Preparation method of Cap protein of parrot beak and feather disease virus

Abstract

The invention relates to the technical field of veterinary biological products, in particular to a preparation method of Cap protein of parrot beak and feather disease virus, which comprises the following steps: S1, on the basis of nucleotide sequences of PBFDV Cap protein, His tag, gp67 signal peptide and eGFP fluorescent protein, constructing expression vectors carrying insect cells pFastBac Dual-eGFP-gp67-His-Cap41-233; s2, pFastBac Dual-eGFP-gp67-His-Cap41-233 is transformed into a DH10BAC competent cell for transposition, a positive strain is obtained through a blue and white spot screening method, and a recombinant stem grain is extracted; s3, the Bacmid-eGFP-gp67-His-Cap41-233 recombinant baculovirus is subjected to transfection with an SF9 insect cell, and the recombinant baculovirus AcMNPV-eGFP-gp67-His-Cap41-233 is obtained, and the recombinant baculovirus AcMNPV-eGFP-gp67-His-Cap41-233 is subjected to transfection with the S4, the SF9 cells subjected to suspension culture are infected with the AcMNPV-eGFP-gp67-His-Cap41-233, the expression condition is optimized, and the target protein gp67-His-Cap41-233 is prepared, and the target protein gp67-His-Cap41-233 is obtained after the SF9 cells are subjected to suspension culture; and S5, purifying the target protein by using a Ni-NTA (nickel-nitrilotriacetic acid) affinity chromatography column, so as to obtain the high-purity target protein gp67-His-Cap41-233.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary biological products, and in particular to a method for preparing Cap protein of psittacine beak and feather disease virus. Background Art

[0002] Psittacine beak and feather disease virus (PBFD) is an infectious disease characterized by feather degeneration, malnutrition and beak deformity. The main clinical symptoms are feather atrophy and loss, beak and claw deformation, and random immunosuppression, and finally the death of the parrot. The disease is caused by Psittacine beak and feather disease virus (PBFDV), which is a member of the Circoviridae family together with porcine circovirus. PBFDV can infect different species of parrots of all ages, as well as other birds, and is widely distributed throughout the world. In 2009, my country first reported the occurrence of psittacine beak and feather disease virus in budgies.

[0003] PBFDV is a single-stranded DNA virus with a genome size of about 1.7 to 2.0 kb. The genome mainly includes two open reading frames in opposite directions, one encoding the replication-related protein (Rep) and the other encoding the capsid protein (Cap). The Cap protein has rich secondary structures and multiple regions with high antigenic index, which have potential B cell antigen epitopes.

[0004] At present, most of the reagents available on the market for psittacine beak and feather disease virus are detection reagents. There are few studies on PBFDV Cap protein, and there is no method for preparing high-purity Cap protein. Therefore, the present invention prepares PBFDV Cap protein, which lays the foundation for the next step of preparing vaccines and diagnostic reagents. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing the Cap protein of psittacine beak and feather disease virus.

[0007] (II) Technical solution

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing the Cap protein of psittacine beak and feather disease virus, comprising the following steps:

[0009] S1. Based on the nucleotide sequences of PBFDVCap protein, His tag, gp67 signal peptide and eGFP fluorescent protein, the insect cell expression vector pFastBac Dual-eGFP-gp67-His-Cap was constructed. 41～233 ;

[0010] S2. Transfer pFastBacDual-eGFP-gp67-His-Cap 41～233 Transform into DH10BAC competent cells for transposition, obtain positive strains by blue-white screening, and extract Bacmid-eGFP-gp67-His-Cap 41～233 Recombinant bacmid;

[0011] S3. Bacmid-eGFP-gp67-His-Cap 41～233 The recombinant bacmid was transfected into SF9 insect cells to obtain the recombinant baculovirus AcMNPV-eGFP-gp67-His-Cap 41～233 ;

[0012] S4. Transfect AcMNPV-eGFP-gp67-His-Cap 41～233 Infect SF9 cells cultured in suspension, optimize the expression conditions, and prepare the target protein gp67-His-Cap 41～233 ;

[0013] S5. Purify the target protein using Ni-NTA affinity chromatography column to obtain high-purity target protein gp67-His-Cap 41～233 .

[0014] Furthermore, the present invention is improved in that the method of step S1 comprises the following steps:

[0015] Sequence optimization was performed based on the nucleotide sequence of the Cap protein of the PBFDV-WF1 strain;

[0016] The Cap protein was truncated and expressed, and the gp67 signal peptide was designed to be added to the N-terminus of the protein, and the Kozak sequence was added before the start codon;

[0017] His-Cap was constructed by adding His tags to the C-terminus and the middle of the Cap protein nucleotide. 41～233 target sequence; at the same time, EcoRⅠ restriction site and HindⅢ restriction site were added to the 5' and 3' ends of the sequence respectively;

[0018] Connect eGFP-tag to pFastBac Dual vector;

[0019] The target sequence and pFastBacDual vector plasmid were double-digested, and the double-digested products were recovered by gel gel, and ligated with T4 DNA ligase at 16°C overnight to prepare the ligation product;

[0020] Sequencing confirmed the correct insertion of the target sequence, and finally constructed pFastBac Dual-eGFP-gp67-Cap 41～233 Expression plasmids.

[0021] Furthermore, the present invention is improved in that the method of step S1 comprises the following steps:

[0022] Take pFastBac Dual-eGFP-gp67-His-Cap 41～233 In the transposed DH10Bac competent cells, pick the blue-white screening plate containing three antibodies to screen the recombinant colonies, pick the white single colony, and perform dominant verification, and extract the recombinant bacmid after identification using M13 primers.

[0023] Furthermore, the present invention is improved in that the specification of the triple-antibody blue-white spot screening plate contains 50 μg / ml kanamycin sulfate, 7 μg / ml gentamicin sulfate, 7 μg / ml tetracycline hydrochloride, 40 μg / ml X-GAL, and 40 μg / ml IPTG.

[0024] Furthermore, the present invention is improved in that the expression conditions in step S4 are the virus infection MOI and the protein harvesting time.

[0025] Furthermore, the present invention is improved in that the method for purifying the target protein using the Ni-NTA affinity chromatography column comprises the following steps:

[0026] First, balance the nickel affinity chromatography column with Binding Buffer; wash the column with Binding Buffer, and then elute the impurities with Wash Buffer; elute the target protein with Elution Buffer containing 100-500 imidazole.

[0027] Furthermore, the present invention is improved in that the Bacmid-eGFP-gp67-His-Cap 41～233 The method for transfecting SF9 insect cells with recombinant bacmid is to use liposome transfection reagent.

[0028] Furthermore, the present invention has been improved in that the optimized virus infection MOI and protein harvesting time in step S4 are: the virus infection amount is 1 MOI, and the protein collection time is 72 hours.

[0029] (III) Beneficial effects

[0030] Compared with the prior art, the present invention provides a method for preparing the Cap protein of psittacine beak and feather disease virus, which has the following beneficial effects: the present invention is effective for the preparation of the recombinant PBFDV Cap protein. 41～233 The protein was efficiently expressed and purified in a soluble form, with a purity greater than 90%, laying a certain foundation for further serological diagnostic technology, polyclonal or monoclonal antibodies against Cap proteins, and subunit vaccine development, especially virus-like particle subunit vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The recombinant expression plasmid pFastBac Dual-eGFP-gp67-His-Cap 41～233 Structural diagram;

[0032] Figure 2 The plasmid is pFastBac Dual-eGFP-gp67-His-Cap 41～233 Double enzyme digestion map;

[0033] Figure 3 gp67-His-Cap 41～233 Protein expression form identification diagram, where 1 is the cell supernatant and 2 is the cell pellet;

[0034] Figure 4 gp67-His-Cap 41～233 Protein expression condition optimization diagram, in which lanes 1-3 in Figure A and B are 1MOI 72h, 96h, and 120h respectively; lanes 4-6 are 2MOI 72h, 96h, and 120h respectively; lanes 7-9 are 3MOI 72h, 96h, and 120h respectively;

[0035] In Figure C and D, lanes 1-3 are 0.1MOI for 72h, 96h, and 120h, respectively; lanes 4-6 are 0.2MOI for 72h, 96h, and 120h, respectively; lanes 7-9 are 0.3MOI for 72h, 96h, and 120h, respectively;

[0036] Figure 5 Purified gp67-His-Cap 41～233 SDS-PAGE identification diagram of the protein, where lanes 1-5 are the washed proteins; lanes 6-9 are the eluted proteins;

[0037] Figure 6 gp67-His-Cap 41～233 Western-blot identification results of purified protein. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The present invention provides a method for preparing the Cap protein of psittacine beak and feather disease virus, comprising the following steps:

[0040] 1 Materials and methods

[0041] 1.1 Plasmids and main reagents

[0042] Gene synthesis and sequencing services were provided by Sangon Biopharma, primer synthesis was provided by GenScript Biopharma, common reagents and experimental instruments were provided by Nanjing Qianyuanhao, and the sources of other experimental materials are shown in the table below.

[0043] Table 1 Sources of relevant reagents in this experiment

[0044]

[0045]

[0046] 1.2 Construction and identification of recombinant plasmids

[0047] 1.2.1 Gene synthesis

[0048] The designed gene was handed over to Sangon Biotech for optimization according to the codon preference of insect cells and synthesized into the PUC18 vector.

[0049] 1.2.2 Construction of recombinant plasmid

[0050] Based on the nucleotide sequence of the Cap protein of the PBFDV-WF1 strain (41-223 bp), the codon preference of Escherichia coli was optimized, and the nucleotide sequence of the His tag was added to the N-terminus of the Cap protein to construct the His-Cap 41～233 Target sequence and His-Cap 41～233 The gp67 sequence and the translation initiation signal Kozak sequence were added to the N-terminus of the target sequence. At the same time, EcoRⅠ (GAATTC) and HindⅢ (AAGCTT) restriction sites were added to the 5' and 3' ends of the sequence, respectively. In order to facilitate the observation of the growth state of the recombinant baculovirus in insect cells and determine the virus titer, eGFP fluorescent protein was added in this experiment. XhoⅠ (CTCGAG) and Kozak sequence were added to the 5' end of the fluorescent protein. KpnⅠ (GGTACC) was added to the 3' end.

[0051] pFastBacDual and eGFP-PU18 were double-digested with XhoⅠ restriction enzyme and KpnⅠ restriction enzyme, respectively. The digested products were subjected to nucleic acid gel electrophoresis, the target band was cut out, and the target band was recovered by gel extraction kit D2500. The recovered products were connected with T4 DNA Ligase, and the ligation products were transformed into Trans5αChemically Competent Cell using heat shock method. The transformed competent cells were spread on LB agar plates containing Amp (100μg / ml) and cultured at 37℃ overnight. Single colonies were picked and verified by bacterial liquid PCR using eGFP-specific primers. The verified correct strain was shaken overnight to extract the pFastBac Dual-eGFP recombinant plasmid.

[0052] The target fragment and pFastBacDual-eGFP vector plasmid were double-digested with EcoRⅠ and HindⅢ, respectively, and the double-digested products were recovered by gel recovery and ligated overnight at 16°C using T4 DNA ligase. The ligated products were then transformed into Trans5α competent cells and cultured overnight at 37°C. Single colonies were picked and verified by bacterial liquid PCR using Cap-specific primers and eGFP-specific primers, respectively. The verified correct strains were shaken overnight, and the recombinant plasmids were extracted and sequenced. Finally, pFastBacDual-eGFP-gp67-His-Cap was constructed. 41～233 Recombinant plasmid.

[0053] 1.2.3 Construction of recombinant bacmid

[0054] Change pFastBac Dual-eGFP-gp67-His-Cap 41～233 The recombinant plasmid was transferred into DH10BacChemically Competent Cell by heat shock method, and the recombinant colonies were screened using triple-antibody blue-white screening plates (containing 50 μg / ml kanamycin sulfate, 7 μg / ml gentamicin sulfate, 7 μg / ml tetracycline hydrochloride, 40 μg / ml X-GAL, 40 μg / ml IPTG). White single colonies were picked and subjected to dominant verification. The recombinant bacmid was extracted after identification using M13 primers.

[0055] 1.3 Analysis of soluble expression of recombinant protein

[0056] The recombinant bacmid that has been sequenced correctly was transfected into SF9 cells using LipoInsectTM transfection reagent. After 3-5 days of infection, the cells were placed under a fluorescence microscope to observe the fluorescence reaction and cytopathic effect of the cells. The cells were harvested, broken, and then SDS-PAGE and WB were performed to verify the solubility of the protein. At this time, the virus was the P1 generation virus. P1 was inoculated into P2 for storage of the virus solution.

[0057] 1.4 Optimization of recombinant protein expression conditions

[0058] 1.4.1 Optimization of optimal inoculation dose and optimal expression time

[0059] The recombinant baculovirus was inoculated at a density of 2×10 6 The cell supernatants were collected at 72h, 96h and 120h from SF9 suspension cells with a concentration of 100 / ml and verified by SDS-PAGE and WB.

[0060] 1.5 Purification of recombinant protein

[0061] The recombinant gp67-His-Cap 41～233 Protein purification. First, recombinant gp67-His-Cap 41～233 The protein was centrifuged at 1000 rpm / min for 10 min and the supernatant was collected. The nickel affinity column was equilibrated with Binding Buffer, and the protein supernatant was passed through the nickel affinity column. The column was washed with Binding Buffer, and then the impurities were eluted with Wash Buffer. Finally, the target protein was eluted with Elution Buffer containing 100, 150, 200 and 500 imidazoles in sequence. Finally, the recombinant gp67-His-Cap was analyzed by 12% SDS-PAGE. 41～233 Purity of protein.

[0062] 1.6 Western-blot identification

[0063] The purified recombinant gp67-His-Cap was stained with HRP-labeled 6×His-Tag monoclonal antibody as the primary antibody. 41～233 The protein was analyzed by Western-blot, and the results were observed after color development with color developing solution.

[0064] 2 Results

[0065] 2.1 Recombinant expression plasmid pFastBacDual-eGFP-gp67-His-Cap 41～233 Construction and enzyme digestion identification

[0066] Recombinant expression plasmid pFastBac Dual-eGFP-gp67-His-Cap 41～233 After double digestion with EcoRⅠ and HindⅢ, two specific bands were found at approximately 750 bp and 5000 bp, which were consistent with the expected size ( Figure 2 ), and then the bacterial solution was PCR tested and sequenced. The sequencing results showed that the nucleotide sequence was correct and the recombinant expression of pFastBac Dual-eGFP-gp67-His-Cap 41～233 Build successful.

[0067] 2.2 Recombinant gp67-His-Cap 41～233 Protein expression identification

[0068] Recombinant expression plasmid pFastBac Dual-eGFP-gp67-His-Cap 41～233 After transposition into bacmid, SF9 cells were infected, and the cell supernatant and precipitate were collected. A specific band was found at about 25 kDa after 12% SDS-PAGE analysis, and the size was consistent with the theoretical value. Comparison of the supernatant and the precipitate revealed that a large amount of target protein was present in the supernatant, so the Cap protein was expressed soluble ( Figure 3 ).

[0069] 2.3gp67-His-Cap 41～233 Optimization of expression conditions

[0070] The gp67-His-Cap 41～233 The virus was inoculated into SF9 cells, and the expression conditions such as the inoculation amount and harvesting time were optimized. After collecting the cell supernatant, it was identified by 12% SDS-PAGE. The results showed that the optimal inoculation amount was 1 MOI ( Figure 4 A) The best harvest time is 72h ( Figure 4 A).

[0071] 2.4 Soluble recombinant gp67-His-Cap 41～233 Protein purification

[0072] According to the optimal expression conditions, the recombinant gp67-His-Cap 41～233 After the protein was expressed, the supernatant was collected for purification. The target protein was obtained by elution with a buffer solution. The purified sample was identified by 12% SDS-PAGE electrophoresis, and a specific protein band was observed at about 25 kDa ( Figure 5 ), consistent with the expected size, and the purified gp67-His-Cap 41～233 The purity of the protein can reach more than 90% ( Figure 5 ).

[0073] 2.5 Recombinant gp67-His-Cap 41～233 Protein Western-blot identification

[0074] The purified gp67-His-Cap 41～233 Western-blot analysis of proteins showed that the recombinantly expressed gp67-His-Cap 41～233 The protein was able to react with the 6×His tag monoclonal clone, and a specific band was observed at about 25 kDa, which was consistent with the expected result ( Figure 6 ). Therefore, pFastBac Dual-eGFP-gp67-His-Cap 41～233 The bacmid can correctly express gp67-His-Cap 41～233 protein.

[0075] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing the Cap protein of psittacine beak and feather disease virus, characterized in that: The following steps are involved: S1. Based on the nucleotide sequences of PBFDV Cap protein, His tag, gp67 signal peptide and eGFP fluorescent protein, the insect cell expression vector pFastBac Dual-eGFP-gp67-His-Cap was constructed. 41～233 ; S2. Combine pFastBac Dual-eGFP-gp67-His-Cap 41～233 Transform into DH10BAC competent cells for transposition, obtain positive strains by blue-white screening, and extract Bacmid-eGFP-gp67-His-Cap 41～233 Recombinant bacmid; S3. Bacmid-eGFP-gp67-His-Cap 41～233 The recombinant bacmid was transfected into SF9 insect cells to obtain the recombinant baculovirus AcMNPV-eGFP-gp67-His-Cap 41～233 ; S4. Transfect AcMNPV-eGFP-gp67-His-Cap 41～233 Infect SF9 cells cultured in suspension, optimize the expression conditions, and prepare the target protein gp67-His-Cap 41～233 ; S5. Purify the target protein using Ni-NTA affinity chromatography column to obtain high-purity target protein gp67-His-Cap 41～233 .

2. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 1, characterized in that: The method of step S1 comprises the following steps: Sequence optimization was performed based on the nucleotide sequence of the Cap protein of the PBFDV-WF1 strain; The Cap protein was truncated and expressed, and the gp67 signal peptide was designed to be added to the N-terminus of the protein, and the Kozak sequence was added before the start codon; His-Cap was constructed by adding His tags to the C-terminus and the middle of the Cap protein nucleotide. 41～233 target sequence; at the same time, EcoRⅠ restriction site and HindⅢ restriction site were added to the 5' and 3' ends of the sequence respectively; Connect eGFP-tag to pFastBac Dual vector; The target sequence and pFastBac Dual vector plasmid were double-digested, and the double-digested products were recovered by gel gel, and ligated with T4 DNA ligase at 16°C overnight to prepare the ligation product; Sequencing confirmed the correct insertion of the target sequence, and finally constructed pFastBac Dual-eGFP-gp67-Cap 41～233 Expression plasmids.

3. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 1, characterized in that: The method of step S1 comprises the following steps: Take pFastBac Dual-eGFP-gp67-His-Cap 41～233 In the transposed DH10Bac competent cells, pick the blue-white screening plate containing three antibodies to screen the recombinant colonies, pick the white single colony, and perform dominant verification, and extract the recombinant bacmid after identification using M13 primers.

4. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 3, characterized in that: The specifications of the triple-antibody blue-white screening plate are 50 μg / ml kanamycin sulfate, 7 μg / ml gentamicin sulfate, 7 μg / ml tetracycline hydrochloride, 40 μg / ml X-GAL, and 40 μg / ml IPTG.

5. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 1, characterized in that: The expression conditions in step S4 are virus infection MOI and protein harvest time.

6. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 1, characterized in that: The method for purifying the target protein using a Ni-NTA affinity chromatography column comprises the following steps: First, balance the nickel affinity chromatography column with Binding Buffer; wash the column with Binding Buffer, and then elute the impurities with Wash Buffer; elute the target protein with Elution Buffer containing 100-500 imidazole.

7. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 1, characterized in that: Bacmid-eGFP-gp67-His-Cap 41～233 The method for transfecting SF9 insect cells with recombinant bacmid is to use liposome transfection reagent.

8. The method for preparing the Cap protein of psittacine beak and feather disease virus according to claim 5, characterized in that: The optimized virus infection MOI and protein harvesting time in step S4 are: the virus infection amount is 1 MOI, and the protein collection time is 72 hours.

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