively.

In a third aspect, a polyclonal antibody is provided, which is the Bunsen NbAPX₃The recombinant protein is an antigen and is prepared by immunizing animals.

In a fourth aspect, the Bunsen NbAPX₃Recombinant protein/polyclonal antibody in NbAPX₃Application in detection.

The raw cigarette NbAPX₃A method of producing a recombinant protein comprising:

a) sequences such as SED ID NO: 1 of the raw tobacco NbAPX₃A gene;

b) constructing a recombinant plasmid: mixing the raw tobacco NbAPX₃The gene is connected to a prokaryotic expression vector pET-28a to obtain a recombinant plasmid pET-28a-NbAPX₃；

Induced expression of recombinant protein: the recombinant plasmid pET-28a-NbAPX₃Transforming into competent cells of Escherichia coli, culturing, inducing, cracking, and purifying to obtain Ningpo NbAPX₃A recombinant protein.

The bensheng cigarette NbAPX₃The preparation method of the recombinant protein is characterized in that when the PCR method is used for amplification, the total RNA of the Nicotiana benthamiana leaf is used as a template, and primers are used: upstream primer NbAPX₃F is shown as SEQ ID NO.3 and a downstream primer NbAPX₃R is amplified as shown in SEQ ID NO. 4.

In some embodiments, the escherichia coli competent cell is an escherichia coli BL21 competent cell.

The preparation method of the polyclonal antibody comprises the Bunsen NbAPX₃Recombinant protein is used as antigen, antiserum is prepared by adopting a conventional polyclonal antibody preparation method, and the antiserum is separated and purified to obtain the polyclonal antibodyA diabody.

In some embodiments, the method of making the polyclonal antibody comprises: the bensheng cigarette NbAPX₃Injecting the recombinant protein as antigen into rabbit, immunizing for four times, collecting rabbit blood, separating serum, and purifying to obtain Bunsen NbAPX₃Protein anti-rabbit polyclonal antibody.

Further, the bensheng tobacco NbAPX₃Adjusting the concentration of the recombinant protein to 1mg/mL for antigen injection; extracting adjuvant and antigen at a volume ratio of 1: 1; the first-time immunization adopts a complete adjuvant, and the second-time immunization adopts an incomplete adjuvant.

In the application, a bensheng cigarette NbAPX₃The preparation method of the gene polyclonal antibody comprises the following steps:

(1) constructing a recombinant expression vector containing a nucleotide sequence shown in SEQ ID NO.1, and transforming escherichia coli competent cells into the recombinant expression vector to obtain a recombinant protein antigen;

(2) subjecting the NbAPX₃Immunizing New Zealand white rabbit with protein antigen, separating and purifying the serum to obtain NbAPX₃Protein polyclonal antibodies. NbAPX₃Preparing a protein standard substance by taking total RNA of the leaves of the Nicotiana benthamiana as a template and using a primer NbAPX₃F and NbAPX₃R amplification to obtain NbAPX₃The gene is cut and connected, and cloned into pET-28a carrier to obtain recombinant plasmid pET28a-NbAPX₃The recombinant plasmid is transformed into Escherichia coli BL21 strain, cultured overnight at 37 ℃, IPTG induced expression, and then purified by nickel column affinity chromatography to obtain protein with the size of 27 kDa.

Wherein, the NbAPX₃The primers of (a) are designed as follows:

NbAPX₃F:5’-CGGGATCCATGATAACGTATGCAGATTTGTACCAGC-3'; cleavage siteBamH l

NbAPX₃R:5’-CCGCTCGAGCTTCATCCTTTTCCGCACTTCGTAC-3'; cleavage siteXho l

(3) The recombinant expression vector is obtained by cloning a fragment containing a nucleotide sequence shown in SEQ ID NO.1 to a prokaryotic expression vector.

The type of the escherichia coli competent cell used in the present invention is not particularly limited as long as it is suitable for efficient expression of the recombinant expression vector in the cell, and preferably, the escherichia coli competent cell is escherichia coli BL21 competent cell for better expression effect.

In the present invention, prokaryotic expression vectors suitable for recombinant protein antigens, which are conventionally used in the art, may be used, and preferably, when the prokaryotic expression vector is pET-28a, the expression effect of recombinant protein antigens is better.

In the method provided by the invention, the step of immunizing New Zealand white rabbits comprises 4 times of immunization, firstly, the 14 th immunization is carried out after the purified recombinant protein antigen is mixed with complete Freund's adjuvant, then, the interval time between the second immunization and the fourth immunization is 7d after the purified recombinant protein antigen is mixed with the incomplete Freund's adjuvant, and the immunization dose is the same as that of the first immunization.

In the present invention, the dose of the immunization and the mixing ratio with the adjuvant used for immunizing an animal can be performed according to a method conventional in the art. The immunized animals include, but are not limited to, New Zealand white rabbits.

And (3) collecting small sample serum from the middle ear artery on the 7 th day after the rabbit three-immunization, detecting the small sample serum to be qualified, adding the rabbit three-immunization after 7 days, and collecting the whole blood after 7 days of adding the rabbit three-immunization.

The invention also provides the Bunsen NbAPX prepared by the method₃Protein polyclonal antibodies. The polyclonal antibody can specifically recognize NbAPX₃The specific amino acid segment in the protein is SEQ ID NO.2 and is NbAPX₃An antibody specific for the protein. The amino acid sequence shown in SEQ ID No.2 is as follows:

MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMITYADLYQLAGVVAVEVTGGPTIDFVPGRKDSSVSPKEGRLPDAKQGVPHLKDVFYRMGLSDKDIVALSGGHTLGRAHPDRSGFDGPWTKEPLKFDNSYFVELLKGETEGLLKLATDIALLDDPEFREYVELYAKDEDAFFRDYAISHKKLSELGFTPSSGSKATLRDGTILAQSAVGVVVAAAVVALSYWYEVRKRMKLEHHHHHHDPAANKARKEA。

the invention also provides the Bunsen NbAPX₃Application of protein polyclonal antibody is provided. The application comprises the preparation of the compositionThis raw cigarette NbAPX₃Reagent or kit of protein polyclonal antibody for detecting Ningpo NbAPX₃Expression or functional characterization during viral infection.

The method provided by the invention clones the coded NbAPX by a PCR method₃DNA sequence of protein, so as to obtain specific polyclonal antibody. The polyclonal antibody prepared by the method provided by the invention can specifically identify the NbAPX of the Bunsen₃Protein in NbAPX₃The protein has wide application in detection, function identification and research. NbAPX₃Preparation of protein antibody for detecting Ningpo NbAPX₃Expression condition of gene in virus infection process and research of NbAPX₃The function of the protein is of great significance.

Drawings

FIG. 1 is a diagram of agarose gel electrophoresis of total RNA from tobacco leaves.

FIG. 2 shows a vector carrying pET-28a-NbAPX₃The plasmid PCR gel electrophoresis image of (1). M: marker; 1: a fragment of interest.

FIG. 3 is NbAPX₃Protein expression and purification electropherograms.

FIG. 4 is NbAPX₃Titer profile of protein polyclonal antibodies.

FIG. 5 is a specific electrophoresis chart of Western blot detection serum: 1^#Over-expression of NbAPX₃The tobacco of (2); 2-5^#Total protein of tobacco infected with tobacco mosaic virus.

Detailed Description

The invention is further illustrated by the following examples and figures.

(1) Extraction of total RNA of Bunsen tobacco leaves

The raw tobacco seeds are stored in a laboratory, the total RNA of the raw tobacco leaves is extracted by adopting a Trizol method, then 2 percent of agarose gel electrophoresis is carried out, the quality of the extracted total RNA of the raw tobacco leaves is detected, as shown in figure 1, the integrity of the extracted total RNA of the raw tobacco leaves is particularly good, the extracted total RNA is not degraded or polluted by DNA, and the extracted total RNA of the raw tobacco leaves can be used as an ideal reverse transcription template in the next step.

(2) Reverse transcription

The total RNA of the leaves of the Bunge tobacco obtained in the previous step is used as a template for reverse transcription to obtain cDNA.

(3)NbAPX₃Design and Synthesis of Gene primers

Design of primer

According to NbAPX₃Gene sequence, design and synthesis in amplification of NbAPX₃Primers specific for the nucleotide sequence: NbAPX₃F (SEQ ID NO.3) and NbAPX₃R (SEQ ID NO. 4); enzyme cutting sites BamH l and Xho l are respectively introduced to connect with a prokaryotic expression recombinant vector pET-28 a. The primers are synthesized by the Shanghai Bioengineering Co., Ltd in a purification mode of PAGE.

The NbAPX₃The primers of (a) are designed as follows:

NbAPX₃F:5’-CGGGATCCATGATAACGTATGCAGATTTGTACCAGC-3' (SEQ ID NO. 3); cleavage siteBamH l

NbAPX₃R:5’-CCGCTCGAGCTTCATCCTTTTCCGCACTTCGTAC-3' (SEQ ID NO. 4); cleavage siteXho l

②RT-PCR

Using cDNA obtained by reverse transcription as template, and using the above-mentioned primer to obtain target NbAPX₃，NbAPX₃The nucleotide sequence of the gene is shown as SEQ ID NO.1, and specifically comprises the following steps:

ATGATAACGTATGCAGATTTGTACCAGCTTGCAGGAGTTGTTGCAGTTGAAGTCACTGGTGGTCCGACCATTGATTTTGTCCCTGGTAGGAAGGATTCCAGTGTTTCTCCAAAGGAAGGACGGCTGCCAGATGCTAAACAAGGTGTGCCACATCTGAAAGATGTATTTTATAGGATGGGTTTGTCTGACAAAGATATAGTGGCACTATCTGGTGGTCACACACTGGGAAGGGCACATCCAGATAGATCAGGCTTTGATGGTCCATGGACAAAGGAGCCACTGAAATTTGACAATTCATATTTTGTGGAGCTGCTTAAGGGGGAAACTGAGGGCCTGCTGAAACTTGCTACAGACATAGCTTTATTGGATGATCCTGAGTTCAGAGAATATGTTGAGCTGTATGCTAAGGATGAAGATGCCTTCTTTAGGGATTATGCCATATCACACAAGAAACTATCTGAGCTAGGGTTTACCCCAAGTTCTGGTTCCAAGGCTACACTGAGGGATGGCACTATATTAGCACAAAGTGCTGTAGGAGTTGTTGTGGCTGCTGCAGTGGTGGCACTGAGTTACTGGTACGAAGTGCGGAAAAGGATGAAGTGA

taking total RNA of the leaves of the raw tobacco as a template and NbAPX₃R primercDNA was synthesized by reverse transcriptase M-MLV, and the reverse transcription system is shown in Table 1.

TABLE 1 reverse transcription System

Template RNA	600ng
		NbAPX3R	0.5μL
RTase M-MLV	0.5μL
		5×M-MLV Buffer	2μL
dNTP Mixture	2μL
		RNase Inhibitor(40U·μL-1)	0.25μL
ddH2O	To 10 μ L

Mixing the flicked tube walls, slightly centrifuging, keeping the temperature at 42 deg.C for 60min, and placing on ice for use;

the PCR reaction system is shown in Table 2.

TABLE 2 PCR reaction System

cDNA	2μL
			2×Taq Mix	25μL
NbAPX3F	2μL
		NbAPX3R	2μL
ddH2O	To 50 μ L

The PCR procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 60 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, extension at 72 deg.C for 7min, and storage at 4 deg.C;

sucking RT-PCR product, performing 1% agarose gel electrophoresis, cutting gel, and recovering.

(iii) ligation of DNA fragment to vector

Target gene fragment	10μL
		pMD19-T	0.5μL
T4 DNA Ligase	1μL
		10×T4 Ligase Buffer	2μL
ddH2O	To 20 μ L

The reaction system is incubated for 12-16h at 16 ℃. 10. mu.L of transformed E.coli DH 5. alpha. competent cells were obtained. Overnight culture, selecting single colony for PCR detection, finally selecting more than 5 positive single colonies to extract plasmid and sequencing.

Construction of recombinant plasmid

And carrying out double enzyme digestion on the recovered product by using BamH l and Xho l, connecting the enzyme digestion recovered product to a pET-28a vector subjected to corresponding enzyme digestion, transforming the product to escherichia coli DH5 alpha competent cells, and identifying positive recombinant bacteria by bacterial liquid PCR, enzyme digestion and sequencing.

The cleavage system is shown in Table 3.

TABLE 3 enzyme digestion System

Recovering the product	40.5μL
		BamH l	1μL
Xho l	1μL
		10×K Buffer	2.5μL
BSA	5μL

The enzyme was cleaved at 37 ℃ for 2 h.

By AxyPrep^TMExtracting positive recombinant plasmid from the DNA Gel Extraction kit, transforming the positive recombinant plasmid into competent cells of a escherichia coli BL21 strain, and obtaining positive clone through bacteria liquid PCR and enzyme digestion identification.

Prokaryotic expression of protein

The positive strains were selected and inoculated in 2mL LB liquid medium and activated overnight at 37 ℃. Adding the activated bacterial liquid into fresh LB according to the ratio of 1:100, and culturing at 37 ℃ to OD₆₀₀Is 0.6. Induction was carried out at 28 ℃ and 37 ℃ respectively, 2 tubes were taken at each temperature, one tube was added with IPTG to a final concentration of 1mM, and the other tube was incubated for 4-6h without IPTG as a control. 2mL of the induced culture was centrifuged at 12000 rpm for 30 seconds to collect the cells. Adding 200 μ L SDS-PAGE Loading Buffer, boiling for 10min, and centrifuging at 12000 rpm for 10 min. 8mL of the supernatant was subjected to SDS-PAGE.

After protein expression is determined by preliminary experiments, 10mL of activated bacteria solution is added into 1L of liquid LB according to a ratio of 1:100, and the mixture is cultured to OD at 37 DEG C₆₀₀0.6, induced with 1mM IPTG for 18h at 18 ℃. Then, the cells were collected by centrifugation, resuspended in 40mL of protein buffer containing 20mM Tris-HCl (pH 7.0), 500mM NaCl, 10% glycerol, and then treated with a protease inhibitor to a final concentration of 1mM, sonicated, and centrifuged to collect the supernatant and the pellet. The supernatant was purified by High Affinity Ni-NTA Resin from GenScript. 5mL of High Affinity Ni-NTA Resin was passed through a 5 column volume protein buffer, and the supernatant was passed through the column 5 times, eluted with 10mL of protein buffer containing 10mM, 50mM, 100mM, and 200mM imidazole, respectively, and the eluate was subjected to SDS-PAGE (FIG. 3). Concentrating the eluate containing target protein with protein concentration column, subpackaging, quick freezing with liquid nitrogen, and storing at-80 deg.C. (2) NbAPX₃Preparation of protein polyclonal antibody

Firstly, selecting young New Zealand white rabbits with the weight of about 2.5kg for marking. With a concentration of 1mg/mL NbAPX₃Coat protein as immunogen New Zealand white rabbit. In the first immunization, firstlyThe antigen is completely mixed, then the antigen is fully mixed with Freund's complete adjuvant (paraffin oil: amniotic fluid fat 7:1, and additionally 1mg/mL of inactivated Mycobacterium tuberculosis component) according to the volume ratio of 1:1, after complete emulsification, multiple-point subcutaneous injection is carried out, each point is 0.2mL, and the immune dose of each New Zealand white rabbit is 1 mg.

② the second-fourth avoidance adopts Freund incomplete adjuvant (paraffin oil: amniotic fluid fat is 7:1), and the second-fourth avoidance is still fully mixed with antigen according to the volume ratio of 1: 1. Second immunization is carried out 14 days after first immunization, the interval between second immunization and third immunization is 7 days, and the immunization dose is the same as that of the first immunization.

Third, the middle ear artery sampling small sample serum detection is carried out on the 7 th day after the rabbit three-immunity: blood was collected from the ear vein of immunized New Zealand white rabbits (200. mu.L) into sterile centrifuge tubes, incubated at 37 ℃ for 2h, and then at 4 ℃ overnight. The next day, centrifugation was carried out at 12000 rpm for 2min, and serum was separated. And (3) detecting the antibody titer to be more than 1: 20000 by using an indirect ELISA method, and collecting blood.

Fourthly, the immunization is carried out after 7 days, and after 7 days, the immunized New Zealand white rabbit is fasted for one day, and the whole blood can be collected.

Fifthly, placing the blood into a serum separation bottle, incubating for 2h at 37 ℃, and then staying overnight at 4 ℃. The next day, the separated serum was collected by a sterilized capillary tube, the remaining sample was centrifuged at 3000 rpm for 3min, the separated serum was collected continuously, and sodium azide with a mass percentage concentration of 0.02% was added to the collected serum for storage at-20 ℃.

Coarse purity of polyclonal antibody IgG

50g of DEAE-cellulose (DE32 or DE52) was weighed, placed in a 1,000mL beaker, floated with distilled water to remove fine particles, treated with acid and alkali, and then equilibrated with 0.01 to 0.05mol/L of phosphate buffer at a pH of about 8.0. The water is drained or filtered through a buchner filter (two layers of filter paper placed inside) and the wet cellulose is collected to reduce the ionic strength. After being fully stirred, the mixture is placed at 4 ℃ for adsorption for 1h according to the condition that 1mL of serum is added with 5g of DEAE cellulose. The supernatant can be treated once more as such, i.e. a purer IgG is obtained.

(3) Indirect ELISA method for determining antibody titer

Wrapping a board: with coating buffer (Na)₂CO₃And NaHCO3 buffer) to dilute known antigens toMu.g/ml, 50. mu.l per well of the polystyrene plate, overnight at 4 ℃ the next day, the well solutions were discarded and washed 1 time with 180. mu.l per well of 1 XTSST wash buffer.

Sealing: blocking was performed by adding 60. mu.l of 1% BSA (in TBST) to each well, and incubating at 37 ℃ for 1 hour. The blocking solution was then discarded.

Adding sample: adding a diluted sample to be tested (diluting the sample to be tested according to a certain proportion), and placing 50 mu l of the diluted sample in the sealed reaction hole. Positive control wells (positive serum) and negative control wells (BSA) were also set. Incubate at 37 ℃ for 1 hour, then discard the blocking solution, wash 2 times with 180. mu.l per well of 1 × TBST wash buffer.

Adding enzyme labeled antibody: freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) was added to the wells of the ELISA plate at 50. mu.l/well and incubated at 37 ℃ for 45min, after which the blocking solution was discarded and washed 3 times with 180. mu.l per well of 1 × TBST wash buffer.

Adding a substrate solution for color development: 100. mu.l of a TMB substrate solution prepared temporarily was added to each reaction well, and the mixture was allowed to react at 37 ℃ for 5 min.

Sixthly, terminating the reaction: to each reaction well was added 90. mu.l of 2M sulfuric acid.

Seventhly, reading a plate: and (3) placing the ELISA plate in a preheated ELISA reader (450nm) for reading, storing data and analyzing.

FIG. 4 is NbAPX₃Titer profile of protein polyclonal antibodies.

(4) Western blot detection of polyclonal antibodies

Firstly, carrying out 60V electrophoresis, after a sample enters a separation gel, carrying out 150V electrophoresis until bromophenol blue just comes out, and stopping electrophoresis.

Secondly, rotating the die: A. and (5) after electrophoresis is finished, closing the switch and taking down the rubber plate. Placing the gel plate on the other side of the white porcelain plate, lightly pushing in the gel plate by using the tip of a green scraper, opening the small glass plate, removing concentrated gel, cutting a target gel strip by using the green scraper according to a Marker 27kDa strip, and cutting off the upper left corner of the PVDF membrane; B. the sandwich was placed in an electric rotating cell with the black plate against the black side. Film transferring conditions: the film is rotated for 2h at 200 mA.

Sealing: the transferred nitrocellulose membrane was rinsed in TBST buffer and transferred to 10ml blocking solution (5% skimmed milk powder) and blocked at 37 ℃ for 2 h.

Incubation primary antibody: after blocking was completed, the nitrocellulose membrane was rinsed 3 times for 10min each in TBST buffer, and a certain volume of specific antiserum was added to the TBST buffer and reacted at 37 ℃ for 1 h.

Incubating a second antibody: the nitrocellulose membrane was rinsed 3 times in TBST buffer for 10min each, and then added with AP-A secondary antibody diluted (1:5000) with TBST, and reacted at 37 ℃ for 30-60 min.

Sixthly, color development: the membrane was washed 3 times 10min each with TBST. The nitrocellulose membrane was placed in alkaline phosphatase buffer containing 330. mu.g/ml NBT and 165. mu.g/ml BCIP under dark conditions to develop until the band was clear. And (4) after the PVDF membrane is washed by clear water, transferring the PVDF membrane to a gel imager, photographing and storing.

In order to verify the precision and accuracy of the kit, a field sample is detected by Western-Blot, and the over-expressed NbAPX with the number of 1 is extracted according to the steps₃Total protein of tobacco (1), tobacco mosaic virus infected tobacco numbered 2-5. The results are shown in FIG. 5, NbAPX₃The expression level of the protein is enhanced when the virus is infected. From this, NbAPX is known₃The accumulation of protein is closely related to the infection of virus.

Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the invention. However, it is intended that all such modifications and improvements be made without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Yangzhou university

<120>Raw tobacco NbAPX₃Gene polyclonal antibody and preparation method and application thereof

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atgataacgt atgcagattt gtaccagctt gcaggagttg ttgcagttga agtcactggt 60

ggtccgacca ttgattttgt ccctggtagg aaggattcca gtgtttctcc aaaggaagga 120

cggctgccag atgctaaaca aggtgtgcca catctgaaag atgtatttta taggatgggt 180

ttgtctgaca aagatatagt ggcactatct ggtggtcaca cactgggaag ggcacatcca 240

gatagatcag gctttgatgg tccatggaca aaggagccac tgaaatttga caattcatat 300

tttgtggagc tgcttaaggg ggaaactgag ggcctgctga aacttgctac agacatagct 360

ttattggatg atcctgagtt cagagaatat gttgagctgt atgctaagga tgaagatgcc 420

ttctttaggg attatgccat atcacacaag aaactatctg agctagggtt taccccaagt 480

tctggttcca aggctacact gagggatggc actatattag cacaaagtgc tgtaggagtt 540

gttgtggctg ctgcagtggt ggcactgagt tactggtacg aagtgcggaa aaggatgaag 600

tga 603

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Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Met Ile Thr Tyr Ala Asp Leu Tyr Gln Leu Ala Gly Val Val

35 40 45

Ala Val Glu Val Thr Gly Gly Pro Thr Ile Asp Phe Val Pro Gly Arg

50 55 60

Lys Asp Ser Ser Val Ser Pro Lys Glu Gly Arg Leu Pro Asp Ala Lys

65 70 75 80

Gln Gly Val Pro His Leu Lys Asp Val Phe Tyr Arg Met Gly Leu Ser

85 90 95

Asp Lys Asp Ile Val Ala Leu Ser Gly Gly His Thr Leu Gly Arg Ala

100 105 110

His Pro Asp Arg Ser Gly Phe Asp Gly Pro Trp Thr Lys Glu Pro Leu

115 120 125

Lys Phe Asp Asn Ser Tyr Phe Val Glu Leu Leu Lys Gly Glu Thr Glu

130 135 140

Gly Leu Leu Lys Leu Ala Thr Asp Ile Ala Leu Leu Asp Asp Pro Glu

145 150 155 160

Phe Arg Glu Tyr Val Glu Leu Tyr Ala Lys Asp Glu Asp Ala Phe Phe

165 170 175

Arg Asp Tyr Ala Ile Ser His Lys Lys Leu Ser Glu Leu Gly Phe Thr

180 185 190

Pro Ser Ser Gly Ser Lys Ala Thr Leu Arg Asp Gly Thr Ile Leu Ala

195 200 205

Gln Ser Ala Val Gly Val Val Val Ala Ala Ala Val Val Ala Leu Ser

210 215 220

Tyr Trp Tyr Glu Val Arg Lys Arg Met Lys Leu Glu His His His His

225 230 235 240

His His Asp Pro Ala Ala Asn Lys Ala Arg Lys Glu Ala

245 250

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cgggatccat gataacgtat gcagatttgt accagc 36

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ccgctcgagc ttcatccttt tccgcacttc gtac 34

Claims (10)

1. Raw tobacco NbAPX₃A gene having a nucleotide sequence such as SED ID NO: 1 is shown.

2. Raw tobacco NbAPX₃Recombinant protein, characterized in that its amino acid sequence is as defined in SED ID NO: 2, respectively.

3. A polyclonal antibody, characterized in that it is the Bunsen NbAPX of claim 2₃The recombinant protein is an antigen and is prepared by immunizing animals.

4. The Bunsen NbAPX of claim 2₃A recombinant protein/polyclonal antibody of claim 3 in NbAPX₃Application in detection.

5. The Bunsen NbAPX of claim 2₃A method for producing a recombinant protein, comprising:

a) sequences such as SED ID NO: 1 of the raw tobacco NbAPX₃A gene;

b) constructing a recombinant plasmid: mixing the raw tobacco NbAPX₃The gene is connected to a prokaryotic expression vector pET-28a to obtain a recombinant plasmid pET-28a-NbAPX₃；

Induced expression of recombinant protein: the recombinant plasmid pET-28a-NbAPX₃Transforming into competent cells of Escherichia coli, culturing, inducing, cracking, and purifying to obtain Ningpo NbAPX₃A recombinant protein.

6. The Bunsen NbAPX of claim 5₃The preparation method of the recombinant protein is characterized in that when the PCR method is used for amplification, the total RNA of the Nicotiana benthamiana leaves is used as a template, and primers are used: upstream primer NbAPX₃F is shown as SEQ ID NO.3 and a downstream primer NbAPX₃R is amplified as shown in SEQ ID NO. 4.

7. The Bunsen NbAPX of claim 5₃The preparation method of the recombinant protein is characterized in that the escherichia coli competent cell is escherichia coli BL21 competent cell.

8. The method for producing the polyclonal antibody of claim 3, wherein the Bunsen NbAPX of claim 2 is used₃The recombinant protein is used as antigen, antiserum is prepared by adopting a conventional polyclonal antibody preparation method, and the antiserum is separated and purified to obtain the polyclonal antibody.

9. The method for producing a polyclonal antibody according to claim 8, comprising: the bensheng cigarette NbAPX₃Injecting the recombinant protein as antigen into rabbit, immunizing for four times, collecting rabbit blood, separating serum, and purifying to obtain Bunsen NbAPX₃Protein anti-rabbit polyclonal antibody.

10. The method of claim 9, wherein the bunsen NbAPX is prepared₃Adjusting the concentration of the recombinant protein to 1mg/mL for antigen injection; extracting adjuvant and antigen at a volume ratio of 1: 1; the first-time immunization adopts a complete adjuvant, and the second-time immunization adopts an incomplete adjuvant.

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