CN110596381A

CN110596381A - Method for detecting melon aphid-borne yellowed virus and preparation of special polyclonal antibody thereof

Info

Publication number: CN110596381A
Application number: CN201910653356.8A
Authority: CN
Inventors: 韩成贵; 时兴; 张绍康; 王颖; 张宗英; 李大伟; 于嘉林; 尚巧霞; 王献兵; 张永亮
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2019-12-20

Abstract

The invention discloses a method for detecting melon aphid-borne yellows virus and a special polyclonal antibody thereof. The invention firstly prepares MABYV-MP protein with an amino acid sequence shown as a sequence 4 or a sequence 6 in a sequence table, and then uses the MABYV-MP protein as immunogen to strengthen immunity of New Zealand white rabbits to obtain polyclonal antibodies. By adopting Western blot and taking the polyclonal antibody as a moving protein for resisting and detecting melon aphid-borne yellows virus, the titer of MABYV-MP antiserum can reach 1:256000 under the condition of transient expression, and the detection working concentration is recommended to be 1: 10000-1: 80000, the polyclonal antibody not only has high accuracy, high sensitivity, but also has good specificity. The method can detect whether the sample to be detected contains the melon aphid-borne yellow viruses or not, and has great application value and economic significance; in addition, the method lays a foundation for the research of the function of the MABYV-MP protein.

Description

Method for detecting melon aphid-borne yellowed virus and preparation of special polyclonal antibody thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a serological method for detecting melon aphid-borne yellows virus and movement protein thereof and preparation of a special polyclonal antibody thereof.

Background

Melon aphid-borne yellows virus (MABYV) belongs to the flaviviridae family (L uteoviridae) genus potexvirus (Polerovirus) and is transmitted by aphids in a persistent non-proliferative manner. The MABY V can be singly infected or compositely infected with other viruses of potato leafroll virus, the virus infection is limited to the phloem of a host plant, and infected leaves are yellowed and thickened, so that the yield and the quality of field plants are influenced. In recent years, the incidence area of infection of the cucurbit crops with MABYV is continuously enlarged, and great economic loss is caused.

MABYV is a positive-sense single-stranded RNA (+ ssRNA) virus, with virions in the shape of a regular icosahedral sphere, formed by 180 protein subunits arranged according to T-3, with a diameter of 25-30 nm. The viral genome is about 5.7Kb in length, contains 7 open reading frames in total and encodes 7 proteins. The MP protein is expressed by ORF4 by osmoscanning and plays an important role in the transmission, replication and expression of MABYV.

For the detection of MABYV, RT-PCR technology is mainly utilized, but the serological detection of MABYV is not yet reported formally.

Disclosure of Invention

The invention aims to detect and identify melon aphid yellow-transmitted virus and motor protein thereof.

The invention firstly protects a method for detecting and identifying melon aphid transmitted yellow virus, which comprises the following steps of 2): and adopting an antibody prepared by taking MA BYV-MP protein as immunogen to carry out immunoassay.

In the above method, the MABYV-MP can be a1), a2), a3) or a4) as follows:

a1) the amino acid sequence is protein shown as a sequence 4 in a sequence table;

a2) the amino acid sequence is protein shown as a sequence 6 in a sequence table;

a3) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 4 or the sequence 6 in the sequence table;

a4) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown by a1) or a2) or a 3).

The sequence 4 in the sequence table is composed of 191 amino acid residues. Sequence 6 consists of 199 amino acid residues.

In order to facilitate the purification of the protein in a1) or a2), the amino terminal or the carboxyl terminal of the protein shown in the sequence 4 or the sequence 6 in the sequence table can be connected with the label shown in the table 1 (including the label connecting different repeating units and other labels).

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagII	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein according to a4), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a4) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a4) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 3 or sequence 5 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 at the 5 'end and/or 3' end.

In the above method, the antibody may be b1) or b 2):

b1) polyclonal antibodies obtained by boosting the immunity of the animal with the immunogen;

b2) the immunogen is used for immunizing animals and then is subjected to somatic cell hybridization to obtain the monoclonal antibody.

In the above method, the animal may be any one of rabbit, mouse, goat, sheep or horse.

The animal may be a rabbit. In one embodiment of the invention, the rabbit may be specifically a new zealand white rabbit.

In the method, the detection by using the antibody prepared by using the MABYV-MP protein as the immunogen can be performed by using a W ester blot.

In the above method, before performing the step 2), the step 1) may be performed: and extracting the total protein of the sample to be detected.

The sample to be tested can be a plant or a plant leaf. The plant may specifically be any one of n1) -n 8):

n1) plants that the MABYV can infect; n2) Cucurbitaceae plants which can be infected by MABYV; n3) cucumber; n4) pumpkin; n5) watermelon; n6) melon; n7) tobacco; n8) this smoke.

The invention relates to an antibody for detecting and identifying melon aphid-borne yellows virus, which is prepared by taking MABYV-MP protein as immunogen.

The antibody is b1) or b 2):

The invention also protects any MABYV-MP protein.

The invention also protects the nucleic acid molecule of any MABYV-MP protein.

The nucleic acid molecule encoding any of the above MABYV-MP proteins may be specifically a DNA molecule represented by q1) or q2) or q3) or q4) as follows:

q1) the nucleotide sequence is a DNA molecule shown as a sequence 3 in the sequence table;

q2) the nucleotide sequence is a DNA molecule shown as a sequence 5 in the sequence table;

q3) has 75% or more identity with the nucleotide sequence defined by q1) or q2) and encodes the DNA molecule of the MABYV-MP protein;

q4) under stringent conditions, and encoding the MABYV-MP protein, with the nucleotide sequence defined by q1) or q 2).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The sequence 3 in the sequence table consists of 573 nucleotides and encodes an amino acid sequence shown as a sequence 4 in the sequence table. The sequence 5 consists of 600 nucleotides and encodes an amino acid sequence shown as a sequence 6 in a sequence table.

The nucleotide sequence encoding the MABYV-MP of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the gene of the MABYV-MP protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the MABYV-MP protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in sequence 4 or sequence 6 of the sequence listing of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The invention also provides the application of any one of the antibodies, the MABYV-MP protein or the nucleic acid molecule, which can be S1) or S2):

s1) detecting the melon aphid-borne yellow virus and the movement protein thereof;

s2) preparing a kit for detecting melon aphid-borne yellows virus and motor proteins thereof.

The invention firstly prepares MABYV-MP protein with an amino acid sequence shown as a sequence 4 or a sequence 6 in a sequence table, and then takes the MABYV-MP protein as immunogen to immunize New Zealand white rabbits to obtain polyclonal antiserum (antibody). The Western blot is adopted, and the polyclonal antibody is used as a primary antibody for detecting the melon aphid-borne yellows virus and the motor protein thereof, so that the polyclonal antibody not only has high accuracy and sensitivity, but also has good specificity. The invention can detect whether the sample to be detected contains the melon aphid-transmitted yellow viruses or not, can also be used for researching the function of the movement protein, and has great application value and economic significance. .

Drawings

FIG. 1 shows the expression and purification of fusion proteins;

FIG. 2 shows the polyclonal antibody detection of MABYV-MP; wherein panel A shows Flag antiserum detecting Flag-tagged MABYV-MP; panel B shows MABYV-MP antiserum detecting MABYV-MP;

FIG. 3 is a measurement of the titer of polyclonal antibodies; panel A shows the potency assay for MABYV-MP antiserum; panel B shows the MABYV-MP antiserum optimal range of use assay;

FIG. 4 is a sensitivity analysis of polyclonal antibodies; panel A shows a sensitivity analysis of MABYV-MP antiserum to endogenously transiently expressed proteins in plants; panel B shows the sensitivity analysis of MABYV-MP antiserum to prokaryotically expressed proteins;

FIG. 5 is an analysis of the specificity of the polyclonal antibody MABYV-MP antiserum;

FIG. 6 shows RT-PCR detection of virus and polyclonal antibody MABYV-MP detection, wherein, panel A shows RT-PCR detection of MABYV virus infected leaves; panel B shows MABYV-MP antiserum detecting virus-infected leaves in agreement with panel A; panel C shows polyclonal antibody detection mimicking natural host cucumber infection by MABYV.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, each set up three replicates.

Coli MC1022 was awarded by professor Salah Bouzubaa, University of Stelas, France, Agrobacterium GV3101 was awarded by professor David Baulcombe, University of Cambridge, UK, publicly available from the Chinese University of agriculture (i.e., the applicant), and this biomaterial was used only for repeating the experiments related to the present invention, and was not used for other purposes.

This raw tobacco (Nicotiana benthamiana) was awarded by professor David Baulcombe of John Innes Centre, UK, publicly available from the Chinese university of agriculture (i.e. the Applicant), and this biomaterial was used only for the repetition of the relevant experiments of the present invention and was not used for other purposes.

The cloning vector pMD19-T (simple) is a product of TaKaRa. The Ni affinity column is QIAGEN.

A recombinant plasmid pCaMA36 (university of Chinese agriculture, doctor's academic thesis 2011, Hiroshima) containing the MABYV-MP gene is publicly available from the university of Chinese agriculture.

A binary expression vector pMDC32(Curtis and Grossniklaus,2003) is publicly available from the university of Chinese agriculture for testing the specificity of the antibodies produced.

The vector pDB. His. MBP (circular) is purchased from DNASU plasmid library, and the nucleotide sequence is shown as sequence 1 in the sequence table.

The TBST buffers in the following examples were all buffers containing 0.05% (v/v) Tween-20, 150mM NaCl, 20mM Tris-HCl (pH 7.5).

Example 1 preparation of polyclonal antibodies

Construction of recombinant plasmid pDB.His.MBP-MABYV-MP

1. pCaMA36 containing MABYV full-length cDNA clone was used as a template, and 5' -TTTACTTCCAGGGC was usedCATATGATGG CATGGGAAGGAGGA-3 '(recognition sites for restriction enzyme Nde I are underlined) and 5' -TGGTGGTGGTGGTGCTCGAGCTACCTATTTCGGGTTCTG-3' (recognition of restriction enzyme Xho I is underlined)Locus) is used as a primer to carry out PCR amplification, and a PCR amplification product of about 615bp is obtained.

2. The vector pDB.His.MBP was digested with restriction enzymes Nde I and Xho I, and a vector backbone of about 6.5kb was recovered.

3. And (3) carrying out homologous recombination on the PCR amplified fragment obtained in the step (1) and the vector skeleton obtained in the step (2) to obtain a recombinant plasmid pDB.

Recombinant plasmid pDB. His. MBP-MABYV-MP were sequenced. According to the sequencing results, the structure of the recombinant plasmid pDB. His. MBP-MABYV-MP is described as follows: a small DNA fragment between restriction enzyme Nde I and Xho I recognition sequences of vector pdb.his. mbp was replaced with a double-stranded DNA molecule shown in sequence 3 of the sequence listing. The nucleotide sequence of the MABYV-MP gene (shown as the sequence 3 in the sequence table) is formed by the sequence 3 in the sequence table and ATG in a recognition sequence (CATATG) of restriction endonuclease Nde I. The MABYV-MP protein shown in a sequence 4 in a sequence table of a MABYV-MP gene coding sequence table.

In the recombinant plasmid pDB.His.MBP-MABYV-MP, a DNA molecule shown in a sequence 3 of a sequence table is fused with a coding sequence of an H is-tag label (consisting of 6 histidine residues) on a vector skeleton to form a fusion gene shown in a sequence 5 of the sequence table, and a fusion protein with the His-tag label shown in a sequence 6 of the sequence table is expressed.

Second, expression and purification of fusion protein

1. The recombinant plasmid pDB.His.MBP-MABYV-MP is introduced into Escherichia coli BL21(DE3) to obtain a recombinant bacterium, and the recombinant bacterium is named as BL21(DE3) -MABYV-MP.

2. After the step 1 is completed, BL21(DE3) -MABYV-MP recombinant bacteria are taken and inoculated into 50mL LB liquid medium (containing 50 ug/mL kanamycin), and shaking culture is carried out at 37 ℃ and 220rpm for 4h to obtain a culture bacterial liquid.

3. After the step 2 is completed, the culture broth is taken and inoculated into LB liquid culture medium (containing 50 mu g/mL kanamycin) according to the volume ratio of 1:200, and the culture broth is subjected to shaking culture at 37 ℃ and 220rpm until OD is reached_600nmThe value is 0.6 to 0.8. Adding IPTG (Sigma-Aldrich, St.Louis, MO, USA) with final concentration of 0.1mM, inducing culture at 18 deg.C and 180rpm under shaking for 16h, centrifuging at 4 deg.C and 5000 rpm for 6min, and collecting thallus precipitate.

4. After completion of step 3, 40ml of high salt buffer (20mM Tris-HCl,500mM NaCl, pH 7.0) was added to suspend the pellet, the pellet suspension was transferred to an empty beaker, high salt buffer was further added to a total volume of 70ml, and after adding 70. mu.l (1:1000) of PMSF, the pellet was sonicated 3 times for 2min each (ultrasonic power 50%, cycle: disruption for 2s, stop for 2 s). Centrifugation was carried out at 16000rpm for 40min at 4 ℃ and the supernatant was collected. The supernatant was applied to a Ni affinity column (Qiagen, Hilde n, Germany) and the protein was eluted with imidazole eluents of different concentrations (20mM, 40mM, 60mM, 100mM, 200mM, 500mM) and the eluates were collected. Finally, the collected eluate was subjected to SDS-PAGE as shown in FIG. 1. The results indicated that the fusion protein present in the supernatant was concentrated in a suitable eluent (pH 7.0, 20mM Tris-HCl buffer containing 150mM NaCl and 200mM imidazole) to obtain pDB. His. MBP-MABYV-MP fusion protein.

5. After the step 4 is completed, the eluent with fusion protein concentration suitable for the above-mentioned concentration is taken for concentration and purification again, and the concentration of the fusion protein in the final concentrated and purified protein solution is about 1.5mg/mL by detection.

Preparation of polyclonal antibody

1. The concentrated protein solution is taken and diluted to 200-.

2. 1 part by volume of the fusion protein solution (containing 400. mu.g of the fusion protein) and 1 part by volume of Freund's complete adjuvant were mixed and emulsified to obtain a mixed solution A. Mixing 1 volume part of fusion protein solution (containing 200 mu g of fusion protein) and 1 volume part of Freund's incomplete adjuvant, and emulsifying to obtain mixed solution B.

3. 1 new zealand white rabbit weighing about 2kg is taken and injected subcutaneously (8-10 points) on the back with the mixed solution A.

4. At 14d, completion of step 3, cocktail b was injected subcutaneously (8-10 points) via the back.

5. On completion of 24d of step 3, cocktail b was injected subcutaneously (8-10 points) via the back.

6. At 34d, completion of step 3, cocktail b was injected subcutaneously (8-10 points) via the back.

7. Completing the 35d of the step 3, collecting blood from carotid artery of New Zealand white rabbit, and separating serum; the serum is the prepared polyclonal antibody. The method specifically comprises the following steps: anaesthetics are used for anaesthetizing New Zealand white rabbits (pentobarbital sodium, 30mg/kg, and intravenous, abdominal and intramuscular injection can be performed), the new Zealand white rabbits are fixed by a fixing frame, the outer skin of the neck is cut off by surgical instruments, the carotid artery is found below the side face of the trachea, and the artery is clamped by a hemostatic forceps and cut off for bloodletting and collection; the polyclonal antibody was obtained by centrifugation twice at 5,000rpm for 10min and serum collection.

The above steps are operated by Beijing Huada protein research and development center, Inc.

Example 2 polyclonal antibody detection of MABYV-MP

And (3) carrying out SDS-PAGE and Western blot detection on the total protein of the raw tobacco leaves and the raw tobacco leaves transiently expressing the MABYV-MP protein, and taking the polyclonal antibody prepared in the third step in the example 1 as a primary antibody to detect the MABYV. The method comprises the following specific steps:

1. extracting sample protein of the leaves of the Bunsen tobacco, comprising: the total protein of the raw tobacco leaf, the total protein of the raw tobacco leaf containing an empty vector pMDC32, and the total protein of the raw tobacco leaf transiently expressing MABYV-MP and MABYV-3 Flag-MP. The preparation method of the Bunsen tobacco leaf for transiently expressing MABYV-MP and MABYV-3Flag-MP comprises the following steps:

replacing a DNA fragment between a restriction enzyme SpeI and a SacI recognition sequence of a vector pMDC32 with a DNA sequence (a sequence shown in a sequence 3) of MABYV-MP and keeping other sequences of pMDC32 unchanged to obtain a recombinant expression vector pMDC32-MA BYV-MP, and infiltrating and inoculating a native tobacco leaf with the recombinant vector pMDC32-MABYV-MP through agrobacterium to obtain a native tobacco leaf for transiently expressing MABY V-MP;

the recombinant expression vector pMDC32-MABYV-3Flag-MP is obtained by replacing the DNA fragment between the restriction enzyme SpeI and SacI recognition sequences of the vector pMDC32 with the DNA sequence of 3Flag-MABY V-MP (the coding sequence of 3 FLAG-tagged proteins in Table 1 are connected to the amino terminal of the protein of sequence 4) and keeping the other sequences of pMDC32 unchanged, and the recombinant expression vector pMDC32-MABYV-3Flag-MP is inoculated to the nicotiana benthamiana leaves through agroinfiltration to obtain the nicotiana benthamiana leaves for transiently expressing MABYV-3Fla g-MP.

2. After completion of step 1, the total protein of each leaf was subjected to SDS-PAGE, and then transferred to a nitrocellulose membrane by electrotransfer (200mA, 90 min).

3. After completion of step 2, the nitrocellulose membrane was placed in TBST buffer containing 5% (w/v) skim milk powder and blocked at 37 ℃ for 1 h.

4. After completion of step 3, a dilution (1:500 dilution) of the polyclonal antibody prepared in three steps in example 1 was added, incubated at 37 ℃ for 1h, and the membrane was washed 3 times with TBST buffer for 10min each.

5. After completing step 4, the nitrocellulose membrane was placed in AP-labeled goat anti-rabbit IgG secondary antibody working solution (1: 20000 dilution), incubated at 37 ℃ for 1h, and washed 3 times with TBST buffer, 10min each time. As a control, the above protein was treated in the same manner with a commercial flag antibody (Sigama Corporation) diluted solution.

6. After completion of step 5, the nitrocellulose membrane was developed in a developing buffer containing NBT at 330. mu.g/mL and BCIP at 165. mu.g/mL with exclusion of light until the band became clear, and the reaction was terminated with distilled water.

As shown in FIG. 2, the total proteins of the nicotiana benthamiana leaves, the nicotiana benthamiana leaves containing empty vector pMDC32, and the total proteins of the nicotiana benthamiana leaves transiently expressing MABYV-MP and 3Flag-MABYV-MP in FIG. 2 are respectively expressed by health, pMDC32, pMDC 32-MP and pMDC32-3FLAG-MP (the same applies hereinafter), and the comparative analysis in FIG. 2 confirms that the polyclonal antibody prepared in step three of example 1 can successfully detect MABYV-MP.

Example 3 measurement of the potency of polyclonal antibody

1. Total proteins were extracted from a nicotiana benthamiana leaf transiently expressing MABYV-MP protein and a nicotiana benthamiana leaf containing empty vector pMDC32 (as a negative control) (prepared in the same manner as in example 2).

2. The MABYV-MP polyclonal antibody prepared in the third step in the example 1 is added into TBST buffer solution for dilution to obtain diluted solution. The dilution factor of the dilution was 1000, 2000, 4000, 8000, 16000, 32000, 64000, 128000, 256000 and 512000, respectively.

3. And (3) performing SDS-PAGE and Western blot on the total protein extracted in the step (1), and using the diluent obtained in the step (2) as a primary antibody to detect MABYV.

The Western blot results are shown in FIG. 3A.

4. Total proteins were extracted from tobacco leaves expressing the MABYV-MP protein transiently and from tobacco leaves injected with pMDC32 empty vector (as a negative control).

5. The MABYV-MP polyclonal antibody prepared in the third step in the example 1 is added into TBST buffer solution for dilution to obtain diluted solution. The dilution times of the dilutions were 10000, 20000, 40000, 60000 and 80000, respectively.

6. And (4) performing SDS-PAGE and Western blot on the total protein extracted in the step (4), and using the diluent obtained in the step (5) as a primary antibody to detect MABYV.

The Western blot results are shown in FIG. 3B.

The result of the combination of FIG. 3 shows that when the polyclonal antibody titer is detected by using the Bunsen tobacco leaf which transiently expresses MABYV, a weak protein band can be detected when the dilution factor of the polyclonal antibody reaches 256000, and a target band cannot be detected when the dilution factor of the polyclonal antibody reaches 512000. Thus, the potency of the MABYV-MP polyclonal antibody prepared in step four of example 1 was about 1: 256000.

the optimal using range titer of the polyclonal antibody is detected by using the Bunsen tobacco leaves for transiently expressing MABYV, and the optimal using range titer is 1: the color development effect is obvious within the range of 10000-1: 80000. When the dilution multiple reaches 80000, the detection of the M ABYV infected Bunsen tobacco is ideal from the aspects of color development effect and economy.

Example 4 sensitivity analysis of polyclonal antibodies

1. 0.1g of a primary tobacco leaf blade transiently expressing MABYV-MP protein was taken, sufficiently ground, and added with 300. mu.L of 2XSDS protein loading buffer to obtain a protein sample 1(1: 4).

2x SDS protein loading buffer: a buffer containing 4% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue, 100mM Tris-HCl (pH 6.8).

2. After step 1 is completed, the protein samples are diluted by 1 and 2 times to obtain protein samples with the dilution times of 8, 16, 32, 64, 128, 256, 512 and 1024 respectively.

3. Another protein sample 1 was taken, and 1 part by volume of the protein sample 1 was mixed with 9 parts by volume of 2XSDS protein loading buffer to obtain a protein sample 2 (i.e., dilution factor 10, 1:40)

4. And (3) after the step 3 is finished, diluting the protein sample by 2 times to obtain protein samples with the dilution times of 80, 160, 320 and 640 respectively.

5. The MABYV-MP polyclonal antibody prepared in the third step in the example 1 is added with TBST buffer solution for dilution to obtain a diluent. The dilution ratio of the diluent is 1000, 10000 and 20000.

6. And (4) performing SDS-PAGE and Western blot on the protein samples obtained in the step (2) and the step (4), and using the diluent obtained in the step (5) as a primary antibody to detect MABYV.

The Western blot results of MABYV-MP are shown in FIG. 4A.

7. And (3) taking a proper amount of the concentrated and purified protein solution with the concentration of 1.5mg/mL prepared in the second step of example 1, and adding a 2XSDS protein extracting solution for dilution to obtain a diluent containing 160ng, 80ng, 40ng, 20ng, 10ng, 5ng, 2.5ng and 1.3ng of prokaryotic expression protein.

8. The polyclonal antibody prepared in the third step of example 1 was diluted with TBST buffer to obtain a diluted solution. The dilution factor of the dilution was 1000 and 2000, respectively.

9. And (3) performing SDS-PAGE and Western blot on the concentrated protein solution obtained in the step (7), and taking the diluent obtained in the step (8) as a primary antibody to detect the prokaryotic expression protein.

The Western blot results are shown in FIG. 4B.

The result shows that 0.1g of the crude tobacco leaf transiently expressing MABYV-MP reacts positively after being diluted by 64 times under the condition that the total protein concentration is 1:1000, and the reaction is positive after being diluted by 32 times under the condition that the total protein concentration is 1:10000, and the reaction is carried out under the conditions that the total protein concentration is 1: protein dilution 16 times under 20000 conditions is positive.

Approximately 2.5ng of the prokaryotic expression protein can be detected by a polyclonal antibody dilution with the dilution factor of 1000; approximately 5ng of the prokaryotically expressed protein could be detected in a dilution of the polyclonal antibody at a dilution of 2000.

Example 5 specificity analysis of polyclonal antibodies

The leaf to be tested is a native tobacco leaf which instantaneously expresses CABYV-MP, MABYV-MP, SABYV-3Flag-MP, BrYV-3Flag-MP, PLRV-3F lag-MP, ScYLV-3Flag-MP and pMDC32 empty vectors.

1. And extracting the total protein of the leaf to be detected.

2. The MABYV polyclonal antibody prepared in step three of example 1 was diluted with TBST buffer to obtain a diluted solution. The dilution ratio of the diluent is 1000, 10000 and 20000.

3. And (3) performing SDS-PAGE and Western blot on the total protein obtained in the step (1), and using the diluent obtained in the step (2) as a primary antibody to detect MABYV. The Western blot results are shown in FIG. 5, and only total proteins of the tobacco leaves expressing MABYV transiently reacted positively under the corresponding serum conditions. The results show that the polyclonal antibody prepared in the third step in example 1 has higher specificity to the MABYV, and does not have serological cross reaction with CABYV and SABYV which can infect melons in a mixed manner. Wherein the Bunsen tobacco leaf transiently expressing SABYV-3Flag-MP, BrYV-3Flag-MP, PLRV-3Flag-MP and ScYLV-3Flag-MP is prepared according to the following method:

SABYV 3Flag-MP is a recombinant expression vector obtained by replacing the DNA fragment between the recognition sequences of the restriction enzymes SpeI and SacI of the vector pMDC32 with the DNA sequence encoding SABYV-3Flag-MP (see the sequence disclosed in Shang Qiao-xia, et al. VirusResearch, 2009,145(2), 341-346) and keeping the other sequences of pMDC32 unchanged. Inoculating the MP transient expression vector SABYV-3Flag-MP with a Flag tag into the natural tobacco leaves through agroinfiltration to obtain the natural tobacco leaves for transient expression of SABYV 3 Flag-MP.

BrYV-3Flag-MP is a recombinant expression vector obtained by replacing the DNA fragment between the recognition sequences of the restriction enzymes SpeI and SacI of the vector pMDC32 with the DNA sequence encoding BrYV-3Flag-MP (see the sequences in Xiaong Hai-Ying et al, Archives of virology, 2011,156(12), 2251-2255) and leaving the other sequences of pMDC32 unchanged. And inoculating the MP transient expression vector BrYV-3Flag-MP with Flag label to the natural tobacco leaf through agroinfiltration to obtain the natural tobacco leaf with transient expression BrYV 3 Flag-MP.

PLRV-3Flag-MP is a recombinant expression vector obtained by replacing the DNA fragment between the recognition sequences of the restriction enzymes SpeI and SacI of the vector pMDC32 with the DNA sequence encoding PLRV 3Flag-MP (see the sequence Fang Yang et al, Phytopathology research, 2019,1:5) while keeping the other sequences of pMDC32 unchanged. Inoculating the MP transient expression vector PLRV-3Flag-MP with Flag label to the natural tobacco leaf through agroinfiltration to obtain the natural tobacco leaf with transient expression PLRV-3 Flag-MP.

ScYLV-3Flag-MP is a recombinant expression vector obtained by replacing the DNA fragment between the recognition sequences of the restriction enzymes SpeI and SacI of the vector pMDC32 with a DNA sequence encoding ScYLV-3Flag-MP (the amino acid sequence of ScYLV-3Flag-MP is shown in SEQ ID NO: 7) while keeping the other sequences of pMDC32 unchanged. Inoculating the MP transient expression vector ScYLV-3Flag-MP with Flag label to the native tobacco leaf through agroinfiltration to obtain the native tobacco leaf transiently expressing ScYLV-3 Flag-MP.

Example 6 use of polyclonal antibodies in Virus detection

The leaf to be tested is a Bunsen tobacco leaf injected with an empty vector pMDC32, and the Bunsen tobacco leaf transiently expressing MABYV-MP and infected with MABYV.

First, RT-PCR detects the infection MABYV's native tobacco leaf

1. Extracting total RNA of the leaves of the MABYV-infected native tobacco by an LiCl precipitation method:

(1) 0.1g of fresh plant material was taken in a 2.0mL centrifuge tube with 0.5mm steel ball, frozen with liquid nitrogen and ground into powder.

(2) Add 600. mu.l of 25:24:1 water-saturated phenol: chloroform: and (3) vigorously shaking and uniformly mixing isoamyl alcohol and the RNA extraction buffer solution with the same volume on an oscillator, standing for 5min at room temperature, and centrifuging for 20min at 12000 rpm.

(3) Mu.l of the supernatant was transferred to a 1.5mL eppendorf centrifuge tube, an equal volume of 4M LiCl solution was added and allowed to settle overnight at 4 ℃ or for 4h at-20 ℃.

(4) Centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant was discarded.

(5) The precipitate was washed once with 75% ethanol solution and once with 100% absolute ethanol, 12000rpm for each wash, 4 ℃ and centrifuged for 5 min.

(6) And discarding the supernatant, drying the precipitate on an ultraclean workbench, and adding 30-60 mu l of DEPC water to dissolve RNA.

Specific primer pairs MAMP-F: 5'-ATGGCATGGGAAGGAGGAGACGGA-3' and MAMP-R: 5'-CTACCTATT TCGGGTTCTGGACCTGGCACTTGA-3' were designed, and RT reaction was performed using M-MLV reverse transcription e (Promega Corporation) using the extracted RNA as a template. The reaction system refers to the instruction book, wherein about 2 mu g of control RNA in a 30 mu L system, and 1 mu L of MA-R (10 mu m/L); the procedure is 75 ℃ denaturation for 10min, 37 ℃ reverse transcription for 2h to synthesize the first strand of cDNA, and then PCR amplification is carried out by taking the synthesized first strand of cDNA as a template. Use of PCRMax DNA Polymerase (T akara Corporation), PCR system reference manual, wherein about 6ng of template is controlled in 50 μ L system, and 1 μ L is added for each of MA-F (10 μm/L) and MA-R (10 μm/L); procedure 98 ° pre-denaturation 2min, 35 cycles: denaturation at 98 ℃ for 10S, annealing at 55 ℃ for 5S, and extension at 72 ℃ for 10S; extension at 72 ℃ for 5min and termination at 25 ℃ for 1 min. The amplification product was electrophoresed through a 1% agarose gel (120V 30min) and the result is shown as A in FIG. 6. Demonstrating that the CABYV full-length cDNA clone was replicated and transcribed in the leaf of Nicotiana benthamiana.

The results show that MABYV infected nicotiana benthamiana leaves can generate target bands consistent with pMDC32 (positive control) through RT-PCR reaction, and the expected size is about 573 bp. The MABYV full-length cDNA clone is replicated and transcribed in the leaf of the nicotiana benthamiana.

Second, polyclonal antibody detection of MABYV infected leaves of the Bunsen tobaccos

1. And extracting the total protein of the leaf to be detected.

2. The MABYV polyclonal antibody prepared in step three of example 1 was diluted with TBST buffer to obtain a diluted solution. The dilution factor of the dilution was 1000.

3. And (3) performing SDS-PAGE and Western blot on the total protein obtained in the step (1), and using the diluent obtained in the step (2) as a primary antibody to detect MABYV. The Western blot results are shown in FIG. 6B, and compared with RT-PCR detection results. The result shows that the MABYV-MP antiserum can detect the MABYV on the bunsen and is consistent with the detection result of RT-PCR. The MABYV-MP antiserum is suitable for detecting MABYV and has high application value.

Example 7 polyclonal antibody assay to mimic natural host cucumber infection with MABYV

The leaves to be tested are respectively a Bunsen tobacco leaf injected with an empty vector pMDC32, a healthy cucumber leaf and a Bunsen tobacco leaf instantly expressing MABYV-M P.

1. And extracting the total protein of the leaf to be detected.

2. The MABYV simulated assay was performed on healthy, natural host cucumber. According to the following steps of 1:4, extracting MABYV-MP protein transiently expressed in the leaf of the Nicotiana benthamiana, and mixing the protein with the protein according to the ratio of 1:4 and 1: the total protein of healthy cucumber leaves extracted at a ratio of 8 was mixed and diluted to 6 gradients: 1:8,1: 16,1: 32,1: 64,1: 128,1: 256. the method takes the native tobacco leaf protein extracted from the injection empty vector P MDC32 and healthy cucumber leaf protein as negative controls, and the extracted native tobacco leaf protein transiently expressing MABYV-MP as a positive control.

3. The MABYV polyclonal antibody prepared in step three of example 1 was diluted with TBST buffer to obtain a diluted solution. The dilution factor of the dilution was 1000.

4. And (3) performing SDS-PAGE and Western blot on the total protein obtained in the step (1), and using the diluent obtained in the step (2) as a primary antibody to detect MABYV. The Western blot results are shown in FIG. 6C, and compared with RT-PCR detection results.

The results show that in the background of the above two cucumber leaf total protein dilution concentrations, the concentration of the total protein of the cucumber leaf is 1: a 1000-ratio dilution of MABYV-MP antiserum detected a dilution of 1: and the MABYV-MP is mixed and diluted in a ratio of 64, the total protein of healthy cucumber leaves does not have serological reaction with the antiserum, and the specific reaction of the antiserum and the MABYV-MP protein is not influenced, so that a theoretical basis is provided for the field detection application of the antiserum and the preparation of a kit for detecting the MABYV and the movement protein thereof.

Sequence listing

<110> university of agriculture in China

<120> method for detecting melon aphid yellow-transmitted virus and preparation of special polyclonal antibody thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6494

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

ggtaccaaaa ctgaagaagg taaactggta atctggatta acggcgataa aggctataac 5160

ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 5220

catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 5280

attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 5340

atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 5400

tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 5460

aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 5520

ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 5580

ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 5640

aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 5700

attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 5760

aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 5820

aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 5880

gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag 5940

ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 6000

ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 6060

gccaccatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 6120

ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 6180

gaagccctga aagacgcgca gactggtacc gattacgata tcccaacgac cgaaaacctt 6240

tacttccagg gccatatggc tagcatgact ggtggacagc aaatgggtcg cggatccgaa 6300

ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga 6360

tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 6420

actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 6480

aactatatcc ggat 6494

<210> 2

<211> 570

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcatgggaag gaggagacgg aaccgtcgac gcacttcaaa gagtaaccgc gtggttgtgg 60

tccaaaccac tggccaacca caacgcggaa gacgacgacg acgaaatcca agacgttctc 120

ctcgaggagg cagagctgga ggacgcccag gtgaaacatt tgtattcagc aaagacaatc 180

tcacgggcag ttcctccgga gcaatcactt tcgggccgtc tctatcagag agcccagcat 240

tcagctctgg aatactcaag gcctaccatg agtataaaat ctcaatggtc aagttggagt 300

tcatctccga ggcctcttcc acctcctcag gttccatctc ttatgagttg gacccccact 360

gcaagcttaa cgccctccaa tccacggtta ataaattcgg aatcacgaag agtggatcta 420

gaacatggag cgcgaagctc atcaacgggc tggaatggca cgacgccacg gaagatcaat 480

tccgcatcct atacaaagga aacgggagct cttcaacggc gggatcgttc aggatcacca 540

tcaagtgcca ggtccagaac ccgaaatagg 570

<210> 3

<211> 573

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcatggg aaggaggaga cggaaccgtc gacgcacttc aaagagtaac cgcgtggttg 60

tggtccaaac cactggccaa ccacaacgcg gaagacgacg acgacgaaat ccaagacgtt 120

ctcctcgagg aggcagagct ggaggacgcc caggtgaaac atttgtattc agcaaagaca 180

atctcacggg cagttcctcc ggagcaatca ctttcgggcc gtctctatca gagagcccag 240

cattcagctc tggaatactc aaggcctacc atgagtataa aatctcaatg gtcaagttgg 300

agttcatctc cgaggcctct tccacctcct caggttccat ctcttatgag ttggaccccc 360

actgcaagct taacgccctc caatccacgg ttaataaatt cggaatcacg aagagtggat 420

ctagaacatg gagcgcgaag ctcatcaacg ggctggaatg gcacgacgcc acggaagatc 480

aattccgcat cctatacaaa ggaaacggga gctcttcaac ggcgggatcg ttcaggatca 540

ccatcaagtg ccaggtccag aacccgaaat agg 573

<210> 4

<211> 191

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Trp Glu Gly Gly Asp Gly Thr Val Asp Ala Leu Gln Arg Val

1 5 10 15

Thr Ala Trp Leu Trp Ser Lys Pro Leu Ala Asn His Asn Ala Glu Asp

20 25 30

Asp Asp Asp Glu Ile Gln Asp Val Leu Leu Glu Glu Ala Glu Leu Glu

35 40 45

Asp Ala Gln Val Lys His Leu Tyr Ser Ala Lys Thr Ile Ser Arg Ala

50 55 60

Val Pro Pro Glu Gln Ser Leu Ser Gly Arg Leu Tyr Gln Arg Ala Gln

65 70 75 80

His Ser Ala Leu Glu Tyr Ser Arg Pro Thr Met Ser Ile Lys Ser Gln

85 90 95

Trp Ser Ser Trp Ser Ser Ser Pro Arg Pro Leu Pro Pro Pro Gln Val

100 105 110

Pro Ser Leu Met Ser Trp Thr Pro Thr Ala Ser Leu Thr Pro Ser Asn

115 120 125

Pro Arg Leu Ile Asn Ser Glu Ser Arg Arg Val Asp Leu Glu His Gly

130 135 140

Ala Arg Ser Ser Ser Thr Gly Trp Asn Gly Thr Thr Pro Arg Lys Ile

145 150 155 160

Asn Ser Ala Ser Tyr Thr Lys Glu Thr Gly Ala Leu Gln Arg Arg Asp

165 170 175

Arg Ser Gly Ser Pro Ser Ser Ala Arg Ser Arg Thr Arg Asn Arg

180 185 190

<210> 5

<211> 600

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggcatggg aaggaggaga cggaaccgtc gacgcacttc aaagagtaac cgcgtggttg 60

tggtccaaac cactggccaa ccacaacgcg gaagacgacg acgacgaaat ccaagacgtt 120

ctcctcgagg aggcagagct ggaggacgcc caggtgaaac atttgtattc agcaaagaca 180

atctcacggg cagttcctcc ggagcaatca ctttcgggcc gtctctatca gagagcccag 240

cattcagctc tggaatactc aaggcctacc atgagtataa aatctcaatg gtcaagttgg 300

agttcatctc cgaggcctct tccacctcct caggttccat ctcttatgag ttggaccccc 360

actgcaagct taacgccctc caatccacgg ttaataaatt cggaatcacg aagagtggat 420

ctagaacatg gagcgcgaag ctcatcaacg ggctggaatg gcacgacgcc acggaagatc 480

aattccgcat cctatacaaa ggaaacggga gctcttcaac ggcgggatcg ttcaggatca 540

ccatcaagtg ccaggtccag aacccgaaat aggctcgagc accaccacca ccaccactag 600

<210> 6

<211> 199

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Ala Trp Glu Gly Gly Asp Gly Thr Val Asp Ala Leu Gln Arg Val

1 5 10 15

Thr Ala Trp Leu Trp Ser Lys Pro Leu Ala Asn His Asn Ala Glu Asp

20 25 30

Asp Asp Asp Glu Ile Gln Asp Val Leu Leu Glu Glu Ala Glu Leu Glu

35 40 45

Asp Ala Gln Val Lys His Leu Tyr Ser Ala Lys Thr Ile Ser Arg Ala

50 55 60

Val Pro Pro Glu Gln Ser Leu Ser Gly Arg Leu Tyr Gln Arg Ala Gln

65 70 75 80

His Ser Ala Leu Glu Tyr Ser Arg Pro Thr Met Ser Ile Lys Ser Gln

85 90 95

Trp Ser Ser Trp Ser Ser Ser Pro Arg Pro Leu Pro Pro Pro Gln Val

100 105 110

Pro Ser Leu Met Ser Trp Thr Pro Thr Ala Ser Leu Thr Pro Ser Asn

115 120 125

Pro Arg Leu Ile Asn Ser Glu Ser Arg Arg Val Asp Leu Glu His Gly

130 135 140

Ala Arg Ser Ser Ser Thr Gly Trp Asn Gly Thr Thr Pro Arg Lys Ile

145 150 155 160

Asn Ser Ala Ser Tyr Thr Lys Glu Thr Gly Ala Leu Gln Arg Arg Asp

165 170 175

Arg Ser Gly Ser Pro Ser Ser Ala Arg Ser Arg Thr Arg Asn Arg Leu

180 185 190

Glu His His His His His His

195

<210> 7

<211> 540

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggactaca aagatgacga taaagactac aaagacgatg acgataaaga ctacaaagac 60

gatgacgata aaggcggagg tactagtatg tcagaagacg cgctaaccgt cgtagacaga 120

ctcggccagt ggtcgtggtc cgggctcccc caggacctag acgagtacga cgacgtagag 180

cacgtgttgg aggaaacgct gtgcgaggac cgggaggaag aagcaaccgg gatgttctca 240

ctttcacggt tgacgatctc aaagccaact caaccgggat cctcaaattc ggaccgaacc 300

tatctcagta cgcagcgttc aacaatggct tactcaaagc ctaccatgag tataaaatca 360

caagtctcac tattcagtat aactcatgct cctccgacgc aactccaggt gcaatcgcac 420

ttgaagtgga tacatcctgc tcccaaacaa caacaggctc caagattact agcttccccg 480

tcaagaggaa cgccaagaaa gtcttcccgg cccccttcat cagggggaaa gactttatag 540

Claims

1. A method for detecting and identifying melon aphid-borne yellows virus is characterized in that detection is carried out by using an antibody prepared by using MABBV-MP protein as an immunogen, wherein the MABBV-MP protein is a1) or a2) or a3) or a 4):

a3) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 4 in the sequence table;

2. The method of claim 1, wherein: the antibody is b1) or b 2):

3. The method of claim 2, wherein: the animal is any one of rabbit, mouse, goat, sheep or horse.

4. The method of claim 3, wherein: the animal is a rabbit.

5. The method of any of claims 1 to 4, wherein: the detection is carried out by Western blot by adopting an antibody prepared by taking MABYV-MP protein as immunogen.

6. An antibody for detecting and identifying melon aphid yellowed virus, which is prepared by taking MABYV-MP protein as immunogen, wherein the MABYV-MP protein is a1) or a2) or a3) or a 4):

7. The antibody of claim 6, wherein the antibody is b1) or b 2):

8. The MABYV-MP protein according to any of claims 1 to 4.

9. A nucleic acid molecule encoding the MABYV-MP protein of claim 8.

10. Use of the antibody of claim 7, the MABYV-MP protein of claim 8 or the nucleic acid molecule of claim 9, S1) or S2):

s1) detecting and identifying the melon aphid-borne yellowed virus and the motor protein thereof;

s2) preparing a kit for detecting and identifying the melon aphid yellowed virus and the motor protein thereof.