CN111748042B

CN111748042B - African swine fever fusion protein containing endotoxin and preparation method and application thereof

Info

Publication number: CN111748042B
Application number: CN202010449917.5A
Authority: CN
Inventors: 王云龙; 李玉林; 张怡青; 王继创; 王国强; 程蕾; 王敏; 王运从
Original assignee: HENAN BIOENGINEERING RESEARCH CENTER
Current assignee: HENAN BIOENGINEERING RESEARCH CENTER
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2023-06-06
Anticipated expiration: 2040-05-25
Also published as: CN111748042A

Abstract

The invention relates to the technical field of vaccines, in particular to an African swine fever fusion protein containing endotoxin, and a preparation method and application thereof. When the endotoxin content in the African swine fever fusion protein containing endotoxin is 50-12800EU/mg, the inventor finds that the endotoxin in the African swine fever fusion protein can play a role in enhancing humoral and cellular immunity, so that the problem of weak immunogenicity of an African swine fever subunit vaccine in the prior art is solved to a certain extent.

Description

African swine fever fusion protein containing endotoxin and preparation method and application thereof

Technical Field

The invention relates to the technical field of vaccines, in particular to an African swine fever fusion protein containing endotoxin, and a preparation method and application thereof.

Background

Currently, research and development of African swine fever vaccines mainly focus on preparing subunit vaccines with better safety by selecting common proteins with stable immunogenicity such as p30, p54, p72, CD2v, p12, p54, p17 and p 30. However, the immunogenicity of the subunit vaccine prepared is relatively weak, so that the cell immune response is not easy to be initiated, and the using effect of the vaccine is affected. For example, studies in the document Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus Vaccine preparation (Sandra et al, vaccine 32 (2014) 3879-3882) have shown that the single addition of B cell activators to African swine fever vaccines showed an Antibody Dependent Enhancement (ADE). The study included 15 hybrid weaned pigs approximately eight weeks of age. The inactivated vaccine preparation is carried out by using the peripheral blood of the infected pigs, and six weaned pigs are immunized twice at intervals of three weeks. Six weeks after the first immunization, animals were challenged with high virulent ASFV homologs. Although ASFV-specific antibodies were detectable in all but one vaccinated animal prior to challenge, no protective effect of immunization was observed. All animals developed acute lethal ASF, with a slight acceleration of the clinical course of the vaccinators and possibly even suggesting an enhancement in antibody dependence.

Therefore, adjuvants that stimulate both B cells and T cells are particularly important in the development of african swine fever.

Microorganisms have been reported earlier as adjuvants to stimulate both B cells and T cells. Lipopolysaccharide from gram negative bacteria was demonstrated to have immunological adjuvant efficacy in the 50 s of the 20 th century. The outer membrane of gram-negative bacteria commonly contains Lipopolysaccharides (LPS), commonly known as endotoxins (endotoxins), which present in mammalian or human cells a cascade of recognition signaling pathways specific for bacterial LPS, picomolar LPS activate the innate immune response. The immune system is activated rapidly, the immune response is enhanced, the continuous release time of the antigen is prolonged, and the like, so that the vaccine has very important value for improving the immune effect of the vaccine on organisms.

The immune recognition mechanism of LPS is that LPS is combined by LPS binding proteins (LPS binding protein, LBP) in body fluid, and is presented on the surface of immune cells by the aid of LBP or is directly spread on the surface of immune cells, and under the action of auxiliary proteins CD14 and MD-2, the LPS is recognized by Toll-like receptor 4 (Toll like receptor, TLR 4) on the surface of host immune cells, activates a series of signal transduction paths in cells, finally induces the release of various pro-inflammatory cytokines, helps to promote the quick fan of the organism to eliminate pathogens and generate immune response, and can be used as an immune system activator to be developed into an endotoxin vaccine adjuvant.

However, because the long-chain part of the polysaccharide of LPS has a plurality of different structures, a standardized extraction method cannot be prepared; because of the large molecular weight of LPS, ionization is difficult during mass spectrometry detection, real-time detection and quantification cannot be realized, and great limitation exists in the aspects of administration control and in-vivo drug metabolite analysis of clinical research. At present, LPS is only applied to animal vaccine systems as an endotoxin adjuvant, for example, certain salmonella LPS adjuvants are beneficial to preventing animal diseases caused by pathogenic salmonella and the like.

Disclosure of Invention

The invention provides an African swine fever fusion protein containing endotoxin, which is found by the research of the inventor that the endotoxin in the African swine fever fusion protein can play a role in enhancing humoral and cellular immunity, and the safety of the African swine fever fusion protein can be ensured at a higher content, so that the problem of weak immunogenicity of an African swine fever subunit vaccine in the prior art is solved.

The African swine fever fusion protein containing endotoxin adopts the following technical scheme: africa containing endotoxin

The endotoxin content of the African swine fever fusion protein is 50-12800EU/mg.

Preferably, the endotoxin content of the African swine fever fusion protein is 200-3200EU/mg. African swine fever fusion egg

When the endotoxin content of white is 200-3200EU/mg, the effect of strengthening the humoral and cellular immunity effects of the African swine fever fusion protein can be achieved, the safety is better, and adverse reactions such as high fever and the like of immunized animals can be effectively avoided.

Preferably, the african swine fever fusion protein comprises the following fragments: at least 1 of the p30 proteins

And epitope peptides, at least 1 neutralizing epitope peptide of p54 protein and at least 1 neutralizing epitope peptide of p72 protein. Humoral and cellular immunity are better when the african swine fever fusion protein has the fragments as described above.

Preferably, the amino acid sequence of the African swine fever fusion protein is shown as SEQ ID NO. 1.

A second object of the present invention is to provide an African swine fever fusion egg comprising endotoxin as defined in any one of the above

The preparation method of the white comprises the following specific technical scheme: preparing the African swine fever fusion protein by adopting a genetic engineering method, crushing engineering bacteria expressing the African swine fever fusion protein, collecting supernatant, and purifying the African swine fever fusion protein by adopting nickel affinity chromatography and DEAE chromatography in sequence. Wherein, the expression system is preferably a prokaryotic expression system, such as using escherichia coli as engineering bacteria to express the African swine fever fusion protein.

Preferably, PB containing 50, 100, 200, 300 and 400 mmol/L imidazole is used for nickel affinity chromatography in sequence

Eluting.

Preferably, the DEAE chromatography is performed by removing impurities from PB solution containing 100mM-200mM NaCl

Eluting PB solution of 1-5MNaCl to obtain the endotoxin-containing African swine fever fusion protein by purification.

The 3 rd object of the invention is to provide an African swine fever vaccine, which comprises the following specific technical scheme: the African swine fever disease

The raw material or the active ingredient of the vaccine comprises the African swine fever fusion protein containing endotoxin as defined in any one of the above. It should be noted that the vaccine may further comprise an excipient, carrier or diluent component in addition to the african swine fever fusion protein. Further, optionally comprising one or more suitable adjuvants, such as: 201 adjuvants (French Sibirk company), chemical immunological adjuvants such as aluminum hydroxide, freund's adjuvant, mineral oil, span, etc.; microbial immunoadjuvants such as mycobacteria, BCC, lipopolysaccharide, muramyl dipeptide, cytopeptide, liposoluble waxy D, and corynebacterium pumilum; the plant immunoadjuvant is polysaccharides extracted from plants or large fungi, such as pachyman, safflower polysaccharide, chinese herbal medicines, etc.

The invention also aims to provide the African swine fever fusion protein containing endotoxin

The specific technical scheme is as follows: the African swine fever fusion protein containing endotoxin is applied to preparation of monoclonal antibodies, polyclonal antibodies, african swine fever detection test strips, detection cards or detection kits.

The beneficial effects of the invention are as follows: the African swine fever fusion protein containing endotoxin has better humoral immunity and cellular immunity effects.

The African swine fever fusion protein containing endotoxin has good safety, and does not cause adverse reactions such as high fever and the like within the range of 200-3200EU/mg.

The preparation method of the African swine fever fusion protein containing endotoxin is simple and efficient.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a graph showing the results of detection of the effect of different endotoxin contents on humoral immunity; wherein: series 1 refers to the results of vena cava blood collection test on day 14 of the experiment, and series 2 refers to the results of vena cava blood collection test on day 28 of the experiment.

FIG. 2 is a graph showing the results of the detection of the effect of different endotoxin contents on cellular immunity; series 1 refers to the results of the test on day 14 peripheral blood lymphocyte test, and series 2 refers to the results of the test on day 28 peripheral blood lymphocyte test.

Description of the embodiments

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Examples

1.1 Amino acid sequence of african swine fever fusion protein-SEQ ID No.1:

MTKPRKKMEVIFKTDLRSSSQVVFHAGKKKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSEN IHEKNDNETNECTSKKEQEPSSEVPKDSKKQYGKAPDFKKTIYGTPLKEEEKEVKKMDSEFFQPVYPRHYGECLSPVTTPSFFSTHMYKKFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTKKLRQRNTYTHKDLENSLKKMASGGAFCLI ANDGKADKIKKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDF FHDKKHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGE RLKKVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDAKKTYQRTRALVGSSSSGSSSGHHHHHHHH

1.2 SEQ ID NO.1 was supplied to the manufacturer (Shanghai) of the company, from which the nucleotide sequence encoding SEQ ID NO.1 was synthesized in terms of amino acid sequence, and the synthesized nucleotide sequence was transformed into the pET28a-ASFV plasmid.

1.3 Expression of recombinant proteins

CaCl is adopted ₂ Competent cells were prepared by the method, the pET28a-ASFV plasmid which was successfully constructed was introduced into BL21 (DE 3) competent cells to prepare recombinant bacteria, and positive recombinant bacteria were selected using kanamycin (Kana) resistance gene on the plasmid and using a selective medium supplemented with kanamycin. Firstly, according to the existing mature condition of recombinant protein expression in the laboratory, carrying out preliminary induced expression on recombinant bacteria, and identifying the expressed recombinant protein through SDS-PAGE gel electrophoresis and Western blot.

1.3.1 optimization of expression conditions: and (3) taking bacteria with identification results meeting expectations, optimizing the concentration, the induction time and the induction temperature of the inducer IPTG for recombinant protein expression by adopting a single-factor variable method, and carrying out large-sample induction expression according to the optimized optimal conditions.

1.3.1.1 optimizing inducer concentration: based on the principle of single factor variable, the induction temperature is 37 ℃, the induction time is 4 h, and the concentration of the inducer IPTG is set to be six levels of 0mmol/L (control), 0.3 mmol/L, 0.7 mmol/L, 1.0 mmol/L, 1.5 mmol/L and 2.5 mmol/L. The recombinant protein expression level of each level group is detected, and the optimal inducer concentration is preferably 1.0 mmol/L.

1.3.1.2 optimization of induction time: based on the principle of single factor variable, the induction temperature is 37 ℃, the optimal concentration of the inducer is selected, the induction time is set to be 2 h, 4 h, 6h and 8 hours, the expression quantity of the recombinant protein in each level group is detected respectively, and the optimal induction time is preferably 6 hours.

1.3.1.3 optimal induction temperature: based on single factor variable as principle, the optimal induction time and the optimal inducer concentration are selected, the induction temperature is set to 25 ℃, 30 ℃ and 37 ℃ to three levels, the recombinant protein expression quantity of each level group is detected respectively, and the optimal induction temperature is preferably 37 ℃.

The method comprises the following specific steps: preparation of competent E.coli BL21 (DE 3) cells: taking a single colony of escherichia coli BL21 (DE 3) in 2ml of LB culture medium, carrying out shaking culture at 37 ℃ and 200rpm for 12-16 hours, and inoculating 1ml of culture in 100ml of LB culture medium, and carrying out culture at 37 ℃ and 200rpm for 3 hours; ice-bath the bacterial liquid for 2 hours, then centrifuging 2500g at low temperature for 20 minutes, and collecting bacterial cells; 100ml ice-cold Triturate buffer (100 mmol/L CaCl) was added ₂ ，70mmol/L MgCl ₂ 40mmol/L sodium acetate, pH 5.5), mixing, placing in ice bath for 45 min, centrifuging at 1800g for 15min, discarding supernatant, adding 10ml ice-cold Trituration buffer solution to suspend cells, sub-packaging according to 200 μl of each portion, and preserving at-70 ℃ for standby.

Conversion: melting 200 mu L of competent cells on ice, adding 3 mu L of DMSO, uniformly mixing, adding 2 mu L of connection reaction liquid, uniformly mixing again, placing on ice for 30 minutes at 42 ℃ for 45 seconds, then rapidly placing back into an ice bath for 1.5 minutes, adding 2ml of LB culture liquid, carrying out shaking culture at 37 ℃ and 200rpm for 1 hour, centrifuging at 4000g for 10 seconds, discarding the supernatant, and re-suspending thalli by using 200 mu L of LB culture medium; the bacterial liquid is spread on an LB agar plate containing ampicillin resistance, evenly spread, placed for 30 minutes at room temperature, and placed in a 37 ℃ incubator for culturing for 12-16 hours. Clones on the plates are screened, plasmids are extracted, ecoR I and HindI I are digested, fragments corresponding to the sizes of genes encoding African swine fever fusion proteins can be cut out, and the fragments are preliminarily determined to be positive clones.

Induction of expression: i.e., positive clones were cultured overnight, following the morning at 1: 100. after 3 hours of transfer and culture, 1mM IPTG was added and the culture was continued at 37℃for 6 hours to prepare a sample; the specific band is found to be the correct clone when the conventional SDS-PAGE detects the expression molecular weight of the target protein; and (3) taking correct clones, culturing in an amplifying way, after SDS-PAGE (sodium dodecyl sulfate-PAGE) proves that the expression is correct, purifying target proteins, and further confirming the expression accuracy by using a conventional western-blot.

1.4 purification of recombinant mycoprotein

With reference to the optimized results, expression was induced under optimal induction conditions (37 ℃ C., induction with IPTG at 1.0 mmol/L for 6 h). The induced bacterial liquid is centrifuged at 10000rpm for 10min, the culture medium is discarded, 200 mL PB (0.05 mmol/L, pH 7.8) is added for washing the precipitate twice, and then 100mL PB (0.05 mmol/L, pH 7.8) is used for carrying out ultrasonic wave at 800V under ice bath condition, the bacteria are broken at 5 s, the interval is 5 s, and the circulation is 99 times. Centrifuging the whole fungus liquid at 10000rpm for 10min, and collecting supernatant for purification.

1) Nickel affinity chromatography column preparation: the regenerated nickel affinity column was equilibrated with 0.05mol/L PB at pH 7.8 until the effluent pH was the same as the buffer pH.

2) Loading: and pumping the treated sample into a nickel affinity chromatographic column by using a constant flow pump, and controlling the rotating speed of the constant flow pump to be 10-15 revolutions per minute.

3) Eluting: eluting with PB (0.05 mol/L, pH 7.8) containing 50, 100, 200, 300, 400 mmol/L imidazole sequentially, and measuring absorbance (OD) of the effluent at 280nm by ultraviolet spectrophotometer ₂₈₀ ). Effluent OD of one concentration ₂₈₀ And (3) changing the next concentration of imidazole for eluting when the concentration is less than or equal to 0.1. Collecting effluent, zeroing with eluent of corresponding concentration, and detecting OD of the effluent ₂₈₀ And recorded.

4) Protein concentration determination: determination of OD ₂₈₀ OD (optical density) ₂₆₀ Protein concentration (mg +.mL)=1.45×OD ₂₈₀ －0.74×OD ₂₆₀ 。

Through calculation, 180-290mg of protein can be obtained after the recombinant engineering bacteria in each liter of fermentation broth are purified through nickel affinity chromatography.

1) DEAE chromatography column preparation: the regenerated DEAE column was equilibrated with 0.05mol/L PB at pH 7.8 to give an effluent having the same pH as the buffer.

2) Loading: and pumping the treated sample into a chromatographic column by using a constant flow pump, and controlling the rotating speed of the constant flow pump to be 10-15 revolutions per minute.

3) Eluting: eluting with PB (0.05 mol/L, pH 7.8) containing 100mM, 200mM and 2000mM NaCl sequentially, and measuring the absorbance (OD) of the effluent at 280nm by ultraviolet spectrophotometer ₂₈₀ ). Effluent OD of one concentration ₂₈₀ And (3) changing the next concentration of imidazole for eluting when the concentration is less than or equal to 0.1. Collecting effluent, zeroing with eluent of corresponding concentration, and detecting OD of the effluent ₂₈₀ And recorded.

4) Protein concentration determination: determination of OD ₂₈₀ OD (optical density) ₂₆₀ Protein concentration (mg/mL) =1.45×od was calculated according to the formula ₂₈₀ －0.74×OD ₂₆₀ 。

The protein recovery rate after DEAE chromatography purification is calculated to be 75-83%, the endotoxin content in each batch of products is quantified by adopting an endotoxin kit, and the endotoxin content in each milligram of protein in the African swine fever fusion protein obtained by purification according to the method is 4000-8000. If the endotoxin content in the African swine fever fusion protein needs to be further reduced, the DEAE chromatography purification step can be repeated again according to the requirement of the subsequent experiment so as to obtain the ideal endotoxin concentration.

Examples

Preparation of african swine fever vaccine: 1128 ml of No. 7 white oil is taken, and span-80 ml is added, and evenly mixed; and weighing 24 g of aluminum stearate, fully dissolving the reagent by using a colloid mill, uniformly mixing, and sterilizing under high pressure to obtain an oil phase. Sterile filtration of the African swine fever fusion protein obtained by purifying the method in the embodiment 2, sterile dilution of the African swine fever fusion protein with sterile physiological saline to 0.6mg/ml, adding 500ml of the solution into 1000 ml of the sterile oil phase, and stirring for 2-5min at 10000r/min to obtain an African swine fever vaccine for later use.

EXAMPLE 4 African swine fever vaccine prepared in example 3 was used to immunize animals

4.1 The immunization method comprises the following steps: the 30 pigs are divided into 10 groups for immunization, and 0.5mL of African swine fever vaccine prepared in the example 3 is respectively injected into the left and right ears by intramuscular injection of the neck behind the ears; the total immunization was performed twice, 14 days apart (first immunization and second immunization were performed on day 1 and day 15 respectively), and the anterior vena cava was sampled on days 14 and 28 for subsequent detection.

Grouping condition: the test groups are 9 groups, and 3 pigs in each group;

control group

1, 3 pigs; pigs of different groups were immunized with african swine fever vaccine made with african swine fever fusion proteins containing different endotoxin contents, respectively, and specific grouping conditions are shown in table 1 below:

TABLE 1

Group of	1	2	3	4	5
						Endotoxin content	50	100	200	400	800
Group of	6	7	8	9	10
						Endotoxin content	1600	3200	6400	12800	<10

Remarks: the endotoxin content in Table 1 above is the endotoxin content of African swine fever fusion protein used in the preparation of vaccines in EU/mg.

Experimental phenomena: in the first three days after the first immunization observations were found: the group with endotoxin content of 12800 (group 9) showed high firing for 3 days; the group with endotoxin content of 6400 (group 8) showed high firing for 1 day; each group with endotoxin content less than 3200 has no adverse reaction such as fever.

Three days before the second immunization, the following observations were: the group with endotoxin content of 12800 (group 9) showed high firing for 5 days; the group with endotoxin content of 6400 (group 8) showed high firing for 2 days; each group with endotoxin content less than 3200 has adverse reaction such as fever.

4.2 Detecting the effect of different groups of vaccines on humoral immunity

The African swine fever fusion proteins used in the different experimental groups are used as antigens, diluted to 0.1ug/ml with CB of pH9.5 and 0.05mol/L, and 100 ul/hole is added into an enzyme plate reaction plate to be coated overnight at 4 ℃; the next day, the plate was washed once with washing solution (pH 7.0 containing 0.01mol/L PB, 0.1mol/L NaCl and 0.1% tween-20) and added at 115 ul/well with pH7.0 containing 5% calf serumSealing with 0.01mol/L PB sealing plate at 4deg.C overnight; the next day the sealing plate liquid is sucked, dried for 1 hour at 37 ℃, and then the sealing plate liquid is packaged in an aluminum foil bag by adding a drying agent, and the coating is finished. In the detection process, 50ul of PBS containing 0.01mol/L PB and 0.1mol/L NaCl with the pH of 7.0 is firstly added into an enzyme-labeled reaction hole, then 50ul of serum to be detected is added, negative and positive control is added, the incubation is carried out for 20 minutes at 37 ℃, the plate is washed five times by using a washing liquid, and the plate is patted dry; diluting goat anti-pig enzyme-labeled antibody with PBS (phosphate buffer solution) at pH7.0 at a ratio of 1:500, adding 100 ul/hole into a reaction plate, incubating at 37deg.C for 20 min, washing the plate, adding 1 drop of developer A, B solution, and developing at 37deg.C for 10min to obtain a developer A containing H ₂ O ₂ The color-developing agent B contains TMB; after the development was completed, 1 drop of 2mol/L H was added to each well ₂ SO ₄ The reaction was terminated and the results were read with an enzyme-labeled instrument at a wavelength of 450 nm. OD450nm>2.1 times negative control OD mean value was positive, OD450nm<The average value of the 2.1 x negative control was negative. Specific detection results are shown in the following table 2 and fig. 1:

TABLE 2

Endotoxin (endotoxin)	Group of	Series 1	Series 2
				50	1	0.2268	0.6915
100	2	0.37364	1.31164
				200	3	0.44016	1.5292
400	4	0.49084	1.8841
				800	5	0.582	1.63504
1600	6	0.512	1.5012
				3200	7	0.56448	1.353
6400	8	0.769	1.4112
				12800	9	0.895	1.7528
<10	10	0.16128	0.63504

Remarks: table 2 and fig. 1: series 1 refers to the results of vena cava blood collection test on day 14, and series 2 refers to the results of vena cava blood collection test on day 28; the endotoxin content is in EU/mg.

4.3 cell immunoassay

Peripheral blood was collected from pigs on day 14 and day 28, peripheral blood lymphocytes were isolated, and detected using the Swine IFN (interferon) Cytoset ELISPOT detection kit from Biosource Europe. The method comprises the following specific steps:

(1) A desired number of well strips were mounted on the support and washed 4 times with sterile PBS (200. Mu.l/well). The remaining strips were kept in a closed bag at room temperature.

(2) Blocking was performed with medium (200 μl/well) containing 10% serum (serum identical to serum of suspension cells). Incubate at room temperature for at least 30 minutes.

(1) The blocking medium is removed and a cell suspension (final volume 100-150. Mu.l/well) containing possible stimulators such as antigen is added. The positive control in the kit was recommended to be used at a final concentration of 100 ng/ml.

(2) The well plate was placed at 37℃with 5% CO ₂ The humidification incubator is provided with the humidification chamber for 12 to 48 hours. The well plate is not moved during incubation, and aluminum foil paper is used for wrapping the well plate to avoid evaporation of water.

(1) The well plate was emptied to remove cells, washed 5 times with sterile PBS, 200. Mu.l/well.

(2) Sterile PBS containing 0.5% fetal bovine serum was used as per 1:200 dilution of one-step reaction detection reagent. Mu.l of each well was added. Incubate 2 h at room temperature.

(3) The well plate was washed 5 times with 200 μl/well PBS.

(4) The substrate lysate (BCIP/NBT-plus) ready for use was filtered with a 0.45 μm filter, 100. Mu.l per well. Imaging until speckles appear; development exceeding 15min can cause background staining. The dyeing reaction was terminated by flushing with tap water. The water inside the slits (soft plastic under the orifice plate) is removed and the back of the membrane is cleaned.

(5) And (5) airing the pore plate. Spots were examined and counted on a microscope (X40) or on an ELISpot counter.

The results of the measurements are shown in Table 3 below

TABLE 3 Table 3

Endotoxin (endotoxin)	Group of	Series 1	Series 2
				50	1	17.41	31.6
100	2	35.21	62.45
				200	3	37.95	75.48
400	4	39.66	79.16
				800	5	46.86	81.44
1600	6	49.39	77.95
				3200	7	53.13	84.81
6400	8	56.32	79.86
				12800	9	63.25	91.7
<10	10	6.1	18.8

Remarks: table 3 and fig. 2: series 1 refers to the results of the peripheral blood lymphocyte test on day 14, and series 2 refers to the results of the peripheral blood lymphocyte test on day 28; the endotoxin content is in EU/mg. .

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

<110> Henan province bioengineering technology research center

<120> an endotoxin-containing African swine fever fusion protein, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 557

<213> Artificial sequence (Artificial Sequence)

<400> 1

MTKPRKKMEVIFKTDLRSSSQVVFHAGKKKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSEN

IHEKNDNETNECTSKKEQEPSSEVPKDSKKQYGKAPDFKKTIYGTPLKEEEKEVKKMDSEFFQPVYPRHYGECLSPVTTPSFFSTHMYKKFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTKKLRQRNTYTHKDLENSLKKMASGGAFCLIANDGKADKIKKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDKKHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLKKVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDAKKTYQRTRALVGSSSSGSSSGHHHHHHHH。

SEQUENCE LISTING

<110>

<120>

<130> 2020.05.25

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 557

<212> PRT

<213>

<400> 1

Met Thr Lys Pro Arg Lys Lys Met Glu Val Ile Phe Lys Thr Asp Leu

1 5 10 15

Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Lys Lys Lys Ser Ala

20 25 30

Arg Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala Gln Glu Glu

35 40 45

Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr Glu Ser Ser

50 55 60

Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp Asn Glu Thr Asn Glu

65 70 75 80

Cys Thr Ser Lys Lys Glu Gln Glu Pro Ser Ser Glu Val Pro Lys Asp

85 90 95

Ser Lys Lys Gln Tyr Gly Lys Ala Pro Asp Phe Lys Lys Thr Ile Tyr

100 105 110

Gly Thr Pro Leu Lys Glu Glu Glu Lys Glu Val Lys Lys Met Asp Ser

115 120 125

Glu Phe Phe Gln Pro Val Tyr Pro Arg His Tyr Gly Glu Cys Leu Ser

130 135 140

Pro Val Thr Thr Pro Ser Phe Phe Ser Thr His Met Tyr Lys Lys Phe

145 150 155 160

Ser Ser Arg Lys Lys Lys Ala Ala Ala Ile Glu Glu Glu Asp Ile Gln

165 170 175

Phe Ile Asn Pro Tyr Gln Asp Gln Gln Trp Val Glu Val Thr Pro Gln

180 185 190

Pro Gly Thr Ser Lys Pro Ala Gly Ala Thr Thr Ala Ser Val Gly Lys

195 200 205

Pro Val Thr Gly Arg Pro Ala Thr Asn Arg Pro Ala Thr Asn Lys Pro

210 215 220

Val Thr Asp Asn Pro Val Thr Asp Arg Leu Val Met Ala Thr Gly Gly

225 230 235 240

Pro Ala Ala Ala Pro Ala Ala Ala Ser Ala Pro Ala His Pro Ala Glu

245 250 255

Pro Tyr Thr Thr Val Thr Thr Gln Asn Thr Ala Ser Gln Thr Lys Lys

260 265 270

Leu Arg Gln Arg Asn Thr Tyr Thr His Lys Asp Leu Glu Asn Ser Leu

275 280 285

Lys Lys Met Ala Ser Gly Gly Ala Phe Cys Leu Ile Ala Asn Asp Gly

290 295 300

Lys Ala Asp Lys Ile Lys Lys Asn Val Asn Lys Ser Tyr Gly Lys Pro

305 310 315 320

Asp Pro Glu Pro Thr Leu Ser Gln Ile Glu Glu Thr His Leu Val His

325 330 335

Phe Asn Ala His Phe Lys Pro Tyr Val Pro Val Gly Phe Glu Tyr Asn

340 345 350

Lys Val Arg Pro His Thr Gly Thr Pro Thr Leu Gly Asn Lys Leu Thr

355 360 365

Phe Gly Ile Pro Gln Tyr Gly Asp Phe Phe His Asp Lys Lys His Ser

370 375 380

Ser Trp Gln Asp Ala Pro Ile Gln Gly Thr Ser Gln Met Gly Ala His

385 390 395 400

Gly Gln Leu Gln Thr Phe Pro Arg Asn Gly Tyr Asp Trp Asp Asn Gln

405 410 415

Thr Pro Leu Glu Gly Ala Val Tyr Thr Leu Val Asp Pro Phe Gly Arg

420 425 430

Pro Ile Val Pro Gly Thr Lys Asn Ala Tyr Arg Asn Leu Val Tyr Tyr

435 440 445

Cys Glu Tyr Pro Gly Glu Arg Leu Lys Lys Val Ser Val Glu Gly Thr

450 455 460

Ser Gly Pro Leu Leu Cys Asn Ile His Asp Leu His Lys Pro His Gln

465 470 475 480

Ser Lys Pro Ile Leu Thr Asp Glu Asn Asp Thr Gln Arg Thr Cys Ser

485 490 495

His Thr Asn Pro Lys Phe Leu Ser Gln His Phe Pro Glu Asn Ser His

500 505 510

Asn Ile Gln Thr Ala Gly Lys Gln Asp Ile Thr Pro Ile Thr Asp Ala

515 520 525

Lys Lys Thr Tyr Gln Arg Thr Arg Ala Leu Val Gly Ser Ser Ser Ser

530 535 540

Gly Ser Ser Ser Gly His His His His His His His His

545 550 555

Claims

1. An african swine fever vaccine, characterized in that the raw material or active ingredient of the african swine fever vaccine comprises endotoxin and african swine fever fusion protein; the endotoxin content is 50-12800EU/mg; the amino acid sequence of the African swine fever fusion protein is shown as SEQ ID NO. 1.

2. African swine fever vaccine according to claim 1, wherein the endotoxin content is 200-3200EU/mg.