CN107446041B

CN107446041B - Antiserum for resisting apple necrosis mosaic virus and preparation method thereof

Info

Publication number: CN107446041B
Application number: CN201710790136.0A
Authority: CN
Inventors: 李世访; 邢飞; 王红清
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2017-09-05
Filing date: 2017-09-05
Publication date: 2021-04-06
Anticipated expiration: 2037-09-05
Also published as: CN107446041A

Abstract

The invention discloses antiserum for resisting apple necrosis mosaic virus and a preparation method thereof. The method for preparing the antiserum for resisting the apple necrosis mosaic virus comprises the following steps: the coat protein of apple necrosis mosaic virus is used as immunogen to immunize animals. The invention utilizes molecular biology means to efficiently express coat protein gene of apple necrosis mosaic virus in escherichia coli, purifies target protein, and prepares and obtains specific polyclonal antiserum by immunizing New Zealand rabbit with the target protein.

Description

Antiserum for resisting apple necrosis mosaic virus and preparation method thereof

Technical Field

The invention belongs to the field of plant virology and immunology, and relates to antiserum for resisting apple necrosis mosaic virus and a preparation method thereof.

Background

The apple is one of important economic crops in China, FAO data show that in 2014, the total cultivation area of the apple in China is about 227 ten thousand hectares, the total yield is about 4100 ten thousand tons, and the apple accounts for about 50% of the apples in the world and is the first worldwide. The apple cultivation in China is mainly distributed in provinces such as Shaanxi, Shandong, Hebei, Henan, Gansu, Shanxi, Liaoning and Xinjiang, becomes an important way for driving regional development and gradually becomes an important component part for industrial structure adjustment and economic development. However, the incidence rate of apple virus diseases in apple producing areas in China is high and can reach 40-100%, and once infected, the fruit trees are infected with the virus for the whole life. The apple mosaic disease is one of the most common viral diseases in apple production, and seriously restricts the healthy development of the apple industry in China.

A recent study showed that a new virus, Apple necrotic mosaic virus (ApNMV), was detected in Apple mosaic samples, and that ApNMV belongs to the third Subgroup (Subgroup 3) of the equiaxed unstable ringspot virus (Ilarvirus) of the brommosaic virus family (Bromoviridae), and has a genomic structure of a triad including RNA1, RNA2, and RNA3, wherein the 5' end of RNA3 encodes a CP consisting of 219aa, and the coding region is 660nt in length. In addition, the detection rate of the new virus ApNMV in apple mosaic samples in China is as high as 82.6%, and the new virus ApNMV has high correlation with the occurrence of mosaic symptoms, is possibly an important pathogen causing apple mosaic diseases in China, and is a serious threat to apple cultivation in China.

Pathogen detection is the premise and basis for the prevention and control of viral diseases. At present, the detection methods of fruit tree viruses mainly comprise biological, serological, electron microscope observation and molecular biological methods. The biological method has the advantages of long detection period, poor specificity, large workload and higher requirements on field practical experience of detection personnel; although plant viruses can be intuitively detected through electron microscope observation according to the characteristics of the viruses and inclusion bodies and the pathological change condition of host cells, the detection method is limited by experimental conditions and detection equipment as in molecular biology, and the detection of a large number of samples is relatively complicated. On the contrary, the serological method, especially the enzyme-linked immunosorbent assay (ELISA) method, is an important tool for identifying, classifying and detecting plant viruses due to its advantages of rapid, sensitive and accurate detection of a large number of samples, low experimental cost, simple method and the like. However, no related report on preparation and effective detection of ApNMV serum exists at home and abroad.

Disclosure of Invention

The invention aims to provide antiserum for resisting apple necrosis mosaic virus and a preparation method thereof.

The method for preparing the antiserum for resisting the apple necrosis mosaic virus comprises the following steps: the coat protein of apple necrosis mosaic virus is used as immunogen to immunize animals.

Wherein the coat protein of the apple necrosis mosaic virus is any one of the following proteins:

(A1) protein with an amino acid sequence of sequence 1 in a sequence table;

(A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function;

(A3) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in (A1) or (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the method, the animal may be specifically a new zealand rabbit.

In the method, immunizing the new zealand rabbit may be performed as follows: emulsifying coat protein of the apple necrosis mosaic virus with equal volume of Freund complete adjuvant (first time) or Freund incomplete adjuvant (subsequent time), carrying out intradermal multipoint injection immunization by using SPF-grade New Zealand rabbits, and after one week of rest after the first immunization, immunizing for 5-6 times (such as 6 times) in total, wherein the use amount of the coat protein for immunizing the apple necrosis mosaic virus for each time is 0.1 mg.

In the method, the coat protein of apple necrosis mosaic virus as an immunogen can be prepared according to the following method for preparing the coat protein of apple necrosis mosaic virus.

The invention also provides a preparation method of coat protein of apple necrosis mosaic virus, which specifically comprises the following steps:

(1) cloning the coding gene of coat protein of the apple necrosis mosaic virus into a polyclonal site of a prokaryotic expression vector to obtain a recombinant prokaryotic expression vector;

(2) introducing the recombinant prokaryotic expression vector into a prokaryotic cell serving as a receptor to obtain a recombinant prokaryotic cell;

(3) culturing the recombinant prokaryotic cell to obtain the coat protein of the apple necrosis mosaic virus.

In the invention, the prokaryotic expression vector is specifically pET28a (+) vector. Accordingly, the multiple cloning sites are specifically BamH I and Hind III. The prokaryotic cell used as a receptor is in particular Escherichia coli BL21(DE 3).

In the step (3), the method further comprises the steps of adding IPTG to a culture system of the recombinant prokaryotic cell to a final concentration of 0.5mM, and carrying out induction culture at 37 ℃ for 2 h.

In the method, the coding gene of coat protein of apple necrosis mosaic virus can be specifically any one of the following DNA molecules:

(B1) DNA molecule shown in sequence 2 in the sequence table;

(B2) a DNA molecule which hybridizes with the DNA molecule defined in (B1) under stringent conditions and encodes a protein represented by any one of (A1) to (A4) above;

(B3) a DNA molecule which has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology with the DNA sequence defined in (B1) or (B2) and which encodes a protein as shown in any one of (A1) to (A4) above.

The invention also protects two products shown as (A) and (B) respectively.

(A) The antiserum against apple necrosis mosaic virus was prepared by the "method for preparing antiserum against apple necrosis mosaic virus" described above.

(B) The coat protein of apple necrosis mosaic virus is prepared by the method for preparing the coat protein of apple necrosis mosaic virus.

The invention also protects four applications shown in the following (C) to (F) respectively.

(C) Use of the envelope protein of apple necrosis mosaic virus prepared using the "method for preparing envelope protein of apple necrosis mosaic virus" as described above and the readable vector described in the "method for preparing antiserum against apple necrosis mosaic virus" as described above for the preparation of a kit for the production of antiserum or polyclonal antibody against apple necrosis mosaic virus.

(D) The application of the coat protein of apple necrosis mosaic virus prepared by the method for preparing the coat protein of apple necrosis mosaic virus in preparing antiserum for resisting the apple necrosis mosaic virus as immunogen.

(E) The application of the antiserum for resisting the apple necrosis mosaic virus, which is prepared by the method for preparing the antiserum for resisting the apple necrosis mosaic virus, in detection of the apple necrosis mosaic virus is disclosed.

(F) The application of the antiserum for resisting the apple necrosis mosaic virus, which is prepared by the method for preparing the antiserum for resisting the apple necrosis mosaic virus, in preventing and treating the apple necrosis mosaic virus is disclosed.

The invention utilizes molecular biology means to efficiently express coat protein gene of apple necrosis mosaic virus in escherichia coli, purifies target protein, immunizes two SPF (specific pathogen free) grade New Zealand rabbits with the target protein, prepares specific polyclonal antiserum, and provides theoretical basis for serological detection of the virus and prevention and control of the disease in apple production.

Drawings

FIG. 1 shows RT-PCR amplification of the cp gene. M: d2000 Marker; 1-2: all are AM75 apple leaf samples. In both lanes there is a bright, single specific band that matches the expected size of the gene of interest (675 bp).

FIG. 2 is an electrophoretic image of BamHI and HindIII double digests. M: DNA Marker DL 10000; 1: the product is recovered by digesting cp gene glue with BamH I and Hind III; 2: double digestion of pET-28a (+) plasmid by BamH I and Hind III; 3: the plasmid pET-28a (+) which has not been digested.

FIG. 3 shows SDS-PAGE analysis and Western blot analysis of the small expression of recombinant proteins. A is the result of SDS-PAGE analysis; wherein, 1: incubating the lysate overnight at 37 ℃; 2: incubating the pellet fraction overnight at 37 ℃; 3: the supernatant fractions were incubated overnight at 37 ℃; 4: no IPTG induction; 5: inducing all lysates with IPTG; 6: inducing the precipitate fraction with IPTG; 7: inducing the supernatant fraction with IPTG; 8: BSA 0.5. mu.g; 9: BSA 1.0. mu.g; 10: BSA 2.0. mu.g. B is a Western blot analysis result; wherein, 1: incubating the lysate overnight at 37 ℃; 2: incubating the pellet fraction overnight at 37 ℃; 3: the supernatant fractions were incubated overnight at 37 ℃; 4: no IPTG induction; 5: inducing all lysates with IPTG; 6: inducing the precipitate fraction with IPTG; 7: inducing the supernatant fraction with IPTG; 8: negative control; 9: a positive control; anti-His antibody detection.

FIG. 4 shows SDS-PAGE and Western blot analysis of recombinant protein mass expression. A is the result of SDS-PAGE analysis; wherein, 1: purifying protein 1 μ g; 2: BSA 0.5. mu.g; 3: BSA 1.0. mu.g; 4: BSA 2.0. mu.g. B is a Western blot analysis result; purified protein 0.5. mu.g, anti-His antibody detection.

FIG. 5 shows the polyclonal antiserum titer detection reaction.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The main reagents are as follows: the polysaccharide polyphenol plant total RNA extraction kit (Polysaccharades and Polyphenonics-rich RNAprep Pure) is purchased from Tiangen biotechnology (Beijing) company, the restriction enzyme is mainly from TaKaRa company, and the reagent required for RT-PCR amplification is mainly from Prologue-McGr (Beijing) biotechnology limited company (PROMEGA), AxyPrep DNA gel recovery kit and AxyPrep plasmid DNA small-lift kit are purchased from Axygen reagent company, and the prokaryotic expression vector pET-28a (+) is purchased from Yingchang researchers of plant protection research institute of Chinese academy of agricultural sciences, wherein the chemiluminescence color developing solution and PVDF membrane are purchased from Millipore company (USA), and goat anti-rabbit enzyme-labeled secondary antibody (product number 458) is purchased from Beijing Bourmai biotechnology limited company.

The main apparatus is as follows: Bio-Rad PCR apparatus, Bio-Rad membrane transfer apparatus, KCBIO-2800 gel imaging system, DYY-6B type voltage-stabilized electrophoresis apparatus, ATTO AE-6200 vertical electrophoresis tank (Japan), novel TS-1 decolorizing shaking table, constant-temperature water bath, constant-temperature incubator, and micro ultraviolet spectrophotometer

ND-1000UV-Vis Spectrophotometer (NanoDrop, America), AIRTECH SW-CJ-1FD super clean bench, Synergy 4 multifunctional microplate reader (America).

Primer synthesis was performed by Beijing Liuhe Huada Dageno science and technology, Inc., and sequencing was performed by Biotechnology engineering (Shanghai), Inc.

Example 1 prokaryotic expression and identification of coat protein of apple necrosis mosaic Virus

Construction of recombinant prokaryotic expression vector pET-ApNMVCP

1. Plant total RNA extraction

The total plant RNA extraction adopts a polysaccharide polyphenol total plant RNA extraction kit (Tiangen), and the specific operation method is carried out according to the instruction. The test specimens were apple leaves infected with apple necrotic mosaic virus AM 75.

2. Design of ApNMV cp gene sequence amplification specific primer

Based on the RNA3 sequence of apple necrosis mosaic virus AM75 amplified by the previous group of inventors, a cp gene sequence amplification primer is designed, and BamH I and Hind III enzyme cutting sites and corresponding protective bases are introduced into the 5' end of a forward primer (Table 1).

TABLE 1 ApNMV CP sequence amplification specific primers

Note: underlined sections indicate restriction enzyme sites.

3. RT-PCR amplification cp gene and double digestion construction of prokaryotic expression recombinant plasmid

Reverse transcription system:

mixing the mixed solution, incubating for 1h at 37 ℃, and then carrying out PCR reaction (the reaction system is as follows) or storing at-20 ℃ for later use.

PCR cycling parameters: pre-denaturation at 98 ℃ for 30sec, denaturation at 98 ℃ for 10sec, annealing at 65 ℃ for 30sec, and extension at 72 ℃ for 30sec, and circulating for 32 times; further extension at 72 deg.C for 10min, and storage at 4 deg.C. The PCR product was analyzed by 1.5% agarose gel electrophoresis, electrophoresed at 120V for about 30min in a model DYY-6B Steady voltage electrophoresis apparatus, the gel was photographed in a KCBIO-2800 gel image analyzer, the band of interest (FIG. 1) was cut off, and the purification and recovery of the amplification product were carried out using AxyPrep DNA gel recovery kit.

Preparing the following double enzyme digestion reaction system:

after mixing, incubation for 15h at 37 ℃, adding 5 μ l of 10 × loading buffer to terminate the enzyme digestion reaction, analyzing the enzyme digestion product by using 1% agarose gel electrophoresis, performing electrophoresis for about 30min at 120V in an DYY-6B type voltage-stabilized electrophoresis apparatus, photographing an electrophoresis gel in a KCBIO-2800 gel imaging analyzer (figure 2), cutting off a target band, and purifying and recycling the amplification product by using an AxyPrep DNA gel recycling kit.

The following ligation reaction system was prepared:

flick, mix, centrifuge, connect at 4 ℃ overnight. Transforming the connecting liquid into 50 mul DH5 alpha competent cells, coating plates, culturing at 37 ℃, picking single clone in 1ml Kana (kanamycin sulfate, 100mg/L) resistant LB liquid culture medium, shaking and shaking the bacteria at 37 ℃, identifying positive recon by a bacteria liquid PCR method, sending the positive clone to the company Limited in the biological engineering (Shanghai) for sequencing, and verifying the correctness of the connecting sequence and the reading frame. The recombinant plasmid which is shown by sequencing to replace a small fragment between enzyme cutting sites BamH I and Hind III of a pET-28a (+) vector with a DNA fragment shown in the 1 st to 657 th sites of a sequence 2 in a sequence table is named as pET-ApNMVCP. Wherein, the DNA fragment shown in the sequence 2 is the coding gene (namely CP gene) sequence of the coat protein of the apple necrosis mosaic virus obtained by the invention, and codes the coat protein (namely CP protein) of the apple necrosis mosaic virus shown in the sequence 1 in the sequence table.

Then, the bacterial solution with the correct reading frame was selected and expanded, and the recombinant Plasmid pET-ApNMVCP was extracted using a Plasmid DNA Miniprep Kit (AxyPrepTM Plasmid Miniprep Kit 250-prep AxyPrep) and stored at-20 ℃ for future use.

Of course, when preparing the recombinant plasmid pET-ApNMVCP, the DNA fragment shown in sequence 2 can be directly synthesized artificially and inserted between enzyme cutting sites BamH I and Hind III of pET-28a (+) vector by the conventional molecular cloning means.

Second, CP gene induced expression and protein purification

And (2) respectively transforming the recombinant plasmids pET-ApNMVCP and pET-28a (+) with correct reading frames prepared in the step one into an escherichia coli expression strain BL21(DE3), carrying out overnight culture at 37 ℃, selecting a single colony, carrying out overnight culture, preserving glycerol bacteria, inoculating overnight bacteria liquid into a fresh Kana resistant LB culture medium according to a volume ratio of 1:20, and carrying out small-amount expression. After the small amount expression is successful, the large amount expression is carried out, 100 mul of glycerol bacteria is inoculated into 50ml of Kana resistant LB culture medium for overnight culture, then the glycerol bacteria is inoculated into 1000ml of culture medium according to the volume ratio of 1:20, the culture medium is stirred and cultured at 37 ℃ and 160 rpm. When OD600 reached about 0.6, 0.5mM IPTG was added to the cultured bacterial solution to induce protein expression, and the mixture was subjected to shaking culture at 37 ℃ and 160rpm for 2 hours. The cells were centrifuged at 8000rpm for 20min at 4 ℃ to recover the cells. The purified protein was used in Ni Sepharose 6Fast Flow and finally the recovered protein was pooled in 250mM imidazole/8M Urea PBS for subsequent multiple anti-immunizations.

Third, SDS-PAGE and Western blot detection analysis of expression product

When the CP protein is expressed in a small amount, the thalli are collected by centrifugation, 1/10 volume of sample buffer solution is added, the oscillation and suspension are carried out, the boiling is carried out for 5min at 100 ℃, the SDS-PAGE analysis is carried out by 12 percent gel, the voltage of the concentrated gel is 8V/cm, and the separation gel is 15V/cm. Coomassie brilliant blue R-250 was stained for 1h and destained on a shaker at room temperature for 3 h. Western blot analysis of the expression products is described in Towbin et al (Towbin H, Staehelin T, Gordon J. electrophoretic transfer of proteins from polyacylamide gels to nitro cellulose sheets: procedure and sodium applications [ J ]. Proceedings of the National Academy of Sciences of the United States of America,1979,76(9): 4350-. And the large-scale expression detection uses the purified protein as a sample, and the rest is subjected to small-scale expression SDS-PAGE and Western blot analysis.

The results of SDS-PAGE and Western blot analysis of small expression samples (FIG. 3) show that after IPTG induced expression, a band of the same size as the expected size of the recombinant protein appears at a position with a molecular weight of about 25kDa, and no band of the same size is evident in a lane without IPTG induction, which indicates that the cp gene is successfully induced and expressed.

After a large amount of induction expression, the precipitated part after the escherichia coli lysis is subjected to protein purification by Ni Sepharose 6Fast Flow, and the recovered protein is concentrated in 250mM imidazole/8M Urea PBS with the concentration of 1.2mg/ml and the volume of 2ml, and is co-purified to obtain 2.4mg of recombinant protein. In addition, SDS-PAGE and Western blot detection analysis (FIG. 4) were carried out on the obtained purified protein, and a major target protein band appeared around a molecular weight of about 25kDa, indicating that the desired target protein was purified by the assay.

Example 2 preparation and application of antiserum against apple necrosis mosaic virus

First, preparation of antiserum and evaluation of potency

A small amount of normal serum was prepared by bleeding 2mL of the ear vein of SPF New Zealand rabbit as a negative control. Thereafter, an equal volume of complete Freund's adjuvant (first) or incomplete Freund's adjuvant (subsequent) was added to the purified protein product prepared in example 1 for emulsification, and 2 SPF-grade New Zealand rabbits were used for intradermal multi-site injection immunization in a barrier environment. The immunization is carried out five times per week, a rest is carried out for one week after the first immunization, the total immunization is 6 times, and the dosage of the coat protein for immunizing the apple necrosis mosaic virus for each time is 0.1 mg. 2ml of blood is collected one week after the completion of the immunization, and ELISA titer detection is carried out, if the titer is not enough, additional immunization is needed. If the titer reaches the standard, carrying out full blood collection and ELISA detection. The prepared antiserum is filtered, added with 0.09 percent of sodium azide and stored at 4 ℃ for later use. The antiserum thus obtained was diluted at 5-fold intervals in a gradient manner, and the titer of the serum was measured by an indirect ELISA method using the expressed fusion protein of interest as an antigen coating.

2 SPF-grade New Zealand rabbits are immunized by the purified target protein, and finally specific antiserum of the ApNMV is obtained. Expressed target fusion protein is used as antigen coating, and the titer of antiserum is determined by using an indirect ELISA detection method. The results show (figure 5), the serum collected from 2 rabbits still can show obvious positive reaction after being diluted by 62500 times, and simultaneously has no obvious serological reaction with the serum before immunization and a blank control. The results show that the antiserum prepared by the invention has good quality.

Second, indirect ELISA detection of apple leaf samples

According to the methods of Shimantling and Zheng Chengling academic thesis (Shimantling. turnip mosaic virus monoclonal antibody preparation and virus genome variation research (doctor academic thesis) (Hangzhou: Zhejiang university, 2005) and Zheng Chengling. Chinese wheat mosaic virus infectious clone construction and functional analysis of motor protein (doctor academic thesis) (Hangzhou: Zhejiang university, 2012), the ApNMV polyclonal antiserum prepared in the step one is used as a primary antibody, and an indirect ELISA (ID-ELISA) method is adopted to detect the apple leaf samples in the field. The specific operation is as follows:

1. weighing about 0.2g of fresh apple leaves (with the main veins removed), grinding with liquid nitrogen, adding 2mL of coating diluent (10mL/g), fully grinding, and centrifuging at 5000rpm at room temperature for 3 min;

2. sucking 100 mu L of supernatant, adding the supernatant into an enzyme label plate, and standing the mixture at 4 ℃ overnight or in a thermostat at 37 ℃ for 2 hours;

3. PBST is washed for three times, each time standing is carried out for 3min, and residue is flapped and dried on a paper towel;

4. adding 150 μ L of 3% skimmed milk powder, sealing at 37 deg.C for 1 hr;

5. pouring out the sealing liquid, beating and spin-drying the residual liquid, adding 100 mu L of ApNMV polyclonal antiserum diluted by 400 times, and standing at 37 ℃ for 1 h;

6. washing is carried out in the same step 3;

7. adding 100 mu L of goat anti-rabbit enzyme-labeled secondary antibody diluted 8000 times with 3% skimmed milk powder, and standing at room temperature for 1 h;

8. washing is carried out in the same step 3;

9. adding 100 μ L of the mixed solution, and developing at room temperature for 20-30 min;

10. stopping the reaction with 100. mu.L of 2M sulfuric acid, and measuring OD with an enzyme-linked immunosorbent assay (ELISA)_450nmAnd recording.

And (3) performing zero calibration by taking no antigen as a blank control, judging according to the critical value (namely the OD value of the sample to be detected/the OD value of a negative control (healthy apple leaf sample)), and if the critical value is greater than 2, determining that the sample is toxic, otherwise, determining that the sample is not toxic.

In order to further verify the accuracy of the ELISA detection result, the RT-PCR method was used as a control in the experiment. The primers used for RT-PCR were as follows:

ApNMV-F：5’-ATGGTGTGCAATCGCTGTCA-3’；

ApNMV-R：5’-CATCGACCATAAGGATATCA-3’。

reference is made to Noda H, Yamagishi N, Yaegashi H, Xing F, Xie J P, Li S F, Zhou T, Ito T, Yoshikawa N.apple near biological virus, a novel areas from biological-distinct applets tree in Japan and China [ J ]. Journal of General Plant Pathology,2017,83:83-90.

The detection results of the samples show that (Table 2), the P/N values of the samples 1-6 are all larger than 2, and the samples are judged to be ApNMV positive; the P/N values of the 7-9 samples are all less than 2, and the samples are judged to be ApNMV negative. The ELISA detection result is consistent with the early RT-PCR detection result, and the ApNMV antiserum prepared by the invention is shown to be applicable to detection of ApNMV of apple leaves, and has high detection sensitivity and good specificity.

TABLE 2 Indirect ELISA assay of ApNMV on samples

Note that in the Table, the OD is compared_450nmData are mean values of three healthy raw tobacco samples, sample OD_450nmThe assays were all done in duplicate; the P/N value is the OD of the sample to be measured and the reference sample_450nmRatio, if P/N value>2, the sample is considered to be toxic, otherwise, the sample is not toxic.

<110> institute of plant protection of Chinese academy of agricultural sciences

<120> antiserum for resisting apple necrosis mosaic virus and preparation method thereof

<130> GNCLN171369

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<170> PatentIn version 3.5

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<213> apple necrotic mosaic Virus

<400> 1

Met Val Cys Asn Arg Cys His His Thr His Ala Gly Gly Cys Arg Ser

1 5 10 15

Cys Arg Gln Cys His Pro Arg Asp Ala Ala Pro Pro Pro Pro Arg Ala

20 25 30

Arg Ala Arg Ala Gln Asn Val Val Ala Arg Gly Leu Ala Arg Pro Glu

35 40 45

Thr Ser Ala Arg Glu Pro Arg Arg Leu Gln Trp Thr Val Ile Gly Pro

50 55 60

Asn Glu Val Pro Arg Val Pro Arg Gly Tyr Val Ala His Ser Asn Arg

65 70 75 80

Glu Val Val Ala Thr Ser Ala Gly Lys Phe Leu His Val Asn Phe Ser

85 90 95

Thr Thr Phe Pro Gln Leu Leu Gly Leu Asn Leu Arg Ile Leu Ser Val

100 105 110

Val Val Arg Ala Ser Cys Leu Val Ser Ala Gly Trp Val Gly Met Leu

115 120 125

Glu Asp Phe Asp Glu Asn His Leu Arg Gly Pro Ser Ala Leu Ser Arg

130 135 140

Lys Gly Phe Arg Gln Asp Gln Pro Arg Gly Trp Gln Trp Leu Ala Pro

145 150 155 160

Ser Asp Leu Glu Tyr Asp Thr Phe Ala Asn Ser His Arg Leu Val Phe

165 170 175

Glu Val Lys Asn Glu Phe Ala Ala Gly Ala Lys Val Leu Val Arg Asp

180 185 190

Ile Tyr Ile Val Val Asn Asp Leu Pro Arg Ile Val Ile Pro Asn Asp

195 200 205

Ile Leu Met Val Asp Glu Asp Leu Leu Asp Val

210 215

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<220>

<223>

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atggtgtgca atcgctgtca tcacactcac gctggtgggt gtcggtcctg ccgacagtgc 60

cacccgagag atgctgctcc accaccaccg agagctcgag ctagagctca aaacgtggta 120

gcgcgaggtt tagcgcgacc agagacttca gctagggagc cgaggaggct tcaatggacc 180

gtgataggtc cgaatgaggt accacgagta ccgaggggat acgtggcaca tagcaacaga 240

gaagttgttg cgactagcgc tgggaagttt ctacatgtga acttcagcac gactttcccg 300

caactattag gattgaatct taggattctc tccgtggtag ttcgagccag ctgcctggta 360

tccgctgggt gggtgggaat gttggaggac tttgatgaga atcatctcag aggtccgagt 420

gccttgtcca ggaagggttt tcgccaagac caaccgagag gttggcaatg gttggctcct 480

tccgatttag aatacgatac gtttgcgaac tcgcaccgtt tagtattcga agtcaagaac 540

gaattcgcgg caggcgcgaa agttcttgtg agggacatct atatagtggt aaatgattta 600

ccacgaattg tgatcccgaa tgatatcctt atggtcgatg aagacctttt ggatgtctag 660

Claims

1. A method for preparing antiserum against apple necrosis mosaic virus comprises the following steps: using coat protein of apple necrosis mosaic virus as immunogen to immunize animal;

the coat protein of the apple necrosis mosaic virus is a protein with an amino acid sequence of a sequence 1 in a sequence table.

2. The method of claim 1, wherein: the animals were New Zealand rabbits.

3. The method according to claim 1 or 2, characterized in that: the coat protein of the apple necrosis mosaic virus serving as immunogen is prepared according to the method comprising the following steps:

4. The method of claim 3, wherein: the prokaryotic expression vector is a pET28a (+) vector; the prokaryotic cell used as a receptor is E.coli BL21(DE 3).

5. The method of claim 3, wherein: in the step (3), IPTG is added into a culture system of the recombinant prokaryotic cell to a final concentration of 0.5mM, and the induction culture is carried out for 2h at 37 ℃.

6. The method of claim 3, wherein: the coding gene of coat protein of the apple necrosis mosaic virus is a DNA molecule shown as a sequence 2 in a sequence table.

7. Antiserum against apple necrosis mosaic virus, prepared by the method of any one of claims 1-6.

8. The application is any one of the following:

(C) use of coat protein of apple necrosis mosaic virus prepared by the method of any one of claims 3 to 6 and a readable vector carrying the method of claim 1 or 2 for the preparation of a kit for the production of antisera or polyclonal antibodies against apple necrosis mosaic virus;

(D) use of coat protein of apple necrosis mosaic virus produced by the method of any one of claims 3 to 6 as an immunogen in the production of antiserum against apple necrosis mosaic virus;

(E) use of antiserum against apple necrosis mosaic virus, prepared by the method according to any one of claims 1-6, for detecting apple necrosis mosaic virus.