AU2021103418A4

AU2021103418A4 - Preparation method of spvd recombinant double cp fusion protein and method for detecting spvd

Info

Publication number: AU2021103418A4
Application number: AU2021103418A
Authority: AU
Inventors: Guoning Du; Qichao Geng; Qiyan Li; Shunyu Lin; Tao MA; Lixian Qiao; Jiongming Sui; Yanyan TANG; Jingshan Wang; Jie Xiang; Chunmei ZHAO; Hong Zhu
Original assignee: Qingdao Youtian Agricultural Development Co Ltd; Qingdao Agricultural University
Current assignee: Qingdao Youtian Agricultural Development Co Ltd; Qingdao Agricultural University
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-12-16
Anticipated expiration: 2029-06-17

Abstract

The present invention belongs to the field of biotechnology, and disclosing a preparation method of a sweet potato virus disease (SPVD) recombinant double CP fusion protein and a method for detecting SPVD. The SPVD recombinant double CP fusion protein is constructed from the fusion fragment of the CP gene of sweet potato greenish dwarfing virus and sweet potato plume mottled virus to the recombinant expression vector, transformed into the expression strain and induced expression. The existing method for detecting SPVD can only detect sweet potato chlorotic stunt virus and sweet potato feathery mottle virus separately, resulting in low detection efficiency and high cost. By preparing the SPVD recombinant double CP polyclonal antibody, sweet potato chlorotic stunt virus and feathery mottle virus can be simultaneously detected, which improves detection efficiency and reduces detection cost.

Description

PREPARATION METHOD OF SPVD RECOMBINANT DOUBLE CP FUSION PROTEIN AND METHOD FOR DETECTING SPVD BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention belongs to the field of biotechnology, and specifically relates to a preparation method of a sweet potato virus disease (SPVD) recombinant double CP fusion protein and a method for detecting SPVD.

[0003] 2. Description of Related Art

[0004] Virus disease is an important disease of sweet potatoes, and it is one of the main reasons that lead to yield decrease, quality decline and seed degradation of sweet potatoes. Due to long term asexual reproduction, viruses can be spread through potato pieces and seedlings. In addition, effective chemical control methods lack for the sweet potato viruses. Therefore, sweet potato virus diseases have become a problem that hinders the development of sweet potato industry.

[0005] More than 30 kinds of viruses that infect sweet potatoes have been reported currently. Among them, the sweet potato virus disease (SPVD) caused by co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) is one of the most serious viral diseases of sweet potatoes. At present, the common method for virus-free seedling detection is ELISA method. Some research groups have also prepared single virus recombinant CP polyclonal antibodies. This method is only used for single virus detection, with high cost and low efficiency.

BRIEF SUMMARY OF THE INVENTION

[0006] In view of the problems in the prior art, the objective of the present invention is to provide a preparation method of an SPVD recombinant double CP fusion protein and a preparation method of an SPVD recombinant double CP polyclonal antibody, which can be used for ELISA detection of two viruses at the same time, thereby improving detection efficiency and reducing test cost.

[0007] In order to achieve the above objective, the technical solution of the present invention is as follows:

[0008] A preparation method of an SPVD recombinant double CP fusion protein, including the following steps:

[0009] (1) construction of a recombinant expression vector: adding digestion linkers of NdeI and HindIII to two ends of a fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus, and connecting the digestion linkers to a prokaryotic expression vector pET22b after digestion to obtain a recombinant expression vector;

[0010] (2) transformation of expression strains: transforming the recombinant expression vector into Rosetta (DE3) competent cells;

[0011] (3) induced expression: picking positive clones in (2) and inducing the expression of a fusion protein; and

[0012] (4) purification of the fusion protein: purifying the fusion protein by nickel ion affinity chromatography.

[0013] On the basis of the above solution, the nucleic acid sequence of the fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus is shown as SEQ ID No: 1.

[0014] On the basis of the above solution, the fusion protein sequence is shown as SEQ ID No: 2.

[0015] A preparation method of an SPVD recombinant double CPpolyclonal antibody, using the SPVD recombinant double CP fusion protein prepared by the method of claim 3 to immunize rabbits, wherein the obtained antiserum is the SPVD recombinant double CP polyclonal antibody.

[0016] A method for detecting SPVD, including the following steps:

[0017] (1) taking sweet potato leaves to be tested, grinding and crushing with liquid nitrogen, adding a virus extraction buffer based on 10 mL/g and further grinding, centrifuging at 4°C and 12000r for 5 minutes, and taking the supernatant;

[0018] (2) adding 150 pL of the supernatant from (1) to each reaction well, coating overnight at 4°C and washing; adding 200 pL of 3% BSA for confining; and

[0019] (3) adding 150 pL of diluted polyclonal antibody prepared by the method of claim 4, 150 pL of enzyme-labeled secondary antibody diluent and 150 pL of substrate solution to each well, and finally adding 100 pL of 1.5M NaOH to terminate the reaction; and measuring an optical density (OD) value with a microplate reader at a wavelength of 450 nm.

[0020] Advantages of the technical solution of the present invention:

[0021] The existing method for detecting SPVD can only detect sweet potato chlorotic stunt virus and sweet potato feathery mottle virus separately, resulting in low detection efficiency and high cost. By preparing the SPVD recombinant double CP polyclonal antibody, sweet potato chlorotic stunt virus and feathery mottle virus can be simultaneously detected, which improves detection efficiency and reduces detection cost.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0022] Fig. 1 is an SDS-PAGE analysis diagram of a fusion protein on laboratory scale (M: Protein Marker; 1: total protein before induction; 2: 20°C supernatant; 3: 20°C precipitation; 4: 37°C supernatant; 5: 37°C precipitation);

[0023] Fig. 2 is an SDS-PAGE analysis diagram of nickel ion affinity chromatography purification (M: Protein marker; 1: loading; 2: effluent; 3: 20 mM Imidazole elution fraction; 4: mM Imidazole elution fraction; 5: 500 mM Imidazole elution fraction);

[0024] Fig. 3 illustrates antiserum titer detection (the first row repeats 1, the second row repeats 2, well 1 is a negative control, well 2 is a blank control, and wells 3-12 have antibody dilution 1: 1K, 2K, 4K, 8K, 16K, 32K, 64K, 128K, 256K, 512K)

DETAILED DESCRIPTION OF THE INVENTION

[0025] The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

[0026] The present invention will be further described in detail below in conjunction with specific embodiments and with reference to data. The following embodiments are only for illustrating the present invention, but not to limit the scope of the present invention in any way.

[0027] Embodiment 1

[0028] 1. Obtaining of a fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus

[0029] According to the published sweet potato chlorotic stunt virus CP gene (West Africa strain, GenBank accession number: CP gene in FJ807785, 4356-5126 bp) and sweet potato feathery mottle virus CP gene (GenBank accession number: HQ844114) sequence, a fusion fragment of the two CP genes was chemically synthesized, a termination codon was removed, NdeI and HindIII digestion sites within the genes were mutated without changing the encoded amino acid (the original sequence had a Ndel digestion site at 268-273 bp, 938-943 bp, and 1127-1132 bp respectively, and bases at 270 bp, 942 bp, and 1131 bp were respectively changed from T to C when the gene sequence was synthesized, to remove the NdeI digestion site inside the gene, without changing the encoded amino acid sequence; one HindIII digestion site was at 1415-1420 bp, the base at 1419 bp was changed from T to A when the gene sequence was synthesized, to remove the HindIII digestion site inside the gene, without changing the encoded amino acid sequence), the synthesized gene fusion fragment was shown as SEQ ID No: 1, digestion linkers of NdeI and HindIII were added to two ends of the sequence respectively and connected to a pMD18 vector.

[0030] 2. Construction of a prokaryotic expression vector containing the fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus

[0031] The fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus in 1 (SEQ ID No: 1) was identified, digested, and connected to a prokaryotic expression vector pET22b for PCR, digestion and sequencing identification.

[0032] 3. Induction of the expression of fusion protein

[0033] (1) Transform

[0034] 1 1 of the recombinant pET22b vector was transformed into Rosetta (DE3), which was then thermally shocked at 42°C for 90 s, stood on ice for 2 min, and coated on an LB solid plate containing 50 pg/mL ampicillin and 34 pg/mL chloramphenicol. 4 g powder was dissolved in 100 mL dd water in use, followed by culture overnight at 37°C.

[0035] (2) Selection of optimal induction conditions for small-scale culture

[0036] A single colony of the expression strain Rosetta (DE3) was picked to 10 mL of LB medium. 50 g/mL ampicillin and 34 g/mL chloramphenicol were added, followed by culture overnight at 37°C and 220 rpm.

[0037] The overnight cultured strain solution was respectively inoculated into 10 mL of LB medium at a ratio of 1:100, 50 g/mL ampicillin and 34 g/mL chloramphenicol were added, followed by culture at 37°C and 220 rpm. When the OD value reached 0.6, IPTG with a final concentration of 0.5 mM was added for induction overnight at 220 rpm and 20°C; the induction was also carried out at 37°C for 4 h, and the solution not added with the IPTG inducer was used as a negative control. The solution was centrifuged at 4000 rpm for 10 min to collect phage, the supernatant was discarded, the phage was suspended with 500 pL of PBS (pH 7.4) buffer and crushed with ultrasonic for 6 min, the ultrasonic crushing was stopped for 1.5 s every 0.5 s, the supernatant and precipitate were respectively collected by centrifugation, the precipitate was dissolved in 500 pL of inclusion body dissolving solution (8 M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), 40 L of sample was mixed with 10 L of 5xprotein loading buffer, and the mixture was treated in a boiling water bath for 10 min.

[0038] SDS-PAGE detection: 12% SDS-PAGE and Tris-Gly electrophoresis buffer (Tris 3.0 g, glycine 14.4 g, SDS 1.0 g, constant volume to 1 L) were prepared, 10 L of sample was loaded, gel was concentrated at 80 V for 20 min and separated at 120 V for 60 min, the gel electrophoresis was over, Coomassie brilliant blue staining was carried out for 20 min, followed by decoloration, thus obtaining a target protein of about 65 KDa (the protein sequence was shown as SEQ ID No: 2). The results were in line with expectations (Fig. 1).

[0039] (3) Mass induced expression of fusion protein

[0040] The cultured strain solution was inoculated into 3 L of LB liquid medium at a ratio of 1: 100, 50 g/mL ampicillin and 34 g/mL chloramphenicol were added, followed by culture at 37°C and 220 rpm. When the OD value reached 0.6, IPTG with a final concentration of 0.5 mM was added for induction overnight at 20°C and 220 rpm, and cell phage was collected by centrifugation.

[0041] 4. Purification of protein

[0042] (1) Ultrasonic crushing of phage

[0043] The collected phage was dissolved in a crushing Buffer (50 mM Tris, 300 mM NaCl, 0.2% Triton X-100, 0.2 mM PMSF, pH 8.0), and the phage was crushed with ultrasonic in an ice bath under power of 400 W for 20 min. The phage was centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was collected for next step of purification.

[0044] (2) Nickel ion affinity chromatography

[0045] 5 mL of Ni-NTA was taken, and the equilibrium column was washed with 5 times the column bed volume of Binding buffer (50 mM Tris, 300 mM NaCl, pH 8.0) at a flow rate of 5 mL/min. The supernatant collected in (1) was loaded to the column at a flow rate of 2 mL/min, and breakthrough liquid was collected. The column was washed with 5 times the column bed volume of Binding buffer at a flow rate of 5 mL/min. Impurities were first washed with Wash buffer (50 mM Tris, 300 mM NaCl, 20/50 mM Imidazole, pH 8.0) at a flow rate of 5 mL/min, and the eluent was collected; then the impurities were eluted with Elution buffer (50 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0) at a flow rate of 2 mL/min, and the eluent was collected.

[0046] It can be seen from Fig. 2 that first elution with 20 mM and 50 mM Imidazole can remove most of the impurities, and then elution with Elution buffer containing high concentration Imidazole (500 mM) can enrich a lot of target protein.

[0047] The collected fractions were detected by SDS-PAGE, and the purified protein was dialyzed into: 1xPBS, pH 8.0, overnight at 4°C. After the dialysis, the solution was centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was retained. After filtration with a 0.45 m filter membrane, the protein was charged into 1mL/tubes and preserved at -80°C. The protein was quantified by a Bradford method.

[0048] 5. Antigen treatment:

[0049] The total amount of purified protein was 3 mg, the primary immunization dose was 0.5 mg/animal, and the second, third, and fourth immunization doses were 0.25 mg/animal. The primary immunization antigen was a protein antigen mixed and emulsified with an equal volume of Freund's complete adjuvant, and the second, third, and fourth immunization antigens were protein antigens mixed and emulsified with equal volumes of Freund's incomplete adjuvant.

[0050] 6. Animal immunization:

[0051] Animals were healthy female New Zealand white rabbits, 4 months old, 2.1 kg.

[0052] Immunization

[0053] First immunization: day 1, the immunization antigen was Freund's complete adjuvant + protein antigen;

[0054] Second immunization: day 21, the immunization antigen was Freund's incomplete adjuvant + protein antigen;

[0055] Third immunization: day 35, the immunization antigen was Freund's incomplete adjuvant + protein antigen;

[0056] Blood collection after the third immunization: day 42, 1 ml of blood was collected from the ear vein, and the titer of antiserum was detected by ELISA;

[0057] Fourth immunization: day 49, the immunization antigen was Freund's incomplete adjuvant + protein antigen;

[0058] Blood collection after the fourth immunization: day 56, 1 ml of blood was collected from the ear vein, and the titer of antiserum was detected by ELISA;

[0059] Final bloodletting: day 57, the ELISA detected that the titer of antiserum met the requirement, and whole blood was collected from the carotid artery.

[0060] 7. Indirect ELISA detection on activity of recombinant CP polyclonal antibody

[0061] Antigen coating: the purified protein was coated with 0.05 mol/L carbonate (PH=9.6) on a plate at 0.2 g/well, 100 l/well, and incubated overnight at 4°C.

[0062] Plate washing: the plate was taken out and washed with 0.05% Tween-20 (PBST) three times, 3 minutes/time.

[0063] Confining: 150 [ of 5% confining liquid of skimmed milk powder was added to each well for confining at 37°C for 60 minutes.

[0064] Plate washing: the plate was taken out and washed with 0.05% Tween-20 (PBST) three times, 3 minutes/time.

[0065] Adding of primary antibody: serums of the rabbits were diluted at 1:1000 respectively, then diluted by multiples, and incubated at 37°C for 1 hour.

[0066] Plate washing: the plate was taken out and washed with 0.05% Tween-20 (PBST) three times, 3 minutes/time.

[0067] Adding of secondary antibody: horseradish enzyme labeled goat anti-rabbit IgG (H+L), item number: D110058, antibody company: Sangon Biotech (Shanghai) Co., Ltd. 1: 8000 dilution and incubation at 37°C for 45 min were carried out.

[0068] Plate washing: the plate was taken out and washed with 0.05% Tween-20 (PBST) five times, 3 minutes/time.

[0069] Color development: a substrate solution (TMB) was added at 100 l/well, reaction occurred for 15 min, and finally 100 1 of 2 mol/L sulfuric acid was added to terminate the reaction.

[0070] Measurement of OD value: the OD value was measured with a microplate reader (Kehua ST-360) at a wavelength of 450 nm.

[0071] Recombinant CP was used as an antigen to coat the reaction wells. In the indirect ELISA determination, the negative control and the blank control did not develop a color, and the reaction wells containing the purified antibody developed colors. The visual inspection results showed that the reaction was positive, but the colors of the reaction wells became light after the dilution increased (Fig. 3).

[0072] The OD value measurement results showed that the antibody dilution was within a range of 1: 512 k, the polyclonal antibody and antigen (recombinant CP) had strong binding activity, the ratio of OD values of the reaction wells and the negative control was >2.5 (Table 1), and the reaction was determined to be positive

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[0074] 8. Indirect ELISA detection on SPVD virus

[0075] Diseased sweet potato leaves infected with SPVD were taken, crushed by grinding with liquid nitrogen, a virus extraction buffer (PBST containing 2% PVP-40000, 1% BSA) was added at 1: 10 (g: mL), the leaves were further ground, and the supernatant was taken for detection after centrifugation at 4°C and 12000r for 5 min. 150 pL of the supernatant was added to each reaction well, washing was carried out after overnighting at 4°C, and 200 pL of 3% BSA for confining was added after coating. Then 150 pL of diluted antiserum, 150 pL of enzyme-labeled secondary antibody diluent and 150 pL of substrate solution (0.6 mg/mL pNPP, 11.7% diethanolamine) were added to each well, and finally 100 pL of 1.5 M NaOH was added to terminate the reaction. The OD value was measured with a microplate reader at a wavelength of 450 nm.

[0076] Indirect ELISA detection was performed on the diseased leaves by using the prepared antiserum. The color of the reaction wells became yellow, indicating that the diseased leaf juice had a strong positive reaction. When the dilution of the antiserum was less than 1: 1000, the A/C was greater than 2.0 (Table 2). The juice of healthy leaves did not change the color and showed a negative reaction. The results showed that the antiserum prepared from the recombinant CP can be used for the detection of SPVD.

[0077] Table 2 Indirect ELISA detection on diseased sweet potato leaves Absorption Dilution of antiserum value 1: 200 1: 400 1: 1000 1: 2000 A 0.168 0.154 0.141 0.128 A/C 2.58 2.37 2.17 1.97

[0078] Note: A was an average value of reaction wells of recombinant CP antiserum, and C was an average value (0.065) of reaction wells of virus-free sweet potato leaves (negative control).

[0079] It should be understood that although the present description is described in terms of the implementation, not every implementation includes only one separate technical solution, and such a description mode of the description is merely for the sake of clarity. A person skilled in the art should take the description as a whole, and the technical solutions in all the embodiments may be appropriately combined to form other implementations that can be understood by a person skilled in the art.

SEQUENCE LISTING

<110> Qingdao Agricultural University & Qingdao Youtian Agricultural Development Co., Ltd

<120> PREPARATION METHOD OF SPVD RECOMBINANT DOUBLE CP FUSION PROTEIN AND METHOD FOR DETECTING SPVD

<130> 2018

<160> 2

<170> PatentIn version 3.3

<210> 1 <211> 1716 <212> DNA <213> Artificial sequence(Sweet potato chlorotic stunt virus and Sweet potato feathery mottle virus)

<400> 1 atggctgata gcactaaagt cgaagagaag aactttgatt ccgactcaga tttggaaact 60

aacccaaaga ataaactaca gtccgagtat gcgtctcgtc gtgacgtcca gactgaagga 120

acggctggtt tgggaagacg agatatggag ttaactagtg gtattcttac ctcagaccag 180

ttagctcttg caagattggg taagatccaa gtatattcca attctccaga tattatgtca 240

aagagtcaag aggatgagtt taagcgtcac atggagaact tcgcaaaagt cgtgactggt 300

gaggctacaa taacgccaga aatttttgca gcgttttatg catcgttaat tcaggcatgg 360

gcaaatcaga gtacgtccga aaagaatgct tcgaacgtaa atcttgagaa tatgtttatg 420

gtcgacggga aagagtatag ttggaagact cacaacttta taaaccatat tcaatctaac 480 atgccagatg ttaagaacgc cataaggaag tgggcaaggg ctcatgcaaa tgattacaag 540 gtccttgttg gtatgggtat tgttaagccc gattaccatt tacaggcgaa acagggtgtc 600 ttacctgaat attggcattt ggccactgat ttcatgagag gtaatgattt ggcgacaacc 660 gcagatgggc tggctgcgac tttgatgatg aaaaggaacg ctctttgtaa caaggatagt 720 aagaattctg tctacaacgt tactcagttg actggaacag gtcttcactg ttctagtgaa 780 cgtactgaat tcaaagatgc gggagcgaac cctccagccc ctaagcctca gaatatccct 840 ccaccaccca caataactga ggttactgat ccagaagacc caaagcaggc agctttgaga 900 gctgcacgag ctaagcaacc cgcaaccatt ccagaatcat acggacgaga cactagcaag 960 gagaaggaat caatagtggg ggcatcatca aagggtgcga gggataaaga tgtaaacgtt 1020 ggaacagttg gtacgtttgt cgtgccacgt gttaagatga atgcaaacaa gaaaaggcaa 1080 ccaatggtaa atggaagggc cattataaat ttccaacact tgtcaacata cgagccagaa 1140 cagtttgagg ttgcaaacac ccggtcgact caagaacagt ttcaagcatg gtatgaggga 1200 gtgaaagggg actatggtgt tgacgataca ggaatgggga tcttattgaa tggattaatg 1260 gtttggtgca ttgaaaatgg cacatcccca aatataaatg gcgtgtggac tatgatggat 1320 ggtgatgagc aagtgacata tccaattaaa ccattgttgg accatgcagt gcctactttt 1380 aggcagatta tgacgcactt cagtgacgtt gctgaagcat acatagaaat gcgaaaccgt 1440 acaaaggcgt acatgccgag gtatggtcta caacgtaatt tgactgatat gagtcttgcg 1500 cgatatgcat ttgatttcta cgagctgcat tcaaccaccc ctgcacgtgc aaaagaagca 1560 catttacaga tgaaggcagc cgcgcttaag aatgcgaaaa atcggttgtt tggtttggac 1620 ggaaacgtct ccacgcaaga agaagatacg gagaggcaca cgacaactga tgttactaga 1680 aatatacata acctcttagg aatgaggggt gtgcaa 1716

<210> 2 <211> 585 <212> PRT <213> Artificial sequence(Sweet potato chlorotic stunt virus and Sweet potato feathery mottle virus)

<400> 2

Met Ala Asp Ser Thr Lys Val Glu Glu Lys Asn Phe Asp Ser Asp Ser 1 5 10 15

Asp Leu Glu Thr Asn Pro Lys Asn Lys Leu Gln Ser Glu Tyr Ala Ser 20 25 30

Arg Arg Asp Val Gln Thr Glu Gly Thr Ala Gly Leu Gly Arg Arg Asp 35 40 45

Met Glu Leu Thr Ser Gly Ile Leu Thr Ser Asp Gln Leu Ala Leu Ala 50 55 60

Arg Leu Gly Lys Ile Gln Val Tyr Ser Asn Ser Pro Asp Ile Met Ser 70 75 80

Lys Ser Gln Glu Asp Glu Phe Lys Arg His Met Glu Asn Phe Ala Lys 85 90 95

Val Val Thr Gly Glu Ala Thr Ile Thr Pro Glu Ile Phe Ala Ala Phe 100 105 110

Tyr Ala Ser Leu Ile Gin Ala Trp Ala Asn Gln Ser Thr Ser Glu Lys 115 120 125

Asn Ala Ser Asn Val Asn Leu Glu Asn Met Phe Met Val Asp Gly Lys 130 135 140

Glu Tyr Ser Trp Lys Thr His Asn Phe Ile Asn His Ile Gln Ser Asn 145 150 155 160

Met Pro Asp Val Lys Asn Ala Ile Arg Lys Trp Ala Arg Ala His Ala 165 170 175

Asn Asp Tyr Lys Val Leu Val Gly Met Gly Ile Val Lys Pro Asp Tyr 180 185 190

His Leu Gln Ala Lys Gln Gly Val Leu Pro Glu Tyr Trp His Leu Ala 195 200 205

Thr Asp Phe Met Arg Gly Asn Asp Leu Ala Thr Thr Ala Asp Gly Leu

210 215 220

Ala Ala Thr Leu Met Met Lys Arg Asn Ala Leu Cys Asn Lys Asp Ser 225 230 235 240

Lys Asn Ser Val Tyr Asn Val Thr Gln Leu Thr Gly Thr Gly Leu His 245 250 255

Cys Ser Ser Glu Arg Thr Glu Phe Lys Asp Ala Gly Ala Asn Pro Pro 260 265 270

Ala Pro Lys Pro Gin Asn Ile Pro Pro Pro Pro Thr Ile Thr Glu Val 275 280 285

Thr Asp Pro Glu Asp Pro Lys Gln Ala Ala Leu Arg Ala Ala Arg Ala 290 295 300

Lys Gln Pro Ala Thr Ile Pro Glu Ser Tyr Gly Arg Asp Thr Ser Lys 305 310 315 320

Glu Lys Glu Ser Ile Val Gly Ala Ser Ser Lys Gly Ala Arg Asp Lys 325 330 335

Asp Val Asn Val Gly Thr Val Gly Thr Phe Val Val Pro Arg Val Lys 340 345 350

Met Asn Ala Asn Lys Lys Arg Gln Pro Met Val Asn Gly Arg Ala Ile 355 360 365

Ile Asn Phe Gln His Leu Ser Thr Tyr Glu Pro Glu Gln Phe Glu Val 370 375 380

Ala Asn Thr Arg Ser Thr Gln Glu Gln Phe Gln Ala Trp Tyr Glu Gly 385 390 395 400

Val Lys Gly Asp Tyr Gly Val Asp Asp Thr Gly Met Gly Ile Leu Leu 405 410 415

Asn Gly Leu Met Val Trp Cys Ile Glu Asn Gly Thr Ser Pro Asn Ile 420 425 430

Asn Gly Val Trp Thr Met Met Asp Gly Asp Glu Gln Val Thr Tyr Pro 435 440 445

Ile Lys Pro Leu Leu Asp His Ala Val Pro Thr Phe Arg Gln Ile Met 450 455 460

Thr His Phe Ser Asp Val Ala Glu Ala Tyr Ile Glu Met Arg Asn Arg 465 470 475 480

Thr Lys Ala Tyr Met Pro Arg Tyr Gly Leu Gln Arg Asn Leu Thr Asp 485 490 495

Met Ser Leu Ala Arg Tyr Ala Phe Asp Phe Tyr Glu Leu His Ser Thr 500 505 510

Thr Pro Ala Arg Ala Lys Glu Ala His Leu Gln Met Lys Ala Ala Ala 515 520 525

Leu Lys Asn Ala Lys Asn Arg Leu Phe Gly Leu Asp Gly Asn Val Ser 530 535 540

Thr Gln Glu Glu Asp Thr Glu Arg His Thr Thr Thr Asp Val Thr Arg 545 550 555 560

Asn Ile His Asn Leu Leu Gly Met Arg Gly Val Gln Lys Leu Ala Ala 565 570 575

Ala Leu Glu His His His His His His 580 585

Claims

What is claimed is:

1. A preparation method of an SPVD recombinant double CP fusion protein, including the following steps: (1) construction of a recombinant expression vector: adding digestion linkers of NdeI and HindlI to two ends of a fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus, and connecting the digestion linkers to a prokaryotic expression vector pET22b after digestion to obtain a recombinant expression vector; (2) transformation of expression strains: transforming the recombinant expression vector into Rosetta (DE3) competent cells; (3) induced expression: picking positive clones in (2) and inducing the expression of a fusion protein; and (4) purification of the fusion protein: purifying the fusion protein by nickel ion affinity chromatography.

2. The preparation method of an SPVD recombinant double CP fusion protein according to claim 1, the nucleic acid sequence of the fusion fragment of CP genes of sweet potato chlorotic stunt virus and sweet potato feathery mottle virus is shown as SEQ ID No: 1.

3. The preparation method of an SPVD recombinant double CP fusion protein according to claim 1, the fusion protein sequence is shown as SEQ ID No: 2.

4. A preparation method of an SPVD recombinant double CP polyclonal antibody, using the SPVD recombinant double CP fusion protein prepared by the method of claim 3 to immunize rabbits, wherein the obtained antiserum is the SPVD recombinant double CP polyclonal antibody.

5. A method for detecting SPVD, including the following steps: (1) taking sweet potato leaves to be tested, grinding and crushing with liquid nitrogen, adding a virus extraction buffer based on 10 mL/g and further grinding, centrifuging at 4°C and 12000r for 5 minutes, and taking the supernatant; (2) adding 150 pL of the supernatant from (1) to each reaction well, coating overnight at 4°C and washing; adding 200 pL of 3% BSA for confining; and (3) adding 150 pL of diluted polyclonal antibody prepared by the method of claim 4, 150 pL of enzyme-labeled secondary antibody diluent and 150 pL of substrate solution to each well, and finally adding 100 pL of 1.5M NaOH to terminate the reaction; and measuring an optical density (OD) value with a microplate reader at a wavelength of 450 nm.