CN110041411A - Stable atypical classical swine fever virus subunit protein, vaccine thereof, preparation method and application - Google Patents
Stable atypical classical swine fever virus subunit protein, vaccine thereof, preparation method and application Download PDFInfo
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- CN110041411A CN110041411A CN201810036856.2A CN201810036856A CN110041411A CN 110041411 A CN110041411 A CN 110041411A CN 201810036856 A CN201810036856 A CN 201810036856A CN 110041411 A CN110041411 A CN 110041411A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a stable atypical classical swine fever virus subunit protein, a vaccine, a preparation method and an application thereof, wherein the amino acid sequence of the atypical classical swine fever virus subunit protein is shown as SEQ ID NO.3, and the subunit protein has good immunogenicity. The subunit protein is prepared by the following steps: 1) cloning a gene sequence shown in SEQ ID NO.1 into a eukaryotic expression vector to obtain a recombinant plasmid containing an atypical classical swine fever virus subunit protein coding gene; 2) then, transfecting the recombinant plasmid containing the atypical classical swine fever virus subunit protein coding gene into a CHO cell; 3) obtaining a highly expressed cell line by culturing, screening and acclimating the CHO cell line described in 2); 4) fermenting and culturing the cell strain in the step 3), and purifying to obtain the atypical classical swine fever virus subunit protein. The method for preparing the subunit protein is simple, low in cost and easy to amplify. And mixing a proper amount of subunit protein with an adjuvant to obtain the subunit vaccine with good immunogenicity.
Description
Technical field
The present invention relates to a kind of non-typical swine fever subunit proteins and its preparation method and application, belong to animal vaccine and beast
Use biological product technical field.
Background technique
2015-2017 American-European countries successively reports, the congenital pig of piglet is trembled (CT) phenomenon, and is separated and identified the virus
A kind of atypical classical swine fever for flaviviridae, pestivirus is viral (APPV).It is also separated in Guangdong Province, China in May, 2017
To the virus.
Non-typical swine fever virus (Atypical porcine pestivirus, APPV) mainly causes congenital piglet to shake
Quiver (congenital tremor, CT), i.e. piglet topically or systemically jerk occurs after a few hours of being born.To current
Until, the isolated virus in only 2 laboratories, and virus titer is very low.It is clear there is no parsing at present for the virus
Chu does not know which albumen of the virus has good immunogenicity yet.Therefore up to the present, for the virus, do not have also
There is good vaccine to prevent epidemic.So studying a kind of vaccine that can prevent the disease is top priority instantly, and working as
It is lower may be in the case where large scale preparation inactivated vaccine or attenuated vaccine, a kind of immunogenic protein of the virus of determination,
To study a kind of vaccine that can prevent the disease or to there is great meaning with the subunit protein that can prevent the disease
Justice.
Summary of the invention
The technical problem to be solved in the present invention is to provide the stable Asias that one kind can prevent non-typical swine fever virus infection
Unit-protein and preparation method and application.
In order to solve this problem, institute getable non-typical swine fever is viral at present in abundant analysis, research by the present inventor
It on the basis of data and pestivirus correlated virus data, and verifies, proposes a kind of with good through a large number of experiments
Immunogenicity and stable non-typical swine fever viral sub-units albumen, the subunit protein amino acid sequence such as SEQ ID NO3
It is shown.
In order to the industrialization production subunit protein, the present inventor is sufficiently analyzing, is studying the subunit protein
On the basis of original gene coded sequence, propose optimization after can in Chinese hamster ovary celI efficient secretory expression subunit's egg
White gene order, the gene order after the optimization is as shown in SEQ ID NO.1.
According to the technique and scheme of the present invention, it may be preferable that the non-typical swine fever viral sub-units albumen is included in SEQ ID
By replacing, missing or adding an amino acid or several amino acid and there is immunogenicity in amino acid sequence in NO3
Derivative protein.
According to the technique and scheme of the present invention, it may be preferable that the expression system packet of the non-typical swine fever viral sub-units albumen
Include but be not limited to Escherichia coli, yeast, mammalian cell and insect cell.
According to the technique and scheme of the present invention, it may be preferable that the mammalian cell is Chinese hamster ovary celI.
According to another aspect of the present invention, Chinese hamster ovary celI efficient secretory expression atypia is used invention further provides a kind of
The preparation method of swine fever virus subunit protein, the preparation method comprises the following steps: 1) by base shown in SEQ ID NO.1
The recombinant plasmid containing non-typical swine fever viral sub-units protein coding gene is obtained in carrier for expression of eukaryon because sequence is cloned into；
2) again by the Transfected Recombinant Plasmid containing non-typical swine fever viral sub-units protein coding gene into Chinese hamster ovary celI；3) pass through training
It supports, screen, Chinese hamster ovary celI strain described in domestication 2) obtains the cell strain that height is expressed；4) fermented and cultured 3) described in cell strain,
Non-typical swine fever viral sub-units albumen is obtained after purification.
According to the technique and scheme of the present invention, it may be preferable that the carrier for expression of eukaryon is pEE12.4.
According to the technique and scheme of the present invention, it may be preferable that the Chinese hamster ovary celI is CHO-K1 cell.
In accordance with a further aspect of the present invention, the present invention also provides a kind of Asia of non-typical swine fever viral sub-units albumen is single
Position vaccine, the non-typical swine fever subunit viral vaccine contain the non-typical swine fever viral sub-units albumen and adjuvant.
According to the technique and scheme of the present invention, it may be preferable that non-typical swine fever in the non-typical swine fever subunit viral vaccine
The dosage of viral sub-units albumen is g/ parts of 15~200 μ.
According to the technique and scheme of the present invention, it may be preferable that non-typical swine fever in the non-typical swine fever subunit viral vaccine
The dosage of viral sub-units albumen is g/ parts of 30~100 μ.
According to the technique and scheme of the present invention, it may be preferable that non-typical swine fever in the non-typical swine fever subunit viral vaccine
The dosage of viral sub-units albumen is g/ parts of g/ parts of 30 μ or 60 μ.
According to the technique and scheme of the present invention, the adjuvant in the non-typical swine fever subunit viral vaccine can be oil-in-water
Adjuvant (such as 15 VG of ISA) or water-in-oil adjuvant (61 VG of ISA etc.) or water adjuvant (aluminium glue adjuvant, Montanide GEL
01 adjuvant, 1313 adjuvant of IMS etc.) or W/O/W adjuvant.
According to the technique and scheme of the present invention, it may be preferable that adjuvant is water packet in the non-typical swine fever subunit viral vaccine
Water-in-oil adjuvant, the W/O/W adjuvant are 201 VG adjuvant of ISA.
The present invention also provides the non-typical swine fever viral sub-units albumen described in one kind in the pre- of non-typical swine fever virus
Application in anti-and treatment.
According to another aspect of the invention, the present invention also provides the non-typical swine fever viral sub-units albumen described in one kind
Application in non-typical swine fever virus dependent diagnostic reagent, it may be preferable that by the non-typical swine fever viral sub-units albumen
Application in Elisa antibody test reagent.
The subunit protein of non-typical swine fever virus provided by the invention has good stability and immunogenicity, and the Asia is single
Position albumen is still stable in 37 DEG C of processing 20h, still stable when saving 10 weeks for 4 DEG C, raw when this much meets as antigen
Needed for production；The subunit protein can be specifically bound with non-typical swine fever virus hyper-immune serum (see in embodiment
Werstern Blot detection and Elisa detection)；And after immune, capable of generating the antibody of high titre, (two exempt from rear serum dilution
OD450 value is up to 3.5 or more after 100 times).The preferential method for preparing the subunit protein of the present invention is using mammal
Cell (mammalian cell can modify posttranslational protein, make it closer to native protein), preferential lactation are dynamic
Object cell is Chinese hamster ovary celI, has yield is high (can reach 400- before fermentation optimization using the expressing cho cell subunit protein
500mg/L), easy purification (secreting, expressing, foreign protein is few, as soon as cross a simple nickel column, SDS-PAGE purity can reach 85% or
More than), immunogenicity it is good (there is posttranslational modification, closer to native state under albumen), the advantages such as production cost is low.
The present invention provides the amino acid sequence of the subunit protein of detailed non-typical swine fever virus, the technologies of this field
Personnel pass through conventional technology, it is easy to protein derived from preparing on the basis of this sequence, the derivative albumen
Amino acid sequence (as shown in SEQ ID NO3) homologous performance of matter and subunit protein of the invention is up to 80% or more, and
Has the function of same immunogenicity, therefore the derivative protein is also fallen within protection scope of the present invention.
The present invention provides the gene coded sequences of the subunit protein of detailed non-typical swine fever virus, including original base
Because of the gene coded sequence after coded sequence and optimization, those skilled in the art passes through conventional technology, it is easy to real
The subunit protein, such as Escherichia coli, yeast, other mammalian cells and insect are prepared in others expression system now
Cell etc. although having certain difference in expression yield, therefore prepares the subunit protein using other expression systems
Preparation method has been also fallen within protection scope of the present invention, and the expression that only others skilled in the art propose can make this
The yield of subunit protein provides significantly or largely reduces the production cost of the subunit protein.
Detailed description of the invention
Fig. 1 shows the three-dimentional mode figures of the AE2 albumen of prediction.
Fig. 2 indicates AE2 gene order optimization front and back comparison result.
Fig. 3 indicates pEE12.4-OPTI-AE2 plasmid map.
Fig. 4 indicates pEE12.4-OPTI-AE2 double digestion qualification result: M is DNA Marker:DL10000Marker；1 is
PEE12.4-OPTI-AE2 double digestion electrophoresis result.
The SDS-PAGE testing result of Fig. 5 expression AE2 albumen: 1 is AE2 albumen, and 2 be Marker.
Fig. 6 indicates the Werstern Blot testing result after AE2 albumen and AE2 albumen deglycosylation: M indicates Marker；
1 is negative control BSA albumen, and applied sample amount is 2 μ g；2 be deglycosylation before AE2 albumen, applied sample amount be 2 μ g；3 be glycosyl
AE2 albumen after change, content is 2 μ g when deglycosylation, all as sample progress WB detection after deglycosylation.
Fig. 7 indicates the Werstern Blot testing result of AE2 albumen the tenth sub-sampling after 37 DEG C and 4 DEG C processing: M table
Show Marker；1 is negative control BSA albumen, and applied sample amount is 2 μ g；2 be 37 DEG C of treated AE2 albumen, and applied sample amount is 2 μ g；3
It is 4 DEG C of treated AE2 albumen, applied sample amount is 2 μ g.
Specific embodiment
Below with reference to drawings and examples, the present invention will be further described, and the embodiment of the present invention is merely to illustrate this
The technical solution of invention, and the non-limiting present invention.
Bacterial strain used in the embodiment of the present invention, plasmid and reagent are commercial product.
The source list of reagent and drug of the present invention is as follows:
Bacterial strain used in the embodiment of the present invention, plasmid and reagent are commercial product.
The source list of reagent and drug of the present invention is as follows:
CHO-K1 cell origin is raw in the American Type Culture Collection committee, Chinese Academy of Sciences cell bank Chinese Academy of Sciences Shanghai
Order Science Institute's cell bank；
Cell culture medium and serum are purchased from Gibco company, the U.S.；
Carrier for expression of eukaryon pEE12.4 is purchased from upper Hailin deep pool Biotechnology Co., Ltd；
Methionine sulfoxide imonium ((L-methioninesulfoximine, MSX)) is purchased from Sigma company；
BCA quantification of protein kit is purchased from U.S. Thermo Fisher company；
Glycosidase F is purchased from New England Biolabs (UK) Ltd；
The goat-anti pig IgG secondary antibody of HRP label is purchased from EarthOx Life Science；
201 VG of ISA is purchased from match BIC Corp, France.
Embodiment 1: the selection of non-typical swine fever viral sub-units albumen and the optimization of gene order
By to non-typical swine fever viral nucleotide sequences (GenBank:KY624591.1), swine fever virus sequence (referring to
Application No. is 201710409930.6 Chinese invention patents), bovine viral diarrhea virus sequence (referring to application No. is
201611239612.1 Chinese invention patent) be compared, discovery sequence 682L-941S be possible for non-typical swine fever
The main antigen protein of virus, is temporarily named as AE2 albumen (Atypical porcine pestivirus E2, AE2), wherein
682L-700G may be the secreting signal peptide of AE2 albumen, and 701S-911H may be the extracellular region of AE2 albumen, and 912L-934L can
It can be the transmembrane region of AE2 albumen, 935S-941S may be the intracellular region albumen of AE2 albumen.In conjunction with us to swine fever virus E2 egg
The experience of white and bovine viral diarrhea virus E2 albumen expression study, we select the extracellular region of expressing cho cell AE2 albumen
As our immunogenic protein, the i.e. amino acid sequence of 701S-911H.We pass through protein tertiary structure and amino acid sheet
The research of body structure finds that the side chain of 4 amino acid (908M-909Y-910R-911H) at C-terminal end is larger, may will affect
The stability of the albumen, and the segment polypeptide is also not the immunogenicity site of AE2 albumen, thus we cut off C-terminal end this 4
A amino acid expresses remaining amino acid sequence, the i.e. amino acid sequence of 701S-907T.The amino acid sequence of 701S-907T is such as
Shown in SEQ ID NO.3, the gene order of this section of amino acid sequence is encoded as shown in SEQ ID NO.2, this section of amino acid sequence
The three-dimentional mode figure of prediction is as shown in Figure 1.
It is optimized by the gene order predicted above-mentioned analysis, to be suitble to the efficient secretory expression in Chinese hamster ovary celI, is obtained
To OPTI-AE2 sequence, as shown in SEQ ID NO.1, the artificial synthesized working delegation Nanjing Jin Sirui biology section of the gene order
Skill Co., Ltd completes.
By after optimization sequence (as shown in SEQ ID NO.1) and optimization before sequence (as shown in SEQ ID NO.2) into
Row compares, as a result as shown in figure 3, total 149/621=24% is different.
Embodiment 2:pEE12.4-OPTI-AE2 construction of recombinant plasmid
2.1 PCR amplification target fragment OPTI-AE2
2.1.1 PCR reacts
(1) design of primers and synthesis
Upstream primer: 5 '-GCAAGCTT GCCGCCACCATGAAGGGCAACCTGATC-3 '
Downstream primer: 5 '-GCGAATTC TCAATGGTGATGGTGATGGTGGGTAGCGGC-3
(2) it is loaded 50 μ L of system, as shown in the table:
PCR amplification program:
2.1.2 PCR product carries out glue recycling
(1) sample collection EP pipe, adsorption column and collecting pipe have been marked；
(2) the empty EP pipe weight marked is weighed, and records numerical value；
(3) single target DNA band is carefully cut from Ago-Gel with scalpel on bale cutting instrument be put into it is dry
In net 1.5mL centrifuge tube；
(4) 600 μ L PC buffer are added in the 1.5mL centrifuge tube in step (3), it is left that 5min is placed in 50 DEG C of water-baths
The right side constantly mildly spins upside down centrifuge tube therebetween, to ensure that blob of viscose sufficiently dissolves；
(5) column equilibration: into adsorption column CB2,500 μ L equilibrium liquid BL, centrifugation is added in (adsorption column is placed in advance in collecting pipe)
12,000rpm/min, 1min outwell the waste liquid in collecting pipe, adsorption column are placed back in collecting pipe；
(6) step (5) acquired solution is added in adsorption column CB2, stands 2min, 10,000rpm/min, centrifugation 30s,
Fall the waste liquid in collecting pipe, then adsorption column CB2 is put into collecting pipe；
(7) 600 μ L rinsing liquid PW buffer are added into adsorption column, stand 3min, are centrifuged 10,000rpm/min, 30s,
The waste liquid in collecting pipe is outwelled, adsorption column CB2 is put into collecting pipe；
(8) step (7) are repeated；
(9) suction attached column is centrifuged, 12,000rpm/min, 2min, as far as possible removing rinsing liquid, and adsorption column is placed in room temperature and is put
10min is set, is thoroughly dried；
(10) adsorption column CB2 is put into collecting pipe, 50 μ L Elution is vacantly added dropwise to adsorbed film middle position
Buffer (65 DEG C of preheatings), stands 3min, is centrifuged 12,000rpm/min, 2min；
(11) from centrifuge tube in step (10) is taken out in centrifuge, intermediate adsorption column CB2 is abandoned, centrifuge tube lid is covered
Son retains the DNA sample in centrifuge tube；
(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue and recycles DNA piece
Section.
2.2 PCR products and carrier double enzyme digestion reaction
(1) label needs well the 1.5mL EP used to manage, and sample-adding is carried out according to the following table in 1.5mL EP pipe, mixes: 50 μ
L reaction system
(2) the 1.5mL EP pipe in step (1) is placed in corresponding enzyme optimum temperature thermostat water bath, water-bath 2-3h.
The recycling of double enzyme digestion product glue: taking out above-mentioned double digestion system, carries out agarose gel electrophoresis to recycle DNA therein
Segment, method are recycled with PCR product glue in 1.2.1.
2.3 connection reactions
(1) it is several to prepare clean 1.5mL EP pipe, marks, is placed on EP pipe support stand-by.
(2) sample-adding, mixing is carried out according to the following table in 1.5mL EP pipe.
(3) after completing sample-adding according to table in step (2), each 10 μ l reaction system is placed in 16 DEG C of cryogenic liquids and is followed
In ring machine, water-bath 10-16h；
(4) EP pipe in step (3) is taken out, is placed it in 65 DEG C of water-baths, water-bath 15min；
(5) the EP pipe in step (4) is taken out, 4 DEG C of preservations are placed in.
1.2.4 conversion reaction
(1) 10 μ L connection reaction solutions are rapidly joined in 100 μ L competent cells, and blows and beats mixing, ice bath 30min；
(2) sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min；
(3) it takes out sample cell and 600 μ L LB liquid mediums is added into sample cell, then by sample in superclean bench
Quality control is placed in 37 DEG C of constant-temperature tables, and 220rpm/min cultivates 1h；
(4) coated plate: taking out sample cell in step (3), and room temperature is centrifuged 8,000rpm/min, 2min, removes 600 μ L supernatants
The thallus of bottom of the tube is resuspended in body, remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be turned with bacteria stick is applied
The bacterium solution for changing plate center is uniformly spread out.
(5) step of converting (4) plate is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, flat-plate inverted will be converted
It sets and carries out culture 15h；
(6) conversion results are observed.
2.5 plasmid extractions and double digestion are identified
2.5.1 plasmid extraction
(1) with 10 μ L liquid transfer gun heads from conversion plate in picking monoclonal to the 5mL resistance of benzyl containing ammonia LB liquid medium
In, 37 DEG C, 220rpm/min shakes bacterium and stays overnight；
(2) bacterium solution is moved in 1.5mL EP pipe, room temperature centrifugation, 12,000rpm/min, 2min abandon supernatant；
(3) 250 μ L plasmids are added into the EP pipe of step (2) and extract reagent P1buffer, thorough suspension thalline；
(4) 250 μ L P2buffer are added into step (3) solution, immediately mildly 5-10 mixing of reverse centrifuge tube, room
Temperature stands 2-4min；
(5) 350 μ L P3buffer are added into step (4) solution, immediately mildly 5-10 mixing of reverse centrifuge tube；Room
Temperature stands 2-4min；
(6) by step (5) solution, room temperature centrifugation, 14,000rpm/min, 10min；
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s are outwelled
Liquid in collecting pipe；
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s outwell receipts
Liquid in collector；
(9) 500 μ L wash solution are added to adsorption column center, room temperature is centrifuged, 12,000rpm/min, 30s,
Fall liquid in collecting pipe, is repeated once；
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5mL centrifuge tube, 30 μ L Elution is added to absorption center membrane
Buffer is stored at room temperature 5min, room temperature centrifugation, 12,000rpm, 2min.Save DNA solution in pipe.
2.5.2 double digestion is identified
(1) label needs well the 1.5mL EP used to manage, and sample-adding: 20 μ L reaction systems is carried out according to the following table
(2) the 20 μ L reaction system of EP pipe in step (1) is placed in 37 DEG C of thermostat water baths, water-bath 2h.
(3) the double digestion system sample in step (2) is subjected to agarose gel electrophoresis, whether checks Insert Fragment size
Just
Really；Experimental result is shown in Fig. 4, skeleton size about 7,197bp, and clip size about 2,266bp, plasmid enzyme restriction is correct.
(4) selection Insert Fragment, which is correctly cloned, send sequencing company to be sequenced.
2.6 endotoxin-free plasmids mention greatly
2.6.1 endotoxin-free plasmid is extracted
(1) correctly clone is seeded in the culture medium of the 100mL resistance of benzyl containing ammonia for sequencing, in 37 DEG C of constant-temperature tables,
220rpm/min cultivates 15h；
(2) bacterium solution cultivated in step (1) is transferred in 50mL centrifuge tube, room temperature 8,000rpm/min, centrifugation 5min,
Thallus is collected, supernatant culture medium is discarded；
(3) 8mL solution P1 is added into the centrifuge tube of step (2), thallus is sufficiently resuspended with pipettor；
(4) 8mL solution P2 is added into the centrifuge tube of step (3), mildly overturns centrifuge tube 6-8 times, is stored at room temperature immediately
5min；
(5) 8mL solution P4 is added into the centrifuge tube of step (4), turns upside down 6-8 times, is mixed well to solution immediately
There is white flock precipitate, is placed at room temperature for 10min or so.8,000rpm/min room temperatures are centrifuged 5-10min, make white precipitate from extremely
Tube bottom；
(6) by supernatant in step (5) all careful immigration filter CS1, slowly push handle filter, filtrate collection exist
In clean 50mL centrifuge tube；
(7) column equilibration: into adsorption column CP6, the equilibrium liquid BL of 2.5mL, room is added in (adsorption column is put into 50mL collecting pipe)
8,000rpm/min of temperature is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；
(8) isopropanol of 0.3 times of filtrate volume is added into step (6) filtrate, is transferred to absorption after mixing of turning upside down
In column CP6.Room temperature 8,000rpm/min are centrifuged 2min, outwell liquid in collecting pipe, adsorption column CP6 is reentered into the same receipts
In collector；
(9) 10mL rinsing liquid PW, room temperature 8 are added into step (8) adsorption column CP6,000rpm/min is centrifuged 2min, abandons and receives
Waste liquid in collector, adsorption column is placed back in collecting pipe；
(10) repetitive operation step (9) is primary；
(11) 3mL dehydrated alcohol, room temperature 8 are added into step (10) adsorption column CP6,000rpm/min is centrifuged 2min,
Fall waste liquid；
(12) step (11) adsorption column CP6 is placed back in collecting pipe, room temperature 8,000rpm/min is centrifuged 5min.It will inhale
Attached column CP6 uncaps, and is placed in and is placed at room temperature for several minutes and dries；
(13) adsorption column in step (12) is put into clean 50mL centrifuge tube, it is slow that 1-2mL is added in adsorbed film center
Fliud flushing TB is stored at room temperature 5min, room temperature 8, and 000rpm/min is centrifuged 2min, and the eluent in 50mL centrifuge tube is all moved into one
A clean 1.5mL centrifuge tube surveys concentration, -20 DEG C of preservations.
(14) it takes the obtained plasmid DNA solution of 1-2 μ L to carry out agarose gel electrophoresis and saves electrophoresis result data.
The foundation of embodiment 3:pEE12.4-OPTI-AE2 Transfected Recombinant Plasmid CHO-K1 cell and monoclonal screening
3.1 CHO-K1 cell transfectings
(1) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；DMEM/F12 (containing 10% serum, 1% is dual anti-), DMEM/F12
37 DEG C of water-baths, which are placed in, with PBS is preheated to 37 DEG C.
(2) cell (10cm Tissue Culture Dish) is taken out from 37 DEG C of incubators, culture medium is discarded supernatant, with the 8mL of pre-temperature
It is primary that PBS washes cell, and discards PBS.
(3) 1-2mL 0.25%trypsin-EDTA is added in each 10cm Tissue Culture Dish, and room temperature digests 2min or so, shows
Micro- microscopic observation cell shrinkage is rounded, and is in individual cells.
(4) 4mL DMEM/F12 (contain 10% serum, 1% is dual anti-) is added and terminates digestion reaction, and with pipettor by cell
It dispels.
(5) cell digested is transferred in 15mL centrifuge tube, room temperature centrifugation, 200g, 5min.
(6) it with DMEM/F12 (containing 10% serum, 1% is dual anti-) again suspension cell, counts.
(7) diluting cells are to 2 × 105A/mL, the cell for taking 2mL to mix are added to six orifice plates, and six orifice plates are placed into 37
DEG C, 5%CO2It is incubated overnight in cell incubator.
(8) step (7) Tissue Culture Dish is taken out, observes cell state: when cell degree of crossing reaches 80%-90%
Start to transfect, culture medium is changed into the DMEM/F12 of antibiotic-free serum-free, the hole 2mL/ before transfection.
(9) it dilutes plasmid: diluting plasmid with OPTI-MEM, 2.5 μ g plasmids are added in 125 μ L OPTI-MEM, are then added
2.5 μ L plus mix, are stored at room temperature 5min.
(10) it dilutes in Lipofectamine LTX:125 μ L OPTI-MEM and 9 μ L Lipofectamine LTX is added,
Then 2.5 μ L plus are added, mixes gently, is stored at room temperature 5min.
(11) step (10) and step (11) mixture are mixed gently.It is placed at room temperature for 5min, six holes are then added dropwise
It is uniformly distributed in plate.
(12) six orifice plates are placed in 37 DEG C, 5%CO24-6h is cultivated in cell incubator.
(13) it changes liquid: discarding supernatant culture medium, 2mL DMEM/F12 (dual anti-containing 10% serum 1%) is added, by six orifice plates
37 DEG C are placed in, 5%CO2It is cultivated in cell incubator.
3.2 pressurization screenings
Start to pressurize for 24 hours after transfection: taking out six orifice plate cells from 37 DEG C of incubators, discard supernatant culture medium, 2mL is added
DMEM/F12 (+25 μM of MSX containing 10% serum), pressurize 7d, and centre observation cell, dead cell changes liquid more.
The screening of 3.3 monoclonals
(1) when death ray basic to negative control cell is screened in pressurization, about 7days starts monoclonal screening.
(2) six orifice plates are taken out, culture medium is discarded, PBS is washed once, 300 μ L 0.25%trypsin-EDTA are then added,
Room temperature digests 2min or so, and 2mL DMEM/F12 (+25 μM of MSX containing 10% serum) are added and terminate digestion reaction, and use pipettor
Cell is dispelled.
(3) cell digested is transferred in 15mL centrifuge tube, room temperature centrifugation, 200g, 5min.
(4) DMEM/F12 (+25 μM of MSX containing 10% serum) suspension cell again is used, is counted.
(5) bed board: diluting cells to 5/mL, the cell for taking 200 μ L to mix are added in 96 orifice plates, are placed into 37 DEG C,
5%CO24-6h is incubated in cell incubator.
(6) hole of individual cells is recorded.
(7) when the hole length of individual cells in 96 orifice plates is got up, culture medium is discarded, PBS is washed once, and 100 μ L are added
0.25%trypsin-EDTA, room temperature digest 2min or so, and 2mL DMEM/F12 (+25 μM of MSX containing 10% serum) are added and terminate
Digestion reaction, and dispelled cell with pipettor.Cell liquid is transferred to 12 orifice plates, when 12 orifice plates cover with, takes supernatant,
Whether ELISA detection clone is the positive, and the positive colony of high efficient expression continues to expand culture, freeze.
The domestication of embodiment 4:CHO-K1 cell strain is cultivated at suspending
(1) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；DMEM/F12 (containing 10% serum, 25 μM of MSX) is placed in 37 DEG C
37 DEG C are preheated in water-bath.
(2) cell (10cm Tissue Culture Dish) is taken out from 37 DEG C of incubators, culture medium is discarded supernatant, with the 8mL of pre-temperature
It is primary that PBS washes cell, and discards PBS.
(3) 1-2mL 0.25%trypsin-EDTA is added in each 10cm Tissue Culture Dish, and room temperature digests 2min or so, shows
Micro- microscopic observation cell shrinkage is rounded, and is in individual cells.
(4) 4mL DMEM/F12 (contain 10% serum, 25 μM of MSX) is added and terminates digestion reaction, and with liquid-transfering gun by cell
It dispels.
(5) cell digested is transferred in 15mL centrifuge tube, room temperature centrifugation, 200g, 5min.
(6) it with 100%DMEM/F12 (containing 10% serum, 25 μM of MSX) suspension cell, counts.
(7) diluting cells are to 5 × 105A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Cell culture
Bottle is placed into 37 DEG C, 5%CO2120rpm/min is incubated overnight on rail mounted oscillator in cell incubator.
(8) bio-safety counter top is sterilized with 75% alcohol wipe, ultraviolet irradiation 30min.
(9) every counting cell density and vigor for 24 hours.
(10) second generation culture is carried out when cell survival rate reaches 94-97% after first generation cell culture is primary.
(11) prepare: Biohazard Safety Equipment ultraviolet sterilization 30min；100%DMEM/F12 (contains 10% serum, 25 μM of MSX),
EX-CELL 302 is placed in CO237 DEG C are preheated in cell incubator.
(12) it takes out cell from 37 DEG C of incubators to be transferred in 50mL centrifuge tube, room temperature 200g is centrifuged 5min.
(13) DMEM/F12 (containing 10% serum, 25 μM of MSX) and EX-CELL 302 are mixed by 1:1, is suspended again thin
Born of the same parents count.
(14) diluting cells are to 5 × 105A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Cell culture
Bottle is placed into 37 DEG C, 5%CO2120rpm/min is incubated overnight on rail mounted oscillator in cell incubator.
(15) bio-safety counter top is sterilized with 75% alcohol wipe, ultraviolet irradiation 30min.
(16) every counting cell density and vigor for 24 hours.
(17) cell survival rate that second generation culture obtains afterwards twice is greater than 95%；Third is commissioned to train to support to six and be obtained afterwards three times
Cell survival rate be greater than 95%.After 7 weeks, cell inoculation breeds three generations after 3 days, and density reaches 1 × 106A cell/mL, simultaneously
Cell survival rate reaches 95%, which is considered being already adapted to the culture that suspends.Inoculum density is reduced to 3 × 105A/mL.
(18) through taming, 12F9 plants, 12C11 plants are all met the requirements, this shows that 12F9 plants, 12C11 plants are all tamed successfully.
Embodiment 5: cell shake flask fermentation
(1) preparation of secondary culture base: the Ex-cell 302 of 60% CD-CHO+40% is placed in 37 DEG C of water-baths in advance
Heat is to 37 DEG C.
(2) from CO2Constant-temperature table takes out shaking flask cell, is counted.
(3) dilution embodiment 4 obtains, 12F9 plants, 12C11 plants of cells to 2.5-3.5 × 105A cell/mL inoculation
30mL culture is based in a 125mL shaking flask.Tissue Culture Flask is placed into 37 DEG C, 5%CO2100rpm/min is incubated in constant-temperature table
It educates overnight.
(4) every counting cell density and vigor for 24 hours, glucose is surveyed, when blood glucose is lower than 2g/L, adds glucose
To 4g/L；1mL sample is taken daily, and supernatant is for detecting protein expression situation.
(5) feed supplement (the about the 4th day): supplement 70g/L CB5 adds the 10% of basal medium.
Start within (6) the 5th days, by CO2Incubator temperature is adjusted to 32 DEG C.
(7) the 9th days, 70g/L CB5 is supplemented, the 10% of basal medium is added.
(8) the 12nd days, harvest cell.
Embodiment 6: protein purification
The cell culture fluid (every batch of about 100ml) of embodiment 5 is collected, 4 DEG C, 8,000g centrifugation 30min take supernatant, mistake
0.8 μm of filter membrane, loading are reserved 5 × SDS- sample buffer that 20 μ L are added in 80 μ L samples, are detected for SDS-PAGE.
Column equilibration: with ultrapure 2~3CV of water balance (column volume column volume), ethyl alcohol is discharged and saves liquid；Then it uses
BufferA(20mM NaH2PO4(pH 7.4), 500mM NaCl) balance 2~3CV, 4~7mL/min.
Loading: if 5mL prepacked column one, 1mL/min carries out loading and (adjusts loading flow velocity according to prepackage column volume, retain
Time 5min), it collects Flow through (FT), takes 80 μ L samples that 5 × SDS- sample buffer of 20 μ L is added, be used for SDS-
PAGE detection.
Washing: 4%bufferB (20mM NaH is used2PO4(pH 7.4), 500mM NaCl, 20mM imidazole) column is washed,
Flow velocity is 4mL/min, and the weaker foreign protein of the albumen and binding ability of unbonded upper prop is rinsed well, until OD280nm baseline
Until steady.
Elution: 50%bufferB (20mM NaH2PO4(pH 7.4), 500mM NaCl, 250mM imidazole) elution
Destination protein is collected until baseline washes flat, 2mL/min: 10mL/ pipe；(Elutethrough-ET) takes 80 μ after collecting sample mixing
5 × SDS- sample buffer of 20 μ L is added in L sample, detects for SDS-PAGE.
Washing: 100%bufferB (20mM NaH2PO4 (pH 7.4), 500mM NaCl, 500mM imidazole),
4mL/min is not collected, rinse 2-3 column volume, until UV baseline wash it is flat.Ultrapure 2~3CV of water balance.Save HisTrap
Excel column can save liquid with 20% ethyl alcohol and balance 2~3CV.
Liquid is changed in dialysis: the imidazole elution containing destination protein being poured into bag filter, with 1 × PBS dialysis at least 1,000
Times, take 80 μ l to keep sample detection.
Aseptic filtration: in Biohazard Safety Equipment, 0.22 μm of low protein binding syringe needle filter is crossed, or a large amount of protein solutions are crossed and gone out
The filter of the Nalgene of 0.22 μm of filter membrane of bacterium, filtered protein solution sample deposit in -80 DEG C of refrigerators.
Determination of protein concentration: protein concentration is measured using BCA method, this batch of protein concentration is respectively 1.2mg/ml, 1.28mg/
Ml, volume are each about 38ml；By calculating (albumen yield=protein concentration * albumen volume/taken fermentation supernatant volume), 12F9
Strain, 12C11 plants of albumen yield are each about 400-500mg/L.
The identification of embodiment 7:AE2 albumen
7.1 SDS-PAGE detection
The albumen of embodiment 6 after purification is subjected to SDS-PAGE detection, AE2 protein concentration is 2 holes μ g/ in sample used,
As a result as shown in Figure 5: it can be calculated that AE2 protein SDS-PAGE purity after purification is 85%, molecular weight is about Cong Tuzhong
40kDa。
7.2 Werstern Blot detection
The albumen of embodiment 6 after purification is subjected to Werstern Blot detection, AE2 albumen (marks in figure in sample used
It is 2 holes μ g/ for 2) concentration, BSA albumen (label is in figure) concentration is 2 holes μ g/；Primary antibody used is from non-typical swine fever disease
The pig serum of poison infection, dilution ratio 1:100；Secondary antibody is the goat-anti pig IgG secondary antibody of HRP label, thinner ratio 1:5000.
As a result as shown in Figure 6: the serum can be in conjunction with AE2 protein-specific, and not in conjunction with negative control BSA.
7.3 deglycosylations detection
The albumen of embodiment 6 after purification is first handled with deglycosylation enzyme: 1) taking the AE2 albumen (about 2 μ g) of 9 μ l after purification
In 200 μ l EP pipes, 1 μ 10 × glycoprotein of l denaturation buffer (commercialization enzyme self-contained reagent) is added；2) it boils for 100 DEG C
10min makes albuminous degeneration；3) 2 μ 10 × G7 of l buffers (commercialization enzyme self-contained reagent), 2 μ l 10%NP-40 (commodity are added
Change enzyme self-contained reagent), 1-2 μ l PNGaseF (glycosidase F), moisturizing makes reaction system to 20 μ l；4) 37 DEG C of incubation 1h；5) it reacts
After, 5 μ l 5 × loading buffer are added, boil 10min, for use；
Above-mentioned sample is subjected to Werstern Blot detection, the AE2 albumen in sample used before deglycosylation is (in figure
It is 2 holes μ g/ labeled as 2) concentration, BSA albumen (label is in figure) concentration is 2 holes μ g/, the AE2 albumen after deglycosylation
(in figure label be) is glycosylase treated sample, and when loading is all used as a sample to be added in a hole and carries out WB
Detection；Primary antibody used derives from the pig serum of non-typical swine fever virus infection, dilution ratio 1:100；Secondary antibody is HRP label
Goat-anti pig IgG secondary antibody, thinner ratio 1:5000.As a result as shown in Figure 6: molecular weight is about 27kD after AE2 albumen deglycosylation,
Smaller than before deglycosylation 13kD, AE2 albumen has about 13/40*100%=32.5% when this explanation is expressed in Chinese hamster ovary celI
Glycosylation；Poorer than before deglycosylation with the specific binding of serum after the deglycosylation of AE2 albumen, this illustrates to glycosylate
Served in the immunogenicity of AE2 albumen it is very important, therefore preferably in lactation when using AE2 albumen as immunogene
It is expressed in zooblast.
7.4 Elisa detection
(1) it is coated with: with coating buffer (50mM carbonate buffer solution, pH 9.5) respectively by the AE2 albumen of purifying, BSA albumen
It is diluted to 0.5 μ g/ml, every kind of antigen is coated with 8 holes (4 hole increase serum samples, 4 holes add confining liquid as control), every kind of antigen
100 holes μ l/ are added, 4 DEG C of refrigerators are stood overnight after sealed membrane is sealed；
(2) it washs: after refrigerator taking-up ELISA Plate, with PBST board-washing 5 times；
(3) close: 200 μ l confining liquids (5% defatted milk), 37 DEG C of incubation 2h after sealed membrane is sealed are added in every hole；
(4) serum dilutes: the pig serum of non-typical swine fever virus infection confining liquid is diluted 100 times, and (such as 495 μ l are dilute
It releases and 5 μ l serum, mixing is added in liquid)；
(5) it washs: with (2)；
(6) it is loaded: dilute serum is added, while being negative control, 37 DEG C of incubation 1h with confining liquid；
(7) it washs: with (2)；
(8) add secondary antibody: the 100 μ l of goat-anti pig IgG secondary antibody of the HRP label of dilution (thinner ratio 1:5000) is added in every hole,
37 DEG C of incubation 0.5h；
(9) it washs: with (2)；
(10) develop the color: the TMB developing solution of 100 μ l, 37 DEG C of incubation 10min are added in every hole under the conditions of being protected from light；
(11) terminate: 50 μ l terminate liquid (2M H are added in every hole2SO4), terminate reaction；
(12) it detects: measuring sample OD value in 450nm wavelength, analyze data；
(13) result is as shown in the table: coating AE2 albumen can with serum specificity ining conjunction with, OD450 mean value be 0.955,
And be coated with BSA cannot in conjunction with serum specificity, OD450 mean value be 0.05；It is coated with AE2 albumen and BSA albumen and confining liquid is equal
It does not specifically bind, OD450 mean value is 0.05.This explanation, AE2 albumen can be used as the antigen of Elisa kit, groping
After suitable peridium concentration and serum thinner ratio, a kind of detection non-typical swine fever virus infection and immune diagnosis examination can be developed
Agent box.
The verifying of 7.5 stability
The albumen of embodiment 6 after purification is diluted to 1mg/ml, total 10ml with PBS, is divided into 2 parts, every part of 5ml；Portion is set
In 37 DEG C of water-baths, every two hours sampling is primary, each 0.5ml, and continuous sampling 10 times；Portion is placed in 4 DEG C of refrigerators, weekly
Sampling is primary, each 0.5ml, and continuous sampling 10 times；Protein concentration is detected with BCA after every sub-sampling, as a result as shown in the table:
From the point of view of the variation of protein concentration, albumen kept stable in two groups of experimentations.In order to further verify place
Whether the immunogenicity of the albumen after reason changes, we carry out Werstern Blot detection with the tenth sample, specific to detect
For method as described in 7.2, concrete outcome is as shown in Figure 7: wherein M indicates Marker；1 is negative control BSA albumen, and applied sample amount is 2 μ
g；2 be 37 DEG C of treated AE2 albumen, and applied sample amount is 2 μ g；3 be 4 DEG C of treated AE2 albumen, and applied sample amount is 2 μ g；From figure
As can be seen that treated, sample (the tenth sub-sampling) still has good immunogenicity.
Embodiment 8: vaccine preparation and immunization experiment
8.1 vaccine preparation
Water phase prepares: using PBS (or physiological saline) that the dilution of AE2 albumen is appropriate dense according to AE2 protein content in vaccine
Degree, as water phase；
It is oily mutually to prepare: it is 1:1, volume ratio 46:54 according to antigen phase and adjuvant weight ratio according to preparing vaccine total amount,
Measure appropriate 201 VG adjuvant of ISA；
Emulsification: water phase and oil are mutually all preheating to 33 DEG C, water phase is slowly added in oily phase, 200-500rpm stirs 20-
30min is placed in 4 DEG C overnight in 20 DEG C of standing 1h；
Packing, storage: being dispensed as needed, is saved backup after inspection is qualified in 4 DEG C.
8.2 vaccine quality inspections
Physical behavior observes appearance (whether being milky emulsion) using the method that eye is seen；
A small amount of vaccine drop is drawn in cold water using cleaning suction pipe, observation (in addition to the 1st drop), vaccine should be cloud expansion
It dissipates, is determined as W/O/W dosage form；
Vaccine 10ml is added in centrifuge tube, 15min is centrifuged with 3000r/min, the water phase that tube bottom is precipitated answers≤0.5mL,
It is judged to stablize；
Viscosity measurements are carried out to vaccine using viscosity apparatus, Ying 20-50cp is judged to qualification.
8.3 immunization experiment
In order to grope suitable antigenic content, vaccine (the specific preparation of not synantigen has been done using 201 VG adjuvant of ISA
For method referring to 8.1 and 8.2), specific vaccine information is as shown in the table:
The piglet (APPV antigen negative, negative antibody) for screening 30 30 ages in days or so, is randomly divided into 6 groups, and every group 5,
Group 1- group 5 distinguishes immune vaccine 1- vaccine 5, and group 6 is not immune as a control group, and every group is often inferior to musculi colli injection 1ml vaccine.
Carry out two with identical approach and dosage and exempt within 21st day after immune, after exempting from one and two exempt from after observation pig whether have exception, and
Before acquisition is immune respectively, two exempt from before, two exempt from 14 days serum detection antibody titers.
In 14 days observed after exempting from 21 days observed after exempting from one with two, all pig body temperature are normal, also different without other
Often, therefore 5 batches of vaccines are safe.Antibody titer (tool in immune preceding and Post-immunisation serum is detected using the method for Elisa
Body detecting method is shown in the 7.4 of embodiment 7, this detection is only coated with AE2 albumen, and each blood serum sample does a repetition), knot
Fruit it is as shown in the table (all numerical value are average OD450 value after 5 pigs detections, the STEDV value of every group of data 10% with
Under): before two exempt from, with the increase of immunizing dose, OD450 mean value is also being increased with it, but after two exempt from, OD450 mean value
Increase unobvious；OD450 mean value is 1.25 or more after immune group used is exempted from two, than positive serum used in embodiment 7
Potency is intended to height.
The present invention is illustrated by above embodiment, it is understood, however, that the present invention is not limited to institutes here
The particular example and embodiment of description.Purpose herein comprising these particular examples and embodiment is to help this field
In technical staff practice the present invention.Any those of skill in the art are easy to do not departing from spirit and scope of the invention
In the case of be further improved and perfect, therefore the present invention is only by the content of the claims in the present invention and the limit of range
System, intention, which covers, all to be included the alternative in the spirit and scope of the invention as defined by appendix claim and waits
Same scheme.
Sequence table
<110>Zhejiang oceanic rise Biotechnology Co., Ltd
<120>stable non-typical swine fever viral sub-units albumen and its vaccine and preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 4
<211> 621
<212> DNA
<213>the AE2 gene order () after codon optimization
<400> 4
tcctgtcaca agcgccagga ccattacaat atccagctgg tggtggagga gaagacaggc 60
gtggagaaga ggtctatcat gggcaagtgg accgtgatca caagagaggg aagggagcca 120
aggctgatgg agcagatcaa catggtgtcc aatgactccc tgagcgagac ctactgctat 180
aaccggctga atacatccag ctggggcaga cagcctgcta ggcagagggg atgtggacag 240
accgtgccat actggcctgg cgataacgtg ctggaggagc agtactattc caccggctat 300
tgggtgaatg ctacaggagg atgccagctg agggagggcg tgtggctgag caggaagggc 360
aacgtgcagt gtcagcggaa tggctcttcc ctgatcctgc agctggccat caaggaggag 420
aacgacacca tggagatccc atgcgatccc gtggagacag agagcatggg acctgtggct 480
cagggaacct gcgtgtactc ttgggccttc gctccacggg gctggtacta taacagaaag 540
gacggctact ggctgcagta catcaagaag aatgattacc agtactggac aaagatgcca 600
accacaagct ctgccgctac c 621
<210> 4
<211> 621
<212> DNA
<213>the AE2 gene order () before codon optimization
<400> 4
tcgtgccaca aaagacaaga ccattacaat atccagctag ttgtcgaaga aaaaacaggt 60
gtagaaaaac gatctataat gggcaaatgg actgtgataa ccagggaagg ccgggaacca 120
agattgatgg agcaaataaa tatggtgtca aatgatagcc tgtcagaaac ttactgctat 180
aataggctaa ataccagtag ttgggggcga caaccggcaa gacagagagg gtgtggtcag 240
actgtgccct attggcctgg tgacaatgtt ctagaagaac aatactatag tacaggttac 300
tgggtgaacg caacaggcgg ttgtcaactg agagaaggcg tgtggctgtc aagaaagggc 360
aatgtacagt gccagcgtaa cggctcatcc ttaatactgc aattggcgat aaaagaagag 420
aatgacacta tggaaatacc atgtgatcct gtggaaacag aaagcatggg tccagttgca 480
cagggcactt gtgtatatag ctgggcattc gccccaagag ggtggtatta taataggaaa 540
gacggttatt ggcttcagta cataaagaaa aatgactacc agtactggac aaaaatgcct 600
actacctcgt ccgctgcaac g 621
<210> 4
<211> 207
<212> PRT
<213>AE2 amino acid sequence ()
<400> 4
Ser Cys His Lys Arg Gln Asp His Tyr Asn Ile Gln Leu Val Val Glu
1 5 10 15
Glu Lys Thr Gly Val Glu Lys Arg Ser Ile Met Gly Lys Trp Thr Val
20 25 30
Ile Thr Arg Glu Gly Arg Glu Pro Arg Leu Met Glu Gln Ile Asn Met
35 40 45
Val Ser Asn Asp Ser Leu Ser Glu Thr Tyr Cys Tyr Asn Arg Leu Asn
50 55 60
Thr Ser Ser Trp Gly Arg Gln Pro Ala Arg Gln Arg Gly Cys Gly Gln
65 70 75 80
Thr Val Pro Tyr Trp Pro Gly Asp Asn Val Leu Glu Glu Gln Tyr Tyr
85 90 95
Ser Thr Gly Tyr Trp Val Asn Ala Thr Gly Gly Cys Gln Leu Arg Glu
100 105 110
Gly Val Trp Leu Ser Arg Lys Gly Asn Val Gln Cys Gln Arg Asn Gly
115 120 125
Ser Ser Leu Ile Leu Gln Leu Ala Ile Lys Glu Glu Asn Asp Thr Met
130 135 140
Glu Ile Pro Cys Asp Pro Val Glu Thr Glu Ser Met Gly Pro Val Ala
145 150 155 160
Gln Gly Thr Cys Val Tyr Ser Trp Ala Phe Ala Pro Arg Gly Trp Tyr
165 170 175
Tyr Asn Arg Lys Asp Gly Tyr Trp Leu Gln Tyr Ile Lys Lys Asn Asp
180 185 190
Tyr Gln Tyr Trp Thr Lys Met Pro Thr Thr Ser Ser Ala Ala Thr
195 200 205
Claims (11)
1. a kind of stable non-typical swine fever viral sub-units albumen, which is characterized in that the non-typical swine fever viral sub-units
The amino acid sequence of albumen is as shown in SEQ ID NO3.
2. non-typical swine fever viral sub-units albumen according to claim 1, which is characterized in that the non-typical swine fever disease
Coding gene sequence after malicious subunit protein optimization is as shown in SEQ ID NO.1.
3. any non-typical swine fever viral sub-units albumen according to claim 1~2, which is characterized in that the SARS
The expression system of type swine fever virus subunit protein includes Escherichia coli, yeast, mammalian cell and insect cell.
4. non-typical swine fever viral sub-units albumen according to claim 3, which is characterized in that the mammalian cell
For Chinese hamster ovary celI.
5. a kind of method for preparing any non-typical swine fever viral sub-units albumen of Claims 1 to 4, feature exist
In the described method comprises the following steps:
1) gene order shown in SEQ ID NO.1 is cloned into carrier for expression of eukaryon, is obtained containing non-typical swine fever virus
The recombinant plasmid of subunit protein encoding gene；
2) again by the Transfected Recombinant Plasmid containing non-typical swine fever viral sub-units protein coding gene into Chinese hamster ovary celI；
3) by Chinese hamster ovary celI strain described in culture, screening, domestication step 2), the cell strain that height is expressed is obtained；
4) fermented and cultured 3) described in cell strain obtain non-typical swine fever viral sub-units albumen after purification.
6. according to the method described in claim 5, it is characterized in that, the carrier for expression of eukaryon is pEE12.4.
7. according to the method described in claim 5, it is characterized in that, the Chinese hamster ovary celI is CHO-K1 cell.
8. a kind of subunit vaccine of non-typical swine fever viral sub-units albumen, which is characterized in that the non-typical swine fever virus
Subunit vaccine contains non-typical swine fever viral sub-units albumen and assistant described in Claims 1 to 4 any claim as above
Agent.
9. subunit vaccine according to claim 8, which is characterized in that the non-typical swine fever viral sub-units albumen
Dosage is g/ parts of 15~200 μ.
10. subunit vaccine according to claim 9, which is characterized in that the non-typical swine fever viral sub-units albumen
Dosage be g/ parts of 30~100 μ.
11. a kind of non-typical swine fever viral sub-units albumen according to any one of claims 1 to 4 is in non-typical swine fever virus
Application in prevention and treatment and dependent diagnostic reagent.
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| CN112341525A (en) * | 2019-08-09 | 2021-02-09 | 浙江海隆生物科技有限公司 | Recombinant African swine fever virus pE120R subunit soluble protein and preparation method and application thereof |
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| CN112341525B (en) * | 2019-08-09 | 2024-02-09 | 浙江海隆生物科技有限公司 | Recombinant African swine fever virus pE120R subunit soluble protein and preparation method and application thereof |
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