CN115073558A - Recombinant African swine fever virus DP96R subunit protein and preparation method and application thereof - Google Patents
Recombinant African swine fever virus DP96R subunit protein and preparation method and application thereof Download PDFInfo
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- CN115073558A CN115073558A CN202110261350.3A CN202110261350A CN115073558A CN 115073558 A CN115073558 A CN 115073558A CN 202110261350 A CN202110261350 A CN 202110261350A CN 115073558 A CN115073558 A CN 115073558A
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- african swine
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- 230000003612 virological effect Effects 0.000 description 1
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Abstract
The invention discloses a recombinant African swine fever virus structural protein DP96R subunit protein, a preparation method and an application thereof, wherein the amino acid sequence of the recombinant African swine fever virus structural protein DP96R subunit protein is shown as SEQ ID NO.3, and the preparation method comprises the following steps: 1) cloning the coding gene sequence of the recombinant African swine fever virus DP96R protein shown in SEQ ID No.1 after codon optimization into a prokaryotic expression vector to obtain a recombinant plasmid containing the coding gene of the African swine fever virus DP96R subunit protein; 2) then the recombinant plasmid containing the African swine fever virus DP96R subunit protein coding gene is transformed into an escherichia coli competent cell to obtain recombinant engineering bacteria; 3) obtaining a highly expressed strain by culturing and screening the strain of the recombinant engineering bacteria in the step 2); 4) fermenting and culturing the highly expressed strain in the step 3), and purifying to obtain the recombinant soluble fusion protein of the subunit DP96R of the African swine fever virus. The African swine fever virulence related protein DP96R subunit protein provided by the invention can be produced industrially in a large scale, and the preparation method is simple, the cost is low, and the African swine fever virulence related protein can reach the existing national standard.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock. Relates to a preparation method and application of African swine fever DP96R protein.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African Swine Fever Virus (ASFV), with a 100% incidence and mortality. The swine is infected with African swine fever virus, has the clinical symptoms of skin congestion, organ hemorrhage and high fever, is the only mammalian host of ASFV natural infection, comprises domestic swine and wild swine, particularly domestic swine, has extremely high susceptibility and has great influence on animal husbandry. The world animal health organization classifies the zoonosis as a type A epidemic disease, and the world also classifies the zoonosis as a type infectious disease.
The disease was first identified to occur in kenia countries in africa in 1921, and has been a tremendous shock to the swine industry in africa and even in many countries around the world. Since 8 months in 2018, outbreaks in a plurality of provinces of China bring serious economic losses to the pig industry in China. Although scholars at home and abroad do a lot of research work on African swine fever, the research finds that: the conventional African swine fever inactivated vaccine has a non-obvious effect, and the weak virus vaccine has a poor protection effect and safety and is easy to cause virus dispersion. At present, no vaccine for effectively preventing the African swine fever and a medicament for treating the African swine fever are found in the world, and the development and production of a novel vaccine for preventing the African swine fever are urgently needed.
ASFV virus is an arbovirus DNA virus with an envelope. The virus particles are in an icosahedral symmetrical structure, the average diameter is 200nm, and the surfaces of the virus particles are covered by the sacculus membranes containing glycolipids. The viral genome is double-stranded linear DNA with the size of 170-190kb, and the whole genome has about 150 ORFs and encodes 150-200 proteins. The UK gene is located in the right variable region of the African swine fever virus genome, contains 92-156 amino acids, is a newly discovered gene and comprises 4-10 tandem repeat regions. The UK gene has highly conserved sequence in genomes of different virus strains, belongs to virus virulence related genes and is one of important factors causing the morbidity of domestic pigs, and the pathogenic ASFV deletion UK gene does not influence the growth of viruses in macrophages in vitro, but greatly reduces the virulence of the pigs. Research shows that the toxicity of ASFV is greatly weakened after screening out UK gene, and the ASFV can be used for research and development of attenuated vaccine; the DP96R protein is a virus virulence protein encoded by the UK gene, has a size of about 15kD, and is mainly secreted in the early stage of virus infection, and on the other hand, the DP96R protein is probably a good protective antigen. In the absence of a current possibility for large-scale production of inactivated or attenuated vaccines, it would be of great importance to identify a method for producing the immunogenic protein of the virus in order to study a vaccine capable of preventing the disease or an agent with a rapid diagnosis of the virus for the control of african swine fever.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the subunit protein of African swine fever DP 96R. The invention also provides a preparation method of the African swine fever DP96R subunit protein.
In order to achieve the purpose, the invention provides a recombinant African swine fever virus DP96R subunit protein, wherein the recombinant African swine fever virus DP96R subunit protein is an African swine fever virus virulence protein, and the amino acid sequence of the recombinant African swine fever virus DP96R subunit protein is shown as SEQ ID NO. 3.
According to one aspect of the present invention, the present invention provides an optimized OPTI-DP96R nucleotide sequence for expressing DP96R protein in E.coli. In order to be able to express DP96R protein in Escherichia coli with high efficiency, the invention analyzes DP96R gene according to the published sequence on GenBank: FR682468.1, firstly optimizes the codon preference of the codon coded by DP96R gene in prokaryotic expression, and secondly optimizes the GC content of DP96R gene and the stability of mRNA in Escherichia coli.
In a preferred technical scheme of the invention, the gene sequence (the nucleotide sequence of OPTI-DP96R) of the recombinant African swine fever virus DP96R protein after codon optimization is shown as SEQ ID NO 1.
The gene for encoding the subunit protein of the African swine fever virus DP96R as described above, preferably, the gene sequence of the African swine fever virus DP96R protein before codon optimization is shown in SEQ ID NO. 2.
To facilitate purification of the fusion protein using affinity chromatography, it is preferred according to the present invention that one of the tags poly-His, FLAG, c-myc, HA and poly-Arg is attached to the amino terminus or the carboxy terminus of the amino acid sequence shown in SEQ ID NO. 3.
According to another aspect of the invention, the invention provides a preparation method of a recombinant subunit protein of African swine fever virus DP96R, which comprises the following steps:
1) cloning the coding gene sequence of the recombinant African swine fever virus DP96R protein shown in SEQ ID No.1 after codon optimization into a prokaryotic expression vector to obtain a recombinant plasmid containing the coding gene of the African swine fever virus DP96R subunit protein;
2) then the recombinant plasmid containing the African swine fever virus DP96R subunit protein coding gene is transformed into an escherichia coli competent cell to obtain recombinant engineering bacteria;
3) obtaining a highly expressed strain by culturing and screening the strain of the recombinant engineering bacteria in the step 2);
4) fermenting and culturing the highly expressed strain in the step 3), and purifying to obtain the recombinant soluble protein of the subunit DP96R of the African swine fever virus.
In a preferred technical scheme of the invention, the prokaryotic expression vector is selected from one of pET30, pET28a, pBAD, pcold, pQE and pKK vectors. More preferably, the prokaryotic expression vector is selected from pET 30.
In a preferred embodiment of the invention, in step 2), the E.coli strain is selected from one of BL21(DE3), BL21star (DE3) and arcticixpress. More preferably, the E.coli strain used in step 2) is arcticixpress.
In a preferred embodiment of the invention, in step 3), IPTG is used in a concentration of 0.2 mmol/L.
In a preferred technical scheme of the invention, in the step 4), a lysis solution is used: 20mM Tris (pH 8.7), 500mM NaCl, 0.2% Triton X-114, 5% glycerol, 1mM β -ME.
According to a further aspect of the invention, the invention provides an application of the recombinant soluble fusion protein of the subunit DP96R of the African swine fever virus in preparation of vaccines for diagnosis, prevention and treatment of African swine fever.
Compared with the prior art, the expression sequence, the expression vector and the corresponding purification preparation method disclosed by the invention overcome the defects in the prior art, and solve the problems that a large amount of DP96R protein cannot be directly and soluble expressed in Escherichia coli and the yield is low. The invention can directly express DP96R in Escherichia coli in a soluble way, overcomes a plurality of problems in the prior art, and has simple preparation method and low cost.
Drawings
FIG. 1 shows the alignment results before and after the optimization of the DP96R gene sequence.
FIG. 2 shows a map of the plasmid pET30-OPTI-DP 96R.
FIG. 3 shows the results of double-restriction enzyme identification of pET30-OPTI-DP 96R: m is a DNA Marker: DL5000 Marker; the plasmid No. 1-2 NdeI/PvuI is subjected to double enzyme digestion, the sizes of the bands are 4133bp and 1417bp respectively, and the enzyme digestion result is correct.
FIG. 4 shows SDS-PAGE detection of recombinant DP96R protein expression before and after induction: m is Marker, 1 is pre-induction supernatant, 2 is pre-induction precipitate, 3 is post-induction supernatant, 4 is post-induction precipitate, and the arrow points to DP96R protein. As can be seen from the figure, the protein of interest is expressed in soluble form.
FIG. 5 shows the SDS-PAGE purification assay of recombinant DP 96R: 1 is a protein Marker, and 2 is a purified DP96R protein.
FIG. 6 shows the Western-blot detection results after DP96R protein purification: 1 is a protein Marker and 2 is a DP96R protein.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.
The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.
Example 1 DP96R protein expression and preparation
1.1 selection of African Swine fever DP96R protein
The African swine fever virulence related protein DP96R is a polypeptide encoded by the virulence gene UK and is secreted mainly in the early stages of viral infection. It is indicated that the deletion of UK gene in pathogenic ASFV does not affect the growth of African swine fever virus in macrophage in vitro, but its toxicity to pig is greatly reduced. Therefore, UK is a new virulence related gene, and the DP96R protein expressed by the UK is used as an antigen, so that the UK gene is deleted to prepare a attenuated vaccine with weaker virulence, and the infection of African swine fever can be well prevented and controlled, although the DP96R protein is reported to be expressed in a prokaryotic expression system. However, the protein can be obtained by soluble expression and purification in a prokaryotic expression system, which is an important technical problem to be solved by the invention.
In order to facilitate the purification of the subunit DP96R protein, a tag as shown in table one can be attached to the amino terminus or the carboxy terminus of the amino acid sequence as shown in SEQ ID No.3, specifically in this example Poly-His is added to the carboxy terminus, which is attached to the carboxy terminus of the amino acid sequence as shown in SEQ ID No. 3.
TABLE 1 tags and their amino acid sequences
1.2 African Swine fever DP96R protein codon optimization
In the laboratory, the nucleotide sequence of UK for coding African swine fever DP96R protein is subjected to codon optimization by taking an African swine fever strain subtype reported in China in 2018 and referring to a Georgia 2007/1 complete gene sequence (GenBank: FR682468.1) as a template to obtain an OPTI-DP96R sequence, as shown in SEQ ID NO.1, and the sequence synthesis work is finished by Nanjing Kingjinsri Biotech Co. As shown in FIG. 1, the nucleotide sequences before and after optimization were 27.4% different.
1.3 pET30-OPTI-DP96R recombinant plasmid construction
1.3.1 PCR amplification of fragment of interest OPTI-DP96R
1.3.1.1 PCR reaction
(1) Primer design and Synthesis
Upstream primer 5'-CCATATGGCGAGCACCCACGAC-3'
Downstream primer 5'-CTCGAGTTAGTGGTGGTGGTGGTGGTGGTTGTTTTTCTGGATG-3'
(2) Sample loading system 50 μ L, as shown in the following table:
PCR amplification procedure:
1.3.1.2 PCR products for gel recovery
(1) Marking a sample collection EP tube, an adsorption column and a collection tube;
(2) weighing the weight of the marked empty EP pipe, and recording the numerical value;
(3) a single DNA band of interest was carefully excised from the agarose gel on a gel cutter with a scalpel and placed into a clean 1.5mL centrifuge tube;
(4) adding 600 mu L of PC buffer into the 1.5mL centrifuge tube in the step (3), placing in a water bath at 50 ℃ for about 5min, and turning the centrifuge tube up and down continuously and gently to ensure that the gel block is fully dissolved;
(5) column balancing: adding 500 μ L of balance liquid BL into adsorption column CB2 (the adsorption column is placed into the collection tube in advance), centrifuging at 12,000rpm/min for 1min, pouring off waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(6) adding the solution obtained in the step (5) into an adsorption column CB2, standing for 2min at 10,000rpm/min, centrifuging for 30s, pouring out waste liquid in a collecting pipe, and then putting the adsorption column CB2 into the collecting pipe;
(7) adding 600 mu L of rinsing liquid PW buffer into the adsorption column, standing for 3min, centrifuging at 10,000rpm/min for 30s, pouring off waste liquid in the collecting tube, and putting the adsorption column CB2 into the collecting tube;
(8) repeating the step (7);
(9) centrifuging with an empty adsorption column at 12,000rpm/min for 2min, removing rinsing liquid as much as possible, standing the adsorption column at room temperature for 10min, and completely air drying;
(10) placing adsorption column CB2 in a collecting tube, suspending and dropwise adding 50 μ L of precipitation buffer (preheated at 65 ℃) to the middle position of an adsorption film, standing for 3min, centrifuging at 12,000rpm/min for 2 min;
(11) taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, covering the centrifuge tube with a cover, and keeping the DNA sample in the centrifuge tube;
(12) and (3) storing the DNA sample in the step 11 at 4 ℃, and preparing an agarose gel electrophoresis identification gel to recover the DNA fragment.
1.3.2 PCR product and vector double digestion reaction
(1) The 1.5mL EP tubes needed were labeled and loaded and mixed in the 1.5mL EP tubes according to the following table: 50 μ L reaction System
(2) And (3) placing the 1.5mL EP tube in the step (1) into a corresponding enzyme constant-temperature water bath kettle with the optimal temperature, and carrying out water bath for 2-3 h.
Recovering the double enzyme digestion product glue: taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment in the double enzyme digestion system by the same method as that of the PCR product gel recovery in the 1.2.1.
1.3.3 ligation reactions
(1) A plurality of clean 1.5mL EP tubes are prepared, marked and placed on an EP tube frame for standby.
(2) The sample was loaded and mixed in a 1.5mL EP tube as described in the following table.
(3) After sample adding is finished according to the table in the step (2), placing each 10 mu l reaction system in a low-temperature cooling liquid circulator at the temperature of 16 ℃ for water bath for 10-16 h;
(4) taking out the EP tube in the step (3), placing the EP tube in a water bath kettle at 65 ℃, and carrying out water bath for 15 min;
(5) taking out the EP tube in the step (4), and storing at 4 ℃.
1.3.4 conversion reaction
(1) Quickly adding 10 μ L of the ligation reaction solution into 100 μ L of competent cells, uniformly mixing by blowing, and carrying out ice bath for 30 min;
(2) taking out the sample tube, placing in water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2 min;
(3) taking out the sample tube, adding 600 mu L of liquid LB culture medium into the sample tube in a super-clean workbench, then placing the sample tube in a constant temperature shaking table at 37 ℃, and culturing for 1h at 220 rpm/min;
(4) coating a plate: and (4) taking out the sample tube in the step (3), centrifuging at room temperature for 8,000rpm/min for 2min, removing 600 mu L of supernatant liquid, re-suspending the thalli at the bottom of the tube by the residual supernatant liquid, putting the re-suspended bacterial liquid into the center of a corresponding transformation plate, and uniformly spreading the bacterial liquid in the center of the transformation plate by using a bacteria coating rod.
(5) The flat plate in the transformation step (4) is placed in a biochemical constant-temperature incubator, and is cultured for 1h at 37 ℃, and then the transformation flat plate is inverted and cultured for 15 h;
(6) the transformation results were observed.
1.3.5 plasmid extraction and double restriction enzyme identification
1.3.5.1 plasmid extraction
(1) Picking single clone from the conversion plate by a 10-microliter pipette tip to 5mL LB liquid culture medium containing kanamycin resistance, shaking the strain at 37 ℃ and 220rpm/min overnight;
(2) transferring the bacterial liquid into a 1.5mL EP tube, centrifuging at room temperature at 12,000rpm/min for 2min, and removing the supernatant;
(3) adding 250 mu L of plasmid extraction reagent P1 buffer into the EP tube in the step (2), and completely suspending the thalli;
(4) adding 250 mu L P2 buffer into the solution in the step (3), immediately and gently inverting the centrifuge tube for 5-10 times, uniformly mixing, and standing at room temperature for 2-4 min;
(5) adding 350 mu L P3 buffer into the solution in the step (4), immediately and gently inverting the centrifuge tube for 5-10 times and uniformly mixing; standing at room temperature for 2-4 min;
(6) centrifuging the solution in the step (5) at room temperature, and carrying out centrifugation at 14,000rpm/min for 10 min;
(7) transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging at room temperature for 30s at 12,000rpm/min, and pouring out liquid in a collecting pipe;
(8) adding 500 μ L Buffer DW1 into the center of the adsorption column, centrifuging at room temperature at 12,000rpm/min for 30s, and pouring off the liquid in the collection tube;
(9) adding 500 μ L wash solution into the center of the adsorption column, centrifuging at room temperature, 12,000rpm/min for 30s, pouring off the liquid in the collection tube, and repeating once;
(10) empty adsorption column, centrifuge at room temperature, 12,000rpm, 2 min.
(11) The adsorption column was placed in a clean 1.5mL centrifuge tube, 30. mu.L of Elution buffer was added to the center of the adsorption membrane, and the mixture was allowed to stand at room temperature for 5min, centrifuged at room temperature, 12,000rpm, and centrifuged for 2 min. The DNA solution in the tube was preserved.
1.3.5.2 double restriction enzyme identification
(1) The 1.5mL EP tubes were labeled for use and loaded as follows: 20 μ L reaction System
(2) Putting the EP tube 20 mu L reaction system in the step (1) into a constant-temperature water bath kettle at 37 ℃ and carrying out water bath for 2 h.
(3) Carrying out agarose gel electrophoresis on the double enzyme digestion system sample in the step (2), and checking whether the size of the inserted fragment is correct; the results are shown in FIG. 3: the plasmid No. 1-2 NdeI/PvuI is subjected to double enzyme digestion, the sizes of the bands are 4133bp and 1417bp respectively, and the enzyme digestion identification construction is correct.
(4) Clones with correct inserts were selected for sequencing by the sequencing company. And (4) storing the plasmid with the correct sequencing result for later use.
1.4 African Swine fever DP96R protein expression
1.4.1 transformation of E.coli arcticixpress (DE3)
(1) 1 μ L of plasmid (extracted from 1.3.5.1) was added to 100 μ L of E.coli arcticixpress competent cells and ice-cooled for 30 min;
(2) heat shock at 42 ℃ for 90 s;
(3) ice-bath for 2 min;
(4) adding 500 mu L of non-resistant LB culture solution into a super clean bench;
(5) shaking at 37 ℃ and 220rpm for 1 h;
(6) 100 μ L of the bacterial solution was applied to a kanamycin-resistant LB plate, cultured overnight at 37 ℃, and a single clone (E. coli arctic xpress pET30-OPTI-DP96R) was picked up, activated and stored at-80 ℃ with glycerol.
1.4.2 Small inducible expression
(1) Activation of the glycerol storage tube strain: the E.coli arcticixpress pET30-OPTI-DP96R strain was thawed in a glycerol storage tube, the bacterial suspension in the glycerol tube was picked up with an inoculating loop and streaked on a kanamycin-resistant plate (50. mu.g/mL), and cultured overnight at 37 ℃.
(2) Selecting bacteria and activating: picking the monoclonal on the cultured plate to 3mL kanamycin-resistant LB culture medium, carrying out shake cultivation at 20 ℃ and 220r/min for 8-9 h until OD 600 0.5 to 0.8;
(3) fermentation and inoculation: inoculating 150 mu L of the activated bacterial suspension into 15mL of Carna resistance LB culture medium, and culturing at 20 ℃ and 220 r/min;
(4) cooling and inducing: when OD is reached 600 When the concentration reaches 0.6-0.8, 5mL of bacterial liquid is taken out, centrifuged at 12,000rpm for 5min, and the thalli are preserved at the temperature of-20 ℃, namely before induction. The remaining bacterial solution was placed in an ice-water bath for 10min, and 1. mu.L of 1M IPTG was added to a final concentration of 0.1mM IPTG. Reducing the temperature of the shaking table to 20 ℃, and inducing for 24 hours;
(5) and (3) collecting thalli: after the fermentation was completed, OD was measured 600 Collecting thallus in the same amount as before inducing, centrifuging at 12,000rpm for 5min, collecting thallus and storing at-20 deg.c to obtain the post inducing thallus.
The results of SDS-PAGE for inducible expression are shown in FIG. 4, where M is Marker, 1 is the pre-induction supernatant, 2 is the pre-induction precipitate, 3 is the post-induction supernatant, 4 is the post-induction precipitate, and the arrows point to DP96R protein. As can be seen from the figure, the protein of interest is expressed in soluble form.
1.4.3 Mass inducible expression
(1) Activation of the glycerol storage tube strain: thawing the strain in a glycerol storage tube, picking the bacterial suspension in the glycerol tube by using an inoculating loop, streaking the bacterial suspension on a kanamycin-resistant plate, and culturing at 37 ℃ overnight.
(2) Selecting bacteria and activating: selecting a monoclonal on the cultured plate, adding the monoclonal into 3mL of a kanamycin-resistant LB culture medium, and carrying out shake culture at 37 ℃ and 220r/min for 5-6 h until OD600 reaches 0.5-0.8;
(3) seed liquid culture: inoculating 150 mu L of the activated bacterial suspension into 150mL of a kanamycin-resistant LB culture medium, and culturing at 37 ℃ and 220r/min for 9-10 h;
(4) preparing a fermentation culture medium: preparing a fermentation medium component 1 according to a fermentation medium formula, placing in a 3L fermentation tank, installing an assembled fermentation tank, preparing a fermentation medium component 2 and a supplement medium in a blue-mouthed bottle, and sterilizing with high-pressure steam at 121 deg.C for 20 min.
(5) Setting fermentation parameters: agit 400 r/min; temperature 37 ℃; pH 7.00; DO 40; air 100 percent; gasflow2.0;
(6) fermentation and inoculation: 450mL of fermentation medium fraction 2, 1mL of fermentation medium fraction 3, 200. mu.L of antifoam, 3mL of kanamycin antibiotic (50. mu.g/mL) were added to the fermentor at the inoculation port; after the tank is stabilized, 150mL of cultured seed liquid is inoculated to 3L of fermentationCarrying out fermentation tank amplification culture in the culture medium for 5-6 h to OD 600 The value is 12-14;
(7) cooling and inducing: setting temperature parameters, reducing the temperature of the fermentation tank to 20 ℃, sampling, adding 0.9mL of IPTG (1M) until the final concentration of the IPTG is 0.3mM, and carrying out induced culture at 20 ℃ for 12 h;
(8) fermentation and material supplement: fermenting and culturing to OD 600 And when the temperature reaches 17-19%, continuously supplementing the supplemented medium at a speed of 5% (firstly, uniformly mixing the components 1 and 2 of the supplemented medium).
(9) And (3) collecting thalli: after the fermentation is finished, collecting fermentation liquor, centrifuging for 10min at 8000r/min, collecting thallus, and storing at-20 deg.C.
Wherein, the culture medium used in the process is as follows:
the fermentation medium comprises 1 part of yeast powder 10g/L, tryptone 20g/L and KH 2 PO 4 1.14g/L，K 2 HPO 4 0.9g/L，(NH 4 ) 2 SO 4 3.0g/L，MgSO 4 ·7H 2 O0.3 g/L, NaCl 5g/L, pH 7.0; (3L of each component of the culture medium is weighed, and purified water is added to the culture medium to fix the volume to 2.4L);
fermentation medium component 2: 30g/L of glycerol; (3L of each component of the culture medium is weighed, and purified water is added to the culture medium to reach the constant volume of 450 mL);
fermentation medium component 3: VB 1 2 mg/L; (preparation of 6mg/mL VB 1 0.22 μm filter sterilization);
feed medium component 1: 16.67g/L of yeast powder; tryptone 33.33 g/L; (weighing each component of the culture medium according to the amount of 450mL, and adding purified water to fix the volume to 300 mL);
feed medium composition 2: 100g/L of glycerol; (the components of the culture medium are weighed according to the amount of 450mL, and purified water is added to the mixture to be constant volume of 150 mL).
1.5 African Swine fever DP96R protein purification
1.5.1 resuspension and disruption of the thallus
Weighing a certain amount of thallus, resuspending the lysate, crushing by a homogenizer, centrifuging and collecting the supernatant. 80. mu.L of the supernatant and the precipitate of disrupted cells before loading were each sampled for SDS-PAGE analysis.
1.5.2 Nickel column purification
(1) Column balancing: balancing 2-3 CV (column volume) with ultrapure water, and discharging ethanol preservation solution; and then balancing 2-3 CV by using BufferA.
(2) Loading: and (3) loading the supernatant by using a peristaltic pump, setting a proper flow rate according to the volume of the nickel column, flowing through and collecting flow-through liquid, and uniformly mixing the flow-through liquid, and taking 80 mu L of the flow-through liquid for SDS-PAGE analysis.
(3) Wash: endotoxin was washed with 30CV wash buffer.
(4) And (3) flushing: wash with 10CV BufferA to reduce Triton X-114 residue.
(5) And (3) elution:
elution with 20mM imidazole eluent (buffer a: buffer b ═ 24:1 mix): eluting the hybrid protein by using 20mM imidazole eluent 5CV, and taking 80 mu L of the mixed solution for SDS-PAGE analysis;
elution with 50mM imidazole eluent (buffer a: buffer b ═ 9:1 mix): eluting the hybrid protein by 50mM imidazole eluent with 4CV, mixing and taking 80 mu L of the mixed solution for SDS-PAGE analysis;
elution with 100mM imidazole eluent (buffer a: buffer b ═ 4:1 mix): eluting the hybrid protein with 100mM imidazole eluent 4CV, mixing, and taking 80 μ L of the mixture for SDS-PAGE analysis;
250mM imidazole eluent (buffer a: buffer b ═ 1:1 mix) eluted: eluting the target protein by using 250mM imidazole eluent 3CV, mixing, and taking 80 mu L of the mixture for SDS-PAGE analysis;
buffer B elutes the protein of interest: the target protein was eluted from bufferB containing 500mM imidazole and collected, and 80. mu.L of the mixture was used for SDS-PAGE analysis.
1.5.3 dialysis Change solution
Adding 1mM EDTA into imidazole eluate containing target protein, pouring into dialysis bag, dialyzing with bufferA and 5% glycerol by at least 1,000 times, and collecting 80 μ L sample for detection.
1.5.4 sterilizing and filtering
In a biosafety cabinet, a 0.22 μm low protein binding needle filter, or Nalgene filter with a 0.22 μm filter sterilized with a large volume of protein solution, was passed through and the filtered protein solution sample was stored in a freezer at-80 ℃.
Wherein the above-mentioned purification solutions used were as follows:
(1) lysis solution: 50mM NaH 2 PO 4 ，500mM NaCl，0.2％Triton X-114，0.05％Tween-20，1mMβ-ME，pH 7.8；
(2)wash buffer：50mM NaH 2 PO 4 ，500mM NaCl，0.4％Triton X-114，0.05％Tween-20，pH 7.4；
(3)Buffer A：50mM NaH 2 PO 4 ，500mM NaCl，0.05％Tween-20，pH 7.4；
(4)Buffer B：50mM NaH 2 PO 4 500mM NaCl, 500mM imidazole, 0.05% Tween-20, pH 7.4.
1.6 identification of African Swine fever DP96R protein
1.6.1 SDS-PAGE detection
The purified protein of example 1 was subjected to SDS-PAGE, and the concentration of DP96R protein in the sample was 2. mu.g/well, as shown in FIG. 5: from the figure, it can be calculated that the purified DP96R protein has an SDS-PAGE purity of 75% and a molecular weight of about 17.5 kD.
1.6.2 WESTERN-BLOT assay
The purified protein of example 1 was subjected to WESTERN-BLOT assay with a membrane-turning time of 1h, the antibody used was Anti His-Tag Mouse, the dilution ratio was 1:2000, and the incubation time was 1h, and the results are shown in FIG. 5: as can be seen from the results in the figure, the purified DP96R protein was able to bind efficiently to the antibody.
1.7 African Swine fever DP96R protein stability verification
The purified protein of example 1 was diluted to 0.5mg/ml with PBS and divided into 20 portions of 0.5ml each; ten portions are placed in a refrigerator at 4 ℃, and one portion is sampled every week and is continuously sampled for 10 times; ten portions are placed in a refrigerator at the temperature of 20 ℃ below zero, one portion is sampled every week, and 10 times of continuous sampling are carried out; protein concentration was measured with BCA after each sampling and the results are shown in the following table:
from the change in protein concentration, the protein remained essentially stable during both experiments.
1.8 immunogenicity of recombinant DP96R protein
1.8.1 vaccine preparation
1.8.1.1 adjuvant by oil: the proportion of the water phase (v: v) is 54: 46.
1.8.1.2 antigen preparation: and (3) performing sterile filtration on the purified recombinant African swine fever DP96R protein by a 0.22 mu m filter membrane, and detecting the concentration and purity for later use.
1.8.1.3 preparation of water phase: according to the content of DP96R protein in the vaccine, DP96R protein is diluted to a proper concentration by 1XPBS, and stirred for 10min to be fully and uniformly mixed.
1.8.1.4 oil phase preparation: according to the water-oil ratio of 1.8.1.1, a proper amount of ISA201VG adjuvant is measured.
1.8.1.5 emulsification: emulsifying at 33 + -1 deg.C, starting the stirrer at 350rpm/min, adding the water phase into the oil phase at constant speed under stirring, and stirring for 10min to mix the water phase and the oil phase sufficiently to obtain the bidirectional oil emulsion vaccine.
1.8.1.6 stable: after the emulsification is finished, the stirrer is closed, and the emulsified vaccine is placed at 20 ℃ for stabilization for 1 h.
1.8.1.7 subpackaging and storing: subpackaging according to the immune requirement, and storing at 2-8 ℃ for later use after the qualified product is inspected.
1.8.2 immunogenicity experiments
1.8.2.1 mouse immunization experiment
10 healthy female BALB/c mice, about 16-18g, were randomly divided into 2 groups of 5 mice each and the vaccine prepared in 1.8.1 was used for immunization experiments. Blood was collected 14 days after the second immunization, and serum was isolated for ELISA detection of antibodies.
1.8.2.2 ELISA detection experiment
(1) Coating: diluting the purified DP96R protein to 0.5. mu.g/ml with coating solution (50mM carbonate buffer, pH 9.5), adding 100. mu.l/well into 96-well plate, sealing with sealing film, and standing in refrigerator at 4 deg.C overnight;
(2) washing: after the ELISA plate was removed from the refrigerator, the plate was washed 5 times with PBST;
(3) and (3) sealing: adding 200 μ l of sealing liquid (5% skimmed milk) into each well, sealing with sealing film, and incubating at 37 deg.C for 2 hr;
(4) serum dilution: diluting pre-immune serum and secondary immune serum of mice immunized with DP96R protein for 14 days by 5000 times with blocking solution (such as adding 5 μ l serum into 4995 μ l dilution solution, and mixing well);
(5) washing: the same (2);
(6) sample adding: adding diluted serum, simultaneously using confining liquid as negative control, and incubating at 37 ℃ for 1 h;
(7) washing: the same as (2);
(8) adding a secondary antibody: adding 100 μ l of HRP-labeled rabbit anti-mouse IgG secondary antibody diluted (at a dilution ratio of 1:5000) into each well, and incubating at 37 ℃ for 0.5 h;
(9) washing: the same as (2);
(10) color development: adding 100 mul of TMB color development solution into each hole under the condition of keeping out of the sun, and incubating for 10min at 37 ℃;
(11) and (4) terminating: add 50. mu.l stop solution (2M H) to each well 2 SO 4 ) Terminating the reaction;
(12) and (3) detection: measuring the OD value of the sample at the wavelength of 450nm, and analyzing the data;
(13) the results are shown in the following table: the coated DP96R protein can be specifically combined with serum of DP96R protein after immunization, and the OD450 mean value is 1.496; the DP 96R-coated protein did not bind specifically to mouse preimmune serum, and the OD450 mean value was 0.050. The DP96R protein can be used as an antigen of an Elisa kit, and the immunized serum can be specifically combined with the DP96R protein, so that the method lays a foundation for later development of a diagnostic kit for detecting African swine fever infection and immunization and a candidate antigen of subunit vaccine.
The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the particular examples and embodiments described herein. These specific examples and embodiments are included to assist those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention and, accordingly, it is intended that the invention be limited only by the terms of the appended claims, along with the full scope of equivalents to which such terms are entitled.
Sequence listing
<110> Zhejiang Hilon Biotechnology Ltd
<120> recombinant African swine fever virus DP96R subunit protein and preparation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 4
<211> 291
<212> DNA
<213> codon optimized DP96R protein nucleotide sequence (DNA)
<400> 4
atgagcaccc acgactgcag cctgaaggag aaaccggttg acatgaacga tatcagcgaa 60
aagagcgtgg ttgtggataa cgcgccggag aaaccggcgg gtgcgaacca cattccggag 120
aagagcgcgc gtgaaatgac cagcagcgag tggatcgcgg aatactggaa gggtattaaa 180
cgtggcaacg acgttccgtg ctgctgcccg cgtaaaatga ccagcgcgga taagaaattc 240
agcgtgtttg gcaagggcag cctgatgcgt agcatccaga aaaacaacta a 291
<210> 4
<211> 291
<212> DNA
<213> nucleotide sequence (DNA) of DP96R protein before codon optimization
<400> 4
atgtctacac atgattgttc tctaaaagag aaaccggttg atatgaacga tatatctgag 60
aaatcagttg tcgtggataa tgcacccgag aaaccagctg gagcgaatca tatacctgag 120
aagtcggccc gcgaaatgac atcatcagaa tggattgctg aatattggaa aggtataaaa 180
cgtggaaatg acgtgccatg ttgttgtcca agaaaaatga ccagtgcaga caaaaagttt 240
tcagtatttg gtaagggatc cctaatgcgc tccatccaga agaataatta a 291
<210> 4
<211> 102
<212> PRT
<213> amino acid sequence (PRT) of DP96R protein
<400> 4
Met Ser Thr His Asp Cys Ser Leu Lys Glu Lys Pro Val Asp Met Asn
1 5 10 15
Asp Ile Ser Glu Lys Ser Val Val Val Asp Asn Ala Pro Glu Lys Pro
20 25 30
Ala Gly Ala Asn His Ile Pro Glu Lys Ser Ala Arg Glu Met Thr Ser
35 40 45
Ser Glu Trp Ile Ala Glu Tyr Trp Lys Gly Ile Lys Arg Gly Asn Asp
50 55 60
Val Pro Cys Cys Cys Pro Arg Lys Met Thr Ser Ala Asp Lys Lys Phe
65 70 75 80
Ser Val Phe Gly Lys Gly Ser Leu Met Arg Ser Ile Gln Lys Asn Asn
85 90 95
His His His His His His
100
Claims (9)
1. A recombinant African swine fever virus DP96R subunit protein is characterized in that the recombinant African swine fever virus DP96R subunit protein is an African swine fever virus virulence protein, and the amino acid sequence of the recombinant African swine fever virus DP96R subunit protein is shown as SEQ ID NO. 3.
2. The recombinant African swine fever virus DP96R subunit protein of claim 1, wherein one of the tags poly-His, FLAG, c-myc, HA and poly-Arg is linked at the amino terminus or the carboxy terminus of the amino acid sequence as set forth in SEQ ID No. 3.
3. The recombinant African swine fever virus DP96R subunit protein of claim 1 or 2, wherein the gene sequence of the recombinant African swine fever virus DP96R protein after codon optimization is shown in SEQ ID No. 1.
4. A method for producing a recombinant african swine fever virus DP96R subunit protein according to any of claims 1 to 3, comprising the steps of:
1) cloning the coding gene sequence of the recombinant African swine fever virus DP96R protein with optimized codon as shown in SEQ ID NO.1 into a prokaryotic expression vector to obtain a recombinant plasmid containing the coding gene of the African swine fever virus DP96R subunit protein;
2) then the recombinant plasmid containing the African swine fever virus DP96R subunit protein coding gene is transformed into an escherichia coli competent cell to obtain recombinant engineering bacteria;
3) obtaining a highly expressed strain by culturing and screening the strain of the recombinant engineering bacteria in the step 2);
4) fermenting and culturing the highly expressed strain in the step 3), and purifying to obtain the recombinant soluble fusion protein of the subunit DP96R of the African swine fever virus.
5. The method of claim 4, wherein the prokaryotic expression vector is selected from the group consisting of pET30, pET28a, pBAD, pcold, pQE, and pKK vectors.
6. The method of claim 5, wherein the prokaryotic expression vector is selected from the group consisting of pET 30.
7. The method according to claim 4, wherein the Escherichia coli is one selected from the group consisting of arcticixpress, BL21(DE3), BL21star (DE 3).
8. The method according to claim 4, wherein the E.coli strain used in step 2) is arcticixpress.
9. Use of the recombinant soluble fusion protein of the subunit of African swine fever virus DP96R of any one of claims 1 to 3 in the preparation of a vaccine for the diagnosis, prevention and treatment of African swine fever.
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Country or region after: China Address after: 312366 No. 1, Baichuan Road, Binhai New Area, Shaoxing City, Zhejiang Province Applicant after: Zhejiang Hailong Biotechnology Co.,Ltd. Address before: 312366 No. 1, Baichuan Road, Binhai New Area, Shaoxing City, Zhejiang Province Applicant before: NOVO BIOTECH Corp. Country or region before: China |