Preparation method and application of soybean yellow mosaic virus coat protein polyclonal antibody

Abstract

The application provides a preparation method of soybean yellow mosaic virus coat protein polyclonal antibody, which comprises the following steps: extracting total RNA of soybean leaves infected with soybean yellow mosaic virus, using the extracted total RNA as a template, amplifying the soybean yellow mosaic virus coat protein gene by using an upstream primer SbCLRV-CP-F and a downstream primer SbCLRV-CP-R, and connecting the soybean yellow mosaic virus coat protein gene to a pET-28a(+) vector to obtain a recombinant vector; the recombinant vector is transformed into escherichia coli, and after expression induction and purification, the purified recombinant protein is used as an antigen to immunize animals, collect whole blood, collect supernatant, and obtain the soybean yellow mosaic virus coat protein polyclonal antibody after purification. The application also provides an application of the polyclonal antibody prepared by the method, which is used for preparing a kit for detecting the soybean yellow mosaic virus. The soybean yellow mosaic virus coat protein polyclonal antibody prepared by the method has high titer, simple operation, can be used for mass detection of the soybean yellow mosaic virus, and has good application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of polyclonal antibody preparation technology, specifically relating to a method for preparing and applying a polyclonal antibody against the coat protein of soybean yellow leaf curl virus. Background Technology

[0002] Soybean (Glycine max L.) is an important legume and a vital source of protein and lipids for humans and livestock. Soybean chlorotic leafroll virus (SbCLRV) is a virus belonging to the genus *Polerovirus* in the family Luteoviridae, and is one of the emerging viruses that threaten soybean production.

[0003] Currently, the detection of SbCLRV mainly relies on RT-PCR technology, but the operation steps are complicated, RNA extraction is not easy, and it is not suitable for field operation. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing polyclonal antibodies against soybean yellow leafroll virus capsid protein and their application, which addresses the shortcomings of the prior art. The polyclonal antibodies against soybean yellow leafroll virus capsid protein prepared by this method have high titer, are simple to operate, and can be used for large-scale detection of soybean yellow leafroll virus, and have good application value and market prospects.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing polyclonal antibodies against the coat protein of soybean yellowing leafroll virus, the method being as follows: S1. Extract total RNA from soybean leaves infected with soybean yellowing leafroll virus; S2. Using the total RNA extracted from soybean leaves in S1 as a template, the soybean yellowing leafroll virus capsid protein gene was amplified using the upstream primer SbCLRV-CP-F and the downstream primer SbCLRV-CP-R to obtain the SbCLRV-CP target gene fragment. The nucleotide sequence of the upstream primer SbCLRV-CP-F is shown in SEQ ID No:1; the nucleotide sequence of the downstream primer SbCLRV-CP-R is shown in SEQ ID No:2. The nucleotide sequence of the SbCLRV-CP target gene fragment is shown in SEQ ID No:3; the amino acid sequence of the SbCLRV-CP target gene fragment is shown in SEQ ID No:4. S3. The SbCLRV-CP target gene fragment obtained in S2 is ligated into the pET-28a(+) expression vector to obtain the recombinant expression vector pET28a-CP. Then, the recombinant expression vector pET28a-CP is transformed into Escherichia coli, induced to express, and then purified to obtain the purified recombinant protein. S4. Using the purified recombinant protein obtained in S3 as an antigen, immunize animals, collect whole blood, collect the supernatant, and purify it to obtain a polyclonal antibody against the outer shell protein of soybean yellow leafroll virus.

[0006] Preferably, the purification method described in S3 is Ni column affinity chromatography.

[0007] Preferably, the immunized animal in S4 is a rabbit, and the immunization is performed on the rabbit a total of 4 times.

[0008] Preferably, the method for immunizing animals described in S4 is as follows: an antigen with a mass concentration of 1 mg / mL is mixed with an adjuvant at a volume ratio of 1:1. The first immunization uses a complete adjuvant, and the second to fourth immunizations use an incomplete adjuvant.

[0009] Preferably, the purification method described in S4 is dialysis.

[0010] This invention also provides the application of the polyclonal antibody against the outer shell protein of soybean yellowing leafroll virus prepared by the above preparation method, wherein the polyclonal antibody against the outer shell protein of soybean yellowing leafroll virus is used to prepare a kit for detecting soybean yellowing leafroll virus.

[0011] Compared with the prior art, the present invention has the following advantages: The polyclonal antibody against the outer shell protein of soybean yellow leafroll virus prepared by this invention has high titer, overcoming the shortcomings of existing detection technologies that are complex to operate and cannot be used for large-scale detection. It can be used to prepare a kit for detecting soybean yellow leafroll virus and has good application value and market prospects.

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 This is a graph showing the affinity purification SDS-PAGE results from Example 1 of the present invention.

[0014] Figure 2 This is a graph showing the specificity detection results of the SbCLRV-CP polyclonal antibody in Example 2 of the present invention. Detailed Implementation

[0015] Example 1

[0016] This embodiment describes a method for preparing polyclonal antibodies against the coat protein of soybean yellowing leafroll virus. The method is as follows: Total RNA was extracted from soybean leaves infected with soybean yellow leafroll virus using the S1 and Trizol methods. Primers were designed using the soybean yellow leafroll virus capsid protein (SbCLRV-CP) gene sequence downloaded from NCBI as the template sequence for detection. S2. Using total RNA extracted from soybean leaves in S1 as a template, and SbCLRV-CP-F and SbCLRV-CP-R as primers, the SbCLRV-CP gene was amplified by RT-PCR using the Goldenstar™ RT6 cDNA Synthesis Kit (Beijing Qingke Biotechnology Co., Ltd., TSK301S). The PCR products were detected by 1% agarose gel electrophoresis. The PCR products were purified using the MolPure® PCR Purification Kit (Shanghai Yisheng Biotechnology Co., Ltd., 19106ES) to obtain the SbCLRV-CP gene fragment. The nucleotide sequence of the upstream primer SbCLRV-CP-F is shown in SEQ ID No:1; the nucleotide sequence of the downstream primer SbCLRV-CP-R is shown in SEQ ID No:2. The nucleotide sequence of the SbCLRV-CP gene fragment is shown in SEQ ID No:3; the amino acid sequence of the SbCLRV-CP gene fragment is shown in SEQ ID No:4. S3. Construction of the recombinant plasmid: The SbCLRV-CP gene fragment and pET-28a(+) expression vector obtained in S2 were digested with Nde I and Sac I, respectively. Ligation was performed using T4 DNA Ligase (TaKaRa, 2011) to obtain the recombinant expression vector pET28a-CP. The ligated recombinant expression vector pET28a-CP was then transformed into E. coli DH5α competent cells (Beijing Qingke Biotechnology Co., Ltd., TSC-C14). PCR colony identification was performed using the T7 primers (nucleotide sequence shown in SEQ ID No:5) provided with the pET28a prokaryotic expression vector kit. The PCR products were sequenced by Chongqing Qingke Xingye Biotechnology Co., Ltd. The recombinant plasmid was extracted using the MolPure® Plasmid Mini Kit (Shanghai Yisheng Biotechnology Co., Ltd., 19001ES70) and transformed into E. coli Rosetta supercompetent cells (DL-Rosetta). (DE3) Competent cells, Beijing Qingke Biotechnology Co., Ltd., DLC204), a single colony was picked and placed in LB liquid medium, placed on a shaker, and incubated at 37℃; when the bacterial solution became turbid, the OD value was measured, and the OD value was approximately 0.5; 1mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the bacterial solution, and the solution was placed on a shaker and incubated at 37℃ for 8h; the sample was centrifuged at 12000rpm for 3min, and the precipitate was retained; the bacterial cells were resuspended in 30mL of 1×PBS, 30μL of β-mercaptoethanol and 300μL of PMSF (phenylmethylsulfonyl fluoride) were added, and the cells were sonicated in an ice bath, with a 3s sonication pause of 3s, and sonication was repeated 100 times; the cells were centrifuged at 12000rpm and 4℃ for 15min, the supernatant protein was removed, the precipitate was quickly washed three times with 1×PBS, and 30mL of 1×PBS (8M urea) and 30μL of... After β-mercaptoethanol, the precipitate was dissolved by pipetting with a disposable pipette for 45 min, then centrifuged at 12000 rpm and 4°C for 10 min. The supernatant was collected as inclusion body protein. The precipitate was resuspended in 10 mL of imidazole-free 8M urea solution, and sonicated at 100 W for 10 min in an ice-water bath. 20 μL of the sample was retained for electrophoresis. Proteins with His-tag were purified using Ni-NTA Resin. Figure 1 As shown, M: Marker, 1: Inclusion body protein, 2: Flow-through buffer, 3-4: Eluent. The purified recombinant protein was then dialyzed with dialysis buffers containing 6M, 4M, 3M, 2M, 1M, and 0M urea, respectively. The concentration of the purified protein was determined using a BCA protein concentration assay kit (Thermo, 23225).

[0017] S4. Using the recombinant protein purified in S3 as the antigen, rabbits were immunized a total of 4 times. The adjuvant and antigen were extracted at a 1:1 volume ratio. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second to fourth immunizations. Rabbits were injected subcutaneously at multiple sites, with a total injection volume of 0.5 mL per rabbit. The second immunization was performed on the 14th day after the first immunization, and the interval between the second and third immunizations was 7 days. On the 7th day after the third immunization, a small sample of serum was collected from the middle ear artery for testing. If the test was qualified, the rabbits were immunized again 7 days later. Seven days after the immunization, whole blood was collected, centrifuged at 12,000 rpm for 2 minutes, and the supernatant was collected. The antibody was purified by dialysis to obtain the SbCLRV-CP polyclonal antibody.

[0018] This embodiment also determined the titer of the SbCLRV-CP polyclonal antibody prepared above.

[0019] Dilute SbCLRV-CP antigen to 1 μg / mL with 1×PBST buffer, add 50 μL to each well of a polystyrene plate, incubate overnight at 4°C, discard the solution, wash once with 200 μL of 1×PBST wash buffer per well, add 60 μL of 1% BSA (bovine serum albumin, prepared with PBST) per well for blocking, incubate at 37°C for 1 h, discard the blocking solution; add 50 μL of serially diluted SbCLRV-CP polyclonal antibody (prepared in Example 1) from 250 to 1024000 times to the above-blocked wells, and set up positive control wells (positive serum) and negative control wells (BSA), incubate at 37°C for 1 h, then discard the blocking solution, wash twice with 200 μL of 1×PBST wash buffer per well, and add freshly diluted goat anti-rabbit... HRP secondary antibody (Hangzhou Huaan Biotechnology Co., Ltd., HA1001-100) (HRP-labeled, 1:5000, diluted with 1% BSA) was added to each well of an ELISA plate at a dose of 50 μL. The plate was incubated at 37°C for 45 min. The blocking buffer was then discarded, and the plate was washed three times with 200 μL of 1×PBST buffer per well. 100 μL of freshly prepared TMB substrate solution was added to each well, and the plate was incubated at 37°C for 5 min. The reaction was terminated by adding 50 μL of 2M sulfuric acid to each well. The reaction was then measured at OD... 450 The absorbance was measured at nm, and a negative control OD was used. 450 The antibody potency is 2.5 times the absorbance value of nm. Therefore, the SbCLRV-CP polyclonal antibody prepared in this example has a potency of 1:1024000.

[0020] Example 2

[0021] This example demonstrates the application of the SbCLRV-CP polyclonal antibody prepared in Example 1.

[0022] Dot-blot-ELISA (dot immunoenzyme staining method) specific detection: After weighing the soybean leaves, add 0.01 mol / L PBS (phosphate buffer) at a ratio of 1 g: 10 mL and grind them. Centrifuge at 8000 rpm for 3 min. Take 2 μL of supernatant and add it to an NC membrane (nitrocellulose membrane). Let it stand for 10 min. Block the NC membrane in PBST containing 5% skim milk powder for 30 min. Wash the membrane. Add the primary antibody (SbCLRV-CP polyclonal antibody prepared in Example 1) to 1×PBST containing 5% skim milk powder at a ratio of 1:5000. Place the membrane in the NC membrane and incubate at room temperature for 2 h. Wash the membrane. Place the NC membrane in goat anti-rabbit-HRP secondary antibody diluted 1×PBST at a ratio of 1:10000 and incubate at room temperature for 1 h. Wash the membrane. Add TMB chromogenic solution and observe the color development. The result is a qualitative result. Color development indicates a positive result, and no color development indicates a negative result.

[0023] The results of Dot-blot-ELISA are as follows: Figure 2 As shown, 1-34 are the samples to be tested, 35-36 are negative controls, and 37-38 are positive controls. Using soybeans infected with SbCLRV as the positive control, CK+ showed obvious color development, while using healthy soybeans as the negative control, CK- showed no color development. The samples to be tested can be accurately colored, indicating that the prepared SbCLRV-CP polyclonal antibody can specifically recognize soybean samples infected with SbCLRV virus.

[0024] In summary, the SbCLRV-CP polyclonal antibody prepared by this invention has high titer and a simple preparation method. It can be used to prepare a kit for detecting soybean yellow leafroll virus and can effectively overcome the shortcomings of existing detection technologies, such as complex operation and inability to perform large-scale detection. It has good application value and market prospects.

[0025] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a polyclonal antibody against the coat protein of soybean yellow leafroll virus, characterized in that, The method is as follows: S1. Extract total RNA from soybean leaves infected with soybean yellowing leafroll virus; S2. Using the total RNA extracted from soybean leaves in S1 as a template, the soybean yellowing leafroll virus capsid protein gene was amplified using the upstream primer SbCLRV-CP-F and the downstream primer SbCLRV-CP-R to obtain the SbCLRV-CP target gene fragment. The nucleotide sequence of the upstream primer SbCLRV-CP-F is shown in SEQ ID No:1; the nucleotide sequence of the downstream primer SbCLRV-CP-R is shown in SEQ ID No:

2. The nucleotide sequence of the SbCLRV-CP target gene fragment is shown in SEQ ID No:3; the amino acid sequence of the SbCLRV-CP target gene fragment is shown in SEQ ID No:

4. S3. The SbCLRV-CP target gene fragment obtained in S2 is ligated into the pET-28a(+) expression vector to obtain the recombinant expression vector pET28a-CP. Then, the recombinant expression vector pET28a-CP is transformed into Escherichia coli, induced to express, and then purified to obtain the purified recombinant protein. S4. Using the purified recombinant protein obtained in S3 as an antigen, immunize animals, collect whole blood, collect the supernatant, and purify it to obtain a polyclonal antibody against the outer shell protein of soybean yellow leafroll virus.

2. The method for preparing a polyclonal antibody against the coat protein of soybean yellowing leafroll virus according to claim 1, characterized in that, The purification method described in S3 is Ni column affinity chromatography.

3. The method for preparing a polyclonal antibody against the coat protein of soybean yellowing leafroll virus according to claim 1, characterized in that, The immunized animal mentioned in S4 is a rabbit, and the immunization was performed on the rabbit a total of 4 times.

4. The method for preparing a polyclonal antibody against the coat protein of soybean yellowing leafroll virus according to claim 1, characterized in that, The method for immunizing animals described in S4 is as follows: the antigen with a mass concentration of 1 mg / mL is mixed with the adjuvant at a volume ratio of 1:

1. The first immunization uses the complete adjuvant, and the second to fourth immunizations use the incomplete adjuvant.

5. The method for preparing a polyclonal antibody against the coat protein of soybean yellowing leafroll virus according to claim 1, characterized in that, The purification method described in S4 is dialysis.

6. The application of a polyclonal antibody against the coat protein of soybean yellowing leafroll virus prepared by the preparation method according to any one of claims 1-5, characterized in that, The polyclonal antibody against the outer shell protein of soybean yellow leafroll virus is used to prepare a kit for detecting soybean yellow leafroll virus.

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