CN109134668A - Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine - Google Patents

Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine Download PDF

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CN109134668A
CN109134668A CN201811104818.2A CN201811104818A CN109134668A CN 109134668 A CN109134668 A CN 109134668A CN 201811104818 A CN201811104818 A CN 201811104818A CN 109134668 A CN109134668 A CN 109134668A
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fusion protein
expression
vaccine
preparation
fabricius virus
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贺笋
任立松
李延涛
任郭子君
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Tiankang Biological Ltd By Share Ltd
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Abstract

The present invention relates to field of biotechnology, specifically, providing fusion protein of a kind of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine.Fusion protein includes VP2 section and VP3 section, wherein the expression of VP2 section nucleotide sequence as shown in SEQ ID NO.1, the expression of VP3 section nucleotide sequence as shown in SEQ ID NO.2.The fusion protein is that the gene order of the VP2 of chicken infectivity bursa of Fabricius virus and VP3 is carried out expressing in series to obtain, the present invention is analyzed by the gene order to VP2 and VP3, it is wherein antigenic good to choose, it connects in easy highly expressed region, obtained fusion protein has antigenic good, the high advantage of expression quantity.The fusion protein, which is prepared vaccine as antigen, has safety good, titer plateaus, the advantage having no toxic side effect.The present invention has been provided with the preparation method and application of fusion protein and the vaccine of preparation.

Description

Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression System and vaccine
Technical field
The present invention relates to field of biotechnology, in particular to a kind of fusion egg of chicken infectivity bursa of Fabricius virus Bletilla preparation method, application, expression system and vaccine.
Background technique
Bursal Disease (Infectious bursal disease, IBD) is by Bursal Disease disease A kind of urgency of 3-12 week old chick caused by malicious (Infectious bursal disease virus, IBDV) and young chicken chicken Property, high degree in contact immunosupress infectious disease.
IBDV is no togavirus, and binodal section, double-stranded RNA genome are in icosahedral symmetry structure, size 60- 70nm.Its nucleocapsid is made of dsRNA (double stranded RNA) and protein.It is also common seedless in addition to complete virion The virus hollow capsid of sour structure.The observation of negative staining virus three-dimensional structure Electronic Speculum shows IBDV virion and aspherical, is in T =13 is symmetrical, and it is 72nm that five times of axis, which extend diameter, and it is 66nm that three times axis, which extends diameter,.
IBDV is mainly to encroach on chick lymphoid tissue, and especially central immune organ-bursa of farbricius is main feature, leads to chicken Gumboro disease only occurs.Bursal Disease is to endanger one of Important Infectious Diseases of poultry husbandry.Diseased chicken is shown as Peck anus, material reducing, spirit is depressed, and feather is fluffy and disorderly, and coat is inverse vertical, and drowsiness, eyelid closure is reluctant to walk about, white or watery diarrhea, anus Door surrounding feather is dirty.Due to other pathogenic microorganisms of secondary infection, second day after infection starts have the phenomena of mortality successively Occur, peak mortality phase generally after infection the 3-4 days, the starts to restore after 5-7 days.
The vaccine of prevention Bursal Disease is mostly inactivated vaccine, malicious vaccine living, recombination live virus both at home and abroad at present Carrier bacterin etc., discovery has more deficiency in use, is mainly reflected in: poison vaccine living easily causes the bursa of farbricius to damage, can The effect of other vaccines can be interfered；Recombinant vaccine seedling is at high cost, and protective rate is low etc..
In view of this, the present invention is specifically proposed.
Summary of the invention
It is an object of the present invention to provide a kind of fusion protein of chicken infectivity bursa of Fabricius virus, the fusion proteins VP2 the and VP3 albumen of chicken infectivity bursa of Fabricius virus is subjected to expressing in series, to alleviate in the prior art, prevention chicken is infected The vaccine antigen source of property bursal disease is dangerous, the unstable technical problem of effect.
It is another object of the present invention to provide a kind of above-mentioned fusion proteins to answer preparation method, which can be real The above-mentioned fusion protein of existing great expression high quality.
It is another object of the present invention to provide one kind can express the protein expression system of above-mentioned fusion protein, can be with Direct rapid, high volume obtains above-mentioned fusion protein.
It is another object of the present invention to provide the applications of above-mentioned fusion protein.
It is another object of the present invention to provide a kind of vaccine containing above-mentioned fusion protein, the vaccine immunogenicities Good, potency is high, with low, poor efficacy or unstable of alleviating vaccine safety in the prior art, high production cost and protective rate is low Etc. technical problems.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of fusion protein of chicken infectivity bursa of Fabricius virus, including VP2 section and VP3 section；
The expression of VP2 section nucleotide sequence as shown in SEQ ID NO.1, the VP3 section is by SEQ ID NO.2 Shown in nucleotide sequence expression.
Further, VP2 section and VP3 section put in order as VP2-VP3, are connected by Linker；
The Linker has the nucleotide sequence as shown in SEQ ID NO.3.
Further, the fusion protein has the amino acid sequence as shown in SEQ ID NO.4.
A kind of preparation method of above-mentioned fusion protein, the gene of encoding said fusion protein is expressed in host.
Further, using the gene of mammalian expression systems expression encoding said fusion protein；
Preferably, using the gene of expressing cho cell system expression encoding said fusion protein；
Preferably, the expression vector of the gene comprising encoding said fusion protein is provided, the expression vector is imported into CHO In cell, then Chinese hamster ovary celI is screened, obtains stablizing the Chinese hamster ovary celI strain for expressing the fusion protein, the Chinese hamster ovary celI Strain expression obtains the fusion protein；
Preferably, the screening includes pressurization screening and monoclonal screening；
It preferably, further include that will stablize the Chinese hamster ovary celI for expressing the fusion protein to be domesticated for suspending in the preparation method The step of culture.
Further, the Chinese hamster ovary celI of the fusion protein is expressed using glutamine synthelase screening amplification system screening Strain；
Preferably, expression vector used in glutamine synthelase screening amplification system be pEE6.4 or pEE12.4。
A kind of protein expression system that can express above-mentioned fusion protein.
The application for the fusion protein that above-mentioned fusion protein or preparation method are prepared, including following A)-D) at least It is a kind of:
A chicken infectivity bursa of Fabricius virus vaccine) is prepared；
B the antibody of chicken infectivity bursa of Fabricius virus) is prepared；
C) the kit of preparation detection chicken infectivity bursa of Fabricius virus；
D chicken infectivity bursa of Fabricius virus diagnostic antigen) is prepared.
A kind of chicken infectivity bursa of Fabricius virus vaccine containing above-mentioned fusion protein.
Further, the concentration of fusion protein described in chicken infectivity bursa of Fabricius virus vaccine is 20-100 μ g/ml, excellent Select 30-80 μ g/ml, more preferable 50 μ g/ml；
Preferably, the vaccine further includes auxiliary material, the auxiliary material include in vaccine adjuvant, stabilizer and antibiotic at least It is a kind of；
Preferably, the vaccine adjuvant includes aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white oil assistant Agent, MF59 adjuvant or ISA201VG are, it is preferable to use ISA201VG.
Compared with prior art, the invention has the benefit that
The present invention provides a kind of fusion protein of chicken infectivity bursa of Fabricius virus, including VP2 section and VP3 section, In, the expression of VP2 section nucleotide sequence as shown in SEQ ID NO.1, VP3 section nucleotide as shown in SEQ ID NO.2 Sequence expression.The fusion protein is that the gene order of the VP2 of chicken infectivity bursa of Fabricius virus and VP3 is carried out expressing in series to obtain It arrives, the present invention is analyzed by the gene order to VP2 and VP3, and it is wherein antigenic good to choose, and easily highly expressed region carries out Series connection, obtained fusion protein have antigenic good, the high advantage of expression quantity.Vaccine tool is prepared using the fusion protein as antigen Have that safety is good, titer plateaus, the advantage having no toxic side effect.
The present invention provides the preparation method of above-mentioned fusion protein, and this method exists the gene order for encoding above-mentioned fusion protein It is expressed in host.
The present invention, which provides, can express the protein expression system of above-mentioned fusion protein, the expression system can with but unlimited attach most importance to Group carrier, recombinant cell etc., can letters using the protein expression system containing the system for expressing above-mentioned antigen-4 fusion protein gene sequence Just quickly purifying obtains a large amount of above-mentioned fusion protein.
The above-mentioned vaccine containing above-mentioned fusion protein provided by the invention, the vaccine safety is good, will not cause bad anti- It answers, unrelated antibody will not be generated, while potency is high and stability is good, the production cost of reducing property bursal disease vaccine, Protective rate is high.
Detailed description of the invention
Fig. 1 is PEE6.4-VP2-VP3 plasmid map schematic diagram in the embodiment of the present invention 2；
Fig. 2 is that the PCR of PEE6.4-VP2-VP3 plasmid in the embodiment of the present invention 2 identifies gel electrophoresis result figure；
Fig. 3 is that PEE6.4-VP2-VP3 plasmid double digestion identifies gel electrophoresis result figure in the embodiment of the present invention 2；
Fig. 4 is fusion protein S DS-PAGE testing result figure in the embodiment of the present invention 6.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
A kind of fusion protein of chicken infectivity bursa of Fabricius virus, including VP2 section and VP3 section, wherein VP2 section The expression of the nucleotide sequence as shown in SEQ ID NO.1, the expression of VP3 section nucleotide sequence as shown in SEQ ID NO.2.
VP2 and VP3 is the main component for forming virus nucleocapsid in IBDV structural proteins.The outer surface of IBDV nucleocapsid There are 260 VP2 tripolymers to be formed, account for about the 51% of virus protein, forms convex architecture on the surface of nucleocapsid.Nucleocapsid it is interior Surface is made of 200 Y type VP3 tripolymers, accounts for about the 40% of virus protein.
The fusion protein is that the gene order of the VP2 of chicken infectivity bursa of Fabricius virus and VP3 is carried out expressing in series to obtain It arrives, the present invention is analyzed by the gene order to VP2 and VP3, and it is wherein antigenic good to choose, and easily highly expressed region carries out Series connection, obtained fusion protein have antigenic good, the high advantage of expression quantity.Vaccine tool is prepared using the fusion protein as antigen Have that safety is good, titer plateaus, the advantage having no toxic side effect.
In some embodiments of the present invention, putting in order as VP2-VP3 for VP2 section and VP3 section passes through Linker Connection, wherein Linker has the nucleotide sequence as shown in SEQ ID NO.3.By the way that VP2 and VP3 is carried out with Linker Connection, both achieved the purpose that Protein reconstitution, in turn avoid VP2 and VP3 albumen respectively higher structure formed influence each other.
In some embodiments of the present invention, fusion protein has the amino acid sequence as shown in SEQ ID NO.4, by such as It is nucleotide sequence coded shown in SEQ ID NO.5 to obtain.
A kind of preparation method of above-mentioned fusion protein, the gene of encoding said fusion protein is expressed in host.This hair Host in bright can be, but not limited to as in escherichia expression system, yeast expression system, insect expression system, plant expression System or mammalian expression systems.After the translated processing of the albumen of mammalian cell expression, structure and biology Characteristic is learned closer to native protein, therefore present invention preferably uses mammalian expression systems expression to encode above-mentioned fusion protein Gene.
In the present invention, one is preferably carried out in mode, expresses encoding said fusion protein using mammalian expression systems Gene.After the translated processing of the albumen of mammalian cell expression, structure and biological characteristics are closer to natural Albumen, therefore present invention preferably uses the genes that mammalian expression systems expression encodes above-mentioned fusion protein.
In a preferred embodiment of the invention, above-mentioned fusion egg is encoded using expressing cho cell system expression White gene.Chinese hamster ovary celI, that is, Chinese hamster ovary cell (Chinese hamster ovary), has the advantages that
(1) there is folding and rhetorical function after accurately translating, the albumen of expression is in molecular structure, physicochemical property and biology Function aspect is learned closest to native protein molecule.
(2) have the function of product exocytosis, isolated and purified convenient for downstream product.
(3) with the efficient amplification and ability to express of recombination.
(4) there is adherent growth characteristic, tolerance shearing force and osmotic pressure ability with higher.It can also carry out suspension training It supports, expression is higher.
(5) CHO belongs to fibroblast, seldom secretes the endogenous protein of itself, conducive to the separation of foreign protein.
In some embodiments of the present invention, the expression vector of the gene comprising encoding above-mentioned fusion protein is provided, by table Up in vector introduction Chinese hamster ovary celI, then Chinese hamster ovary celI is screened, obtains the Chinese hamster ovary celI strain for stablizing expressed fusion protein, CHO Cell strain expresses to obtain fusion protein.This method is easy, easy to operate, can successfully filter out the avian infectious method of stable, high expression The eucaryotic cell strain of family name's bursal disease virus fusion protein, the eucaryotic cell strain obtained for later use do every research including preparing epidemic disease The application such as seedling provides feasible technical solution, lays a good foundation.
In some embodiments of the present invention, screening includes pressurization screening and monoclonal screening.In the present invention, it can choose Glutamine synthetase gene (glutaminesynthetase, GS) screens amplification system or dihydrofolate reductase gene (dihydrofolatereductase, dhfr) screening system carries out pressurization screening, both systems can be used alone, can also To be used in combination.
In the present invention, one is preferably carried out in mode, further includes that will stablize the expression fusion protein in preparation method Chinese hamster ovary celI is domesticated for the step of the cultivating that suspend.Be adherent growth under CHO general condition, which limit the growth of cell concentration and Attached cell be domesticated for can the suspending cell strain of culture can be efficiently solved this problem, increased by the expression of fusion protein Fusion protein expression reduces vaccine cost.
In the present invention, one is preferably carried out in mode, using in glutamine synthelase screening amplification system screening expression State the Chinese hamster ovary celI strain of fusion protein.It is available to be cultivated in selection after carrying the expression plasmid transfection CHO cell of GS gene The cell clone of basal growth, GS can be inhibited by methionine Asia maple (MSX), in the resisting cell that only a few survives, GS gene is expanded, and as a result will lead to the coamplification of the foreign gene together with GS gene tandem, and copy number can increase Several hundred to thousands of times, to make target gene high level expression, to offset the depression effect of MSX.
In certain embodiments of the present invention, expression vector used in glutamine synthelase screening amplification system is PEE6.4 or pEE12.4.When screening amplification system using glutamine synthetase gene, the base of the fusion protein will be first expressed Recombinant vector is obtained because being cloned on the expression vector with GS selection markers, is then imported recombinant vector in Chinese hamster ovary celI, so It is pressurizeed afterwards by using the culture medium culture Chinese hamster ovary celI containing MSX to Chinese hamster ovary celI, to filter out high expression target gene Cell strain, the present invention in GS selection markers expression vector can with but be not limited to pEE6.4 or pEE12.4.
In the present invention, one is preferably carried out in mode, and the preparation method of above-mentioned fusion protein is specific as follows:
First eukaryotic expression will be connected to by digestion after the chicken infectivity bursa of Fabricius virus VP 2-VP3 gene magnification of optimization Carrier pEE6.4 obtains the positive plasmid containing VP2-VP3 recombination by identification.Then by expanding culture, sun is extracted Property grain is transfected to Chinese hamster ovary celI.
Pressurization screening, transfection at least start to pressurize afterwards for 24 hours, pressurize 7 days；Be forced into negative control cell dead 90% with On, start monoclonal screening.
Cell strain domestication is cultivated at suspending, every counting cell and vigor for 24 hours, when first generation Cell viability reaches 94%- When 97% or so, second generation culture is carried out；The second generation is commissioned to train to the 6th support after obtained Cell viability reach 95% or more, 7 Cell inoculation bred for 3 generations after week after 3 days, and density reaches 1 × 106A/ml thinks that cell strain is tamed successfully at this time.
Cell strain is subjected to shake flask fermentation culture, collects cell culture fluid, centrifuging and taking supernatant, then filter membrane is purified, adopted Protein concentration is measured with BCA method, concentration about can achieve between 0.1-0.8g/L, detect purity using HPLC method, and purity can be with Reach 95% or more.
A kind of protein expression system that can express above-mentioned fusion protein.The protein expression system can with but it is unlimited for recombination Carrier, recombinant cell etc., can be easy using the protein expression system containing the system for expressing above-mentioned antigen-4 fusion protein gene sequence Quickly purifying obtains a large amount of above-mentioned fusion protein.
At least one of the fusion protein that above-mentioned fusion protein or above-mentioned preparation method are prepared is in following A)-D) Using:
A chicken infectivity bursa of Fabricius virus vaccine) is prepared；
B the antibody of chicken infectivity bursa of Fabricius virus) is prepared；
C) the kit of preparation detection chicken infectivity bursa of Fabricius virus；
D chicken infectivity bursa of Fabricius virus diagnostic antigen) is prepared.
It, can since chicken infectivity bursa of Fabricius virus fusion protein provided by the invention has preferable immunogenicity Preferably to prepare VP2-VP3 subunit vaccine to prepare chicken infectivity bursa of Fabricius virus vaccine, can produce after immune animal The titre of higher antibody.Use the antibody and the fusion protein of chicken infectivity bursa of Fabricius virus prepared by the fusion protein It can be applied to prepare a variety of detection reagents and kit, for detecting chicken infectivity bursa of Fabricius virus or antibody.For example, making Chicken infectivity bursa of Fabricius virus is detected with the ELISA kit containing chicken infectivity bursa of Fabricius virus antibody, or is used Chicken infectivity bursa of Fabricius virus antibody in colloidal gold immune chromatography test detection sample to be tested containing the fusion protein contains Amount.
A kind of chicken infectivity bursa of Fabricius virus vaccine containing above-mentioned fusion protein.The vaccine safety is good, Bu Huiyin Adverse reaction is played, unrelated antibody will not be generated, while potency is high and stability is good, the life of reducing property bursal disease vaccine Produce cost.It can produce higher antibody titer after the vaccine immunity animal, animal made to obtain preferable protecting effect.
In the present invention, one is preferably carried out in mode, the concentration of fusion protein in chicken infectivity bursa of Fabricius virus vaccine For 20-100 μ g/ml, preferably 30-80 μ g/ml, more preferable 50 μ g/ml.
In some embodiments of the present invention, vaccine further includes auxiliary material, specifically includes vaccine adjuvant, stabilizer and antibiotic At least one of；
Preferably, the vaccine adjuvant includes aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white oil assistant Agent, MF59 adjuvant or ISA201VG are, it is preferable to use ISA201VG.
In order to help to further understand the present invention, technical solution of the present invention is carried out now in conjunction with preferred embodiment detailed Explanation.
Unless otherwise specified, instrument or reagent commercially obtain in the embodiment of the present invention.The present invention is real It applies test method used in example unless otherwise specified, is conventional method.
Main agents and medicine source of the invention are as follows:
Chinese hamster ovary cell (CHO) is purchased from U.S. ATCC company；
Cell culture medium and serum are purchased from gibcom company of the U.S.；
Carrier for expression of eukaryon PEE6.4 is purchased from U.S. Thermo Fisher company；
Lipofectamine LTX is purchased from Beijing Suo Laibao Science and Technology Ltd；
Aminomethyl petrin (mnethotrexate MTX) is purchased from Sigma company；
Methionine sulfoxide imonium (L-methioninesulfoximine MSX) is purchased from Sigma company；
BCA quantification of protein kit is purchased from U.S. Thermo Fisher company；
ISA201VG is purchased from match BIC Corp of France.
The design of 1 VP2-VP3 gene of embodiment and synthesis
The design of VP2-VP3 gene and synthesis: after VP2, VP3 sequence in GenBank accession number M97346.1 is optimized, if VP2 length 1356bp is counted, there is the sequence as shown in SEQ ID NO.1, VP3 length 870bp has such as SEQ ID NO.2 institute The sequence shown.It is connected between VP2 and VP3 using Linker, Linker sequence has sequence shown in SEQ ID NO.3.VP2-VP3 base Because of overall length 2256bp, there is nucleotide sequence shown in the amino acid sequence as shown in SEQ ID NO.4 and SEQ ID NO.5.VP2- VP3 gene chemical synthesis is completed by Shanghai Sheng Gong biotech firm.
2 PEE6.4-VP2-VP3 construction of recombinant plasmid of embodiment
2.1 addition restriction enzyme sites: the upstream and downstream by PCR amplification in VP2-VP3 gene order adds digestion respectively Site: EcoR I, BamH I, PCR amplification upstream primer is as shown in SEQ ID NO.6, PCR amplification downstream primer such as SEQ ID Shown in NO.7.PEE6.4-VP2-VP3 plasmid map is as shown in Figure 1.
2.2 VP2-VP3 genes and carrier double enzyme digestion reaction
2.2.1 50 μ L reaction systems are constructed, according to following table by each component 37 DEG C of water-bath 2h after mixing.
10×buffer 5μL
DNA sample 2μg
Eco RI 2.5μL
Bam HI 2.5μL
dd H20 38μL
2.2.2 target DNA fragment recycles: DNA gel QIAquick Gel Extraction Kit (being purchased from Beijing Lai Bao Science and Technology Ltd.) is used, Digestion target fragment is recycled, steps are as follows:
(1) after agarose gel electrophoresis, target DNA band is carefully cut from Ago-Gel with blade, is put into In 1.5mL EP pipe, weight is weighed.
(2) 3 times of volume sol solutions are added into EP pipe, during which 50-55 DEG C of water-bath 10min is carefully turned over centrifuge tube, it is ensured that Blob of viscose sufficiently dissolves.
(3) previous step acquired solution is added in adsorption column (adsorption column is put into collecting pipe), 12000rpm is centrifuged 30- 60s outwells the waste liquid in collecting pipe, adsorption column is reentered into collecting pipe.
(4) 600 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collection Guan Zhong.
(5) 600 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collection Guan Zhong.
(6) 12000rpm is centrifuged 2min, as far as possible removing rinsing liquid.Adsorption column opening is placed in room temperature or 50 DEG C of incubators are placed 2min。
(7) adsorption column is put into 1.5mL EP pipe, is preheated in right amount through 65 DEG C of water-baths to the hanging dropwise addition in adsorbed film center Eluent, is placed at room temperature for 2min, and 12000rpm is centrifuged 1min.
2.3 VP2-VP3 genes and carrier connection reaction, construct 10 μ L reaction systems, then mix coupled reaction system Uniformly be placed on 10-16h in 16 DEG C of cold baths, after be put into 65 DEG C of water-bath 15min, last 4 DEG C of preservations.
10×T4buffer 1μL
DNA fragmentation 6μL
Carrier 2μL
T4 ligase 1μL
2.4 conversion reaction
10 μ L connection reaction solutions are added in 100 μ L DH5 α competent cells, ice bath 30min after mixing, 42 DEG C of water-baths 100s, then ice bath 2min.EP pipe is then taken out, 600 μ L LB culture solutions are added, is placed in 37 DEG C of constant-temperature tables, 240rpm cultivates 1h EP pipe is taken out afterwards, and room temperature is centrifuged 8000rpm, 2min, absorbs 500 μ L supernatants, and resuspension blows even thallus, the bacterium solution of resuspension is dripped to It converts on plate, is uniformly spread out bacterium solution with bacteria stick is applied.Then conversion plate is placed in constant incubator, 37 DEG C of culture 1h Afterwards, conversion plate inversion is subjected to culture 15h, observes conversion results after culture.
2.5 plasmid extractions and double digestion are identified
2.5.1 plasmid extracts, and using U.S.'s OMEGA plasmid extraction kit, extracting method is according to kit operation instruction Book extracts.
(1) single bacterium colony is into the LB liquid medium of the 5ml resistance of benzyl containing ammonia in picking reformer plate, and 37 DEG C, 220rpm training Support 8h.
(2) it takes bacterium solution 1.5ml into EP pipe, is centrifuged 10000rpm1min under room temperature, abandon supernatant, 250 μ are then added L solution I, oscillation mix；250 μ L solution IIs are added, carefully reverse EP pipe 4-6 times, are stored at room temperature 2min, until clarification, then 350 μ L solution IIIs are added, carefully reverse centrifuge tube 4-6 times, until there is white flock precipitate, the centrifugation of 10000rpm room temperature 10min。
(3) supernatant solution is carefully drawn, and moves to adsorption column center, 10000rpm room temperature is centrifuged 1min, outwells collecting pipe 500 μ L Buffer HB, 10000rpm are added after middle liquid and are centrifuged 1min, filtrate are abandoned, then plus such as 700 μ L Wash Buffer, 10000rpm are centrifuged 1min, abandon filtrate；It is repeated 1 times.
(4) room temperature is centrifuged suction attached column, and adsorption column is put into clean 1.5ml EP pipe, adds 30 by 10000rpm, 2min μ L deionized water is stored at room temperature 5min, 10000rpm, 2min on filter membrane.Save DNA solution in pipe.
2.5.2 PCR and double digestion identification
Recombinant plasmid is identified with PCR, as a result as shown in Figure 2, wherein M:marker；1:VP2-VP3；
Recombinant plasmid is identified with double digestion, constructs 20 μ L reaction systems:
10×buffer 2μL
DNA sample 1μg
Eco RI 1μL
Bam HI 1μL
Mend ddH2It is uniformly mixed after 0 to 20 μ L, detected through gel electrophoresis is carried out after 37 DEG C of water-bath 2h, and DNA fragmentation will be inserted into Company is sent to be sequenced, double digestion qualification result is as shown in Figure 3, wherein M:marker；1:VP2-VP3 and carrier.
2.6 go endotoxin plasmid to mention greatly, and using a large amount of extracts kits of endotoxin plasmid are gone, (Beijing Suo Laibao science and technology is limited Company)
(1) by sequencing, correctly clone is seeded in the culture medium of the 100ml resistance of benzyl containing ammonia, 240rpm, 37 DEG C of constant temperature trainings After supporting 15h, take 50ml bacterial cultures into 50ml centrifuge tube, 11000rpm is centrifuged 1min, absorbs supernatant.
(2) add 4ml solution P1, oscillator suspended bacterial cell precipitation, then plus 4ml solution P2, mild overturn 6-8 times make Thallus sufficiently cracks, and finally adds 4ml solution P3 again, and temperature is 6-8 times reverse immediately, mixes well, until there is white flock precipitate, After 11000rpm is centrifuged 10min, supernatant is moved on in another clean centrifuge tube.
(3) the Endotoxin removal agent of clear 1/5 volume being pre-chilled on ice is added, oscillation mixes, and ice bath 2min to solution becomes clear Bright, then 37 DEG C of water-bath 5min, vibrate frequently.
(4) 11000rpm room temperature is centrifuged 5min, and solution is divided into two-phase, and upper strata aqueous phase contains Plasmid DNA, and lower layer's oil mutually contains endogenous toxic material Upper strata aqueous phase containing Plasmid DNA is transferred to new pipe, abandons lower layer's oil phase by element；In triplicate.
(5) the combination liquid of 12ml is added, is added in adsorption column after mixing well, is placed at room temperature for 2min, 11000rpm centrifugation 1min outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
(6) 8ml rinsing liquid is then added, 11000rpm is centrifuged 1min, and waste liquid is abandoned, adsorption column is put into collecting pipe, then 6ml rinsing liquid is added, 11000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collecting pipe.Then 11000rpm is centrifuged Adsorption column opening is placed in room temperature or 50 DEG C of incubators places 4-5min by 3min.
(7) finally adsorption column is put into a clean centrifuge tube, is washed to adsorbed film drop 2ml through what 65 DEG C of water-baths preheated De- liquid, is placed at room temperature for 5min, and 11000rpm is centrifuged 2min, -20 DEG C of preservations.
Embodiment 3 transfects CHO-K1 cell
(1) cell is taken out, culture medium is discarded supernatant, is washed once with the 8ml PBS of pre-temperature, discard PBS, then each culture 2ml 0.25%trypsin-EDTA is added in ware, and room temperature digests 2min, and microscopic observation cell rounding is in individual cells.4ml is added DMEM/F12 (containing 10% serum, 1% ammonia benzyl-streptomysin is dual anti-) terminates digestion reaction, shifts after being dispelled cell with pipettor Into 15ml centrifuge tube, 200rpm is centrifuged 5min.
(2) DMEM/F12 (contain 10% serum, 1% ammonia benzyl-streptomysin dual anti-) suspension cell again, after counting, dilution is thin Born of the same parents are to 2 × 105A/ml, the cell for taking 2ml to mix are added to six hole culture dishes, set 37 DEG C, 5%CO2It is incubated in cell incubator Overnight.
(3) it observes cell state: starting to transfect when cell degree of crossing reaches 80%-90%, change culture medium before transfection DMEM/F12 (serum-free is without double antibody), the hole 2mL/.
(4) plasmid is diluted with OPTI-MEM, 2.5 μ g plasmids is added in 125 μ l OPTI-MEM, 2.5 μ l are then added Plus is stored at room temperature 5min after mixing.
(5) it dilutes in Lipofectamine LTX:125 μ l OPTI-MEM and 9 μ l Lipofectamine LTX is added, so After 2.5 μ l plus are added, mix gently, be stored at room temperature 5min.
(6) plasmid will be diluted and dilution Lipofectamine LTX is mixed and mixed, 5min is placed at room temperature for, then adds dropwise Enter and is uniformly distributed in six hole culture dishes.
(7) six hole culture dishes are placed in 37 DEG C, 5%CO2Liquid is changed after cultivating 4-6h in cell incubator: discarding supernatant culture Base is added 2ml DMEM/F12 (dual anti-containing 10% serum, 1% ammonia benzyl-streptomysin), six orifice plates is placed in 37 DEG C, 5%CO2 cell It is cultivated in incubator.
Embodiment 4: monoclonal cell strain screening
(1) start to pressurize for 24 hours after transfecting: taking out six hole culture dishes from 37 DEG C of incubators, discard supernatant, 2ml is added DMEM/F12 (+25 μM of MSX containing 10% serum), pressurize 7d, and during which under the microscope, dead cell changes liquid more.
(2) when pressurization screening to negative control cell death at least 90% or more, start monoclonal screening；
(3) six hole culture dishes are taken out, culture medium is abandoned, is washed once with PBS, 300 μ l 0.25%trypsin-EDTA are added, Room temperature digests 2min, adds 2ml DMEM/F12 (+25 μM of MSX containing 10% serum) to terminate digestion reaction, is blown cell with pipettor It dissipates, then cell is transferred in 15ml centrifuge tube, 200rpm is centrifuged 5min.
(4) again then cell is diluted to 5 to DMEM/F12 (+25 μM of MSX containing 10% serum) by suspension cell, counting A/ml takes 200 μ L to be added in 96 orifice plates, is placed into 37 DEG C, 5%CO2It is marked individually after being incubated for 4-6h in cell incubator The hole of cell.
When the hole of individual cells covers in (5) 96 orifice plates, culture medium is abandoned, PBS is washed once, and 100 μ l 0.25% are added Trypsin-EDTA, room temperature digest 2min, and 2ml DMEM/F12 (+25 μM of MSX containing 10% serum) are added and terminate reaction, with shifting Liquid device dispels cell, and cell liquid is then transferred to 12 orifice plates, when cell covers with, takes supernatant, ELISA detection, high efficient expression Continue to cultivate, freeze.
(6) by screening, 2 plants of cell strains are harvested altogether, number is 05 plant, 16 plants.
5 CHO-K1 cell strain of embodiment is tamed into suspended culture cell strain
(1) Tissue Culture Dish is taken out from 37 DEG C of incubators, discarded supernatant, it is primary to wash cell with 8ml PBS, and discards Then 2ml 0.25%trypsin-EDTA is added to culture dish in PBS, room temperature digests 2min, and microscopic observation cell is rounded by wrinkling, In individual cells.Then 4ml DMEM/F12 (containing 10% serum, 25 μM of MSX) is added and terminates digestion reaction, it will be thin with pipettor Born of the same parents dispel.Then cell liquid is transferred in 15ml centrifuge tube, 200rpm is centrifuged 5min.
(2) using 100%DMEM/F12 (contain 10% serum, 25 μM of MSX) suspension cell, after counting diluting cells to 5 × 105A/ml, inoculation 30ml culture set 37 DEG C, 5%CO based in 125ml shaking flask2On rail mounted oscillator in cell incubator 130rpm is incubated overnight, and counts 1 time every for 24 hours, observes cell density and vigor.
(3) when Cell viability reaches 94%-97% after first generation cell culture once, second generation culture is carried out, by the Generation cell is transferred in 50ml centrifuge tube, and 200rpm is centrifuged 5min, then by DMEM/F12 (containing 10% serum, 25 μM of MSX) It is mixed with EX-CELL302 by 1:1 mixing while after corresponding concentration puromycin is added, suspension cell, dilutes thin after counting again Born of the same parents are to 5 × 105A/ml, inoculation 30ml culture set 37 DEG C, 5%CO based in a 125ml shaking flask2Rail in cell incubator 120rpm is incubated overnight on road formula oscillator, and counts 1 time every for 24 hours, observes cell density and vigor.
(4) when second generation culture twice after obtained cell survival rate be greater than 95%, third to six, which is commissioned to train, supports three times afterwards The cell survival rate arrived is greater than 95%.After 7 weeks, cell inoculation breeds three generations after 3 days, and density reaches 1 × 106A/ml, at the same it is thin Born of the same parents' survival rate reaches 95%, which is considered being already adapted to the culture that suspends, and inoculum density is reduced to 3 × 105A/ml.By Domestication, the cell line that 05 plant of number meet the requirements, show to tame successfully.
6 cell fermentation of embodiment
(1) prepare culture medium: the Ex-cell302 of 60% CD-CHO+40% is placed in 37 DEG C of water-baths and preheats；
(2) from CO2Constant-temperature table takes out shaking flask cell, and diluting cells are to 3.0 × 10 after counting5A/ml, inoculation 30ml training It supports based in 125ml shaking flask, sets 37 DEG C, 5%CO2In constant-temperature table, 100rpm is incubated overnight.
(3) every cell count for 24 hours, density and vigor are observed, monitors concentration of glucose, when concentration of glucose is lower than 2g/L When, addition glucose to 4g/L；1ml Sample supernatants are taken daily, detect protein expression situation.
(4) feed supplement: the about the 4th day, 70g/L CB5 added the 10% of basal medium；5th day, by CO2Incubator temperature It adjusts to 32 DEG C；9th day, 70g/L CB5 is supplemented, the 10% of basal medium is added；12nd day, harvest cell.
(5) SDS-PAGE is detected, as a result as shown in Figure 4, wherein M:marker；1 and 3:control；2:16 plants after purification Express albumen；4:05 plants are expressed albumen after purification；5: not purifying expression albumen.
7 protein purification of embodiment
(1) cell culture fluid is collected, 4 DEG C, 10000rpm is centrifuged 30min, filter membrane (0.45 μm) after supernatant is taken, in preparation Sample.
(2) column equilibration: with 3 column volumes of ultrapure water balance, ethyl alcohol is discharged and saves liquid；BufferA (20mM is added NaH2PO4, 500mM NaCl) and 4-8ml/min, balance 3 column volumes.
(3) loading: using 5ml prepacked column, and 1ml/min carries out loading and (loading flow velocity adjusted according to prepackage column volume, when reservation Between 5min), collect Flow through (FT).
(4) it washs: 4%bufferB (20mM NaH2PO4, 500mM NaCl, 100mM imidazole) wash column, flow velocity is 4ml/min, until OD280nmUntil baseline is steady.
(5) it elutes: 50%bufferB (20mM NaH2PO4, 500mM NaCl, 100mM imidazole) elution purpose egg It is white, until baseline washes flat, 2ml/min, collect 5ml/ pipe.
(6) it washs: 100%bufferB (20mM NaH2PO4, 500mM NaCl, 500mM imidazole) 4ml/min, 2-3 column volume is rinsed, until UV baseline washes flat, ultrapure 3 column volume of water balance.
(7) dialyse: 4 DEG C of Millipore 10KD PBS (pH7.4) dialysis, the secondary liquid imidazoles extension rate that changes is 2, changes liquid Number is 10 times.
(8) aseptic filtration: being filtered with 0.22 μm of filter, and -80 DEG C of refrigerators of protein sample solution save.
(9) protein concentration and purity testing: measuring protein concentration using BCA method, wherein 05 plant of albumen yield is 500mg/ L, other are in 100mg/L or so；Purity is detected using HPLC method, purity is attained by 95% or more.
8 vaccine preparation of embodiment and immunity inoculation
8.1 chicken infectivity bursa of Fabricius virus vaccine preparations: by the recombinant protein VP2-VP3 of expression and ISA201VG adjuvant 45:55 is mixed by volume, emulsification, and final concentration of protein is 50 μ g/ml.
8.2 vaccine immunities and antibody test: chicken infectivity bursa of Fabricius virus VP 2-VP3 fusion protein subunit (5 μ g/ Plumage) immunity inoculation 3-4 week old SPF chicken, it 14 days after being immunized, takes a blood sample respectively within 21st and surveys HI antibody, can be detected within 14th as the result is shown It is not less than 1:64 to HI Geometric mean titers, HI antibody average titer was not less than 1:128 on 21st.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Tian Kang Biological Co., Ltd.
<120>fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine
<130> 2018
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1356
<212> DNA
<213>artificial sequence
<400> 1
atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60
ccaacaaccg gaccggcgtc cattccggac gacaccctgg agaagcacac tctcaggtca 120
gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
cctggattcc ctggctcaat tgtgggtgct cactacacac tgcagagcaa tgggaactac 240
aagttcgatc agatgctcct gactgcccag aacctaccgg ccagctacaa ctactgcagg 300
ctagtgagtc ggagtctcac agtaaggtca agcacactcc ctggtggcgt ttatgcacta 360
aacggcacca taaacgccgt gaccttccaa ggaagcctga gtgaactgac agatgttagc 420
tacaatgggt tgatgtctgc aacagccaac atcaacgaca aaattgggaa cgtcctagta 480
ggggaagggg ttactgtcct cagcttaccc acatcatatg atcttgggta tgtgaggctt 540
ggtgacccca tacccgctat agggcttgac ccaaaaatgg tagcaacatg tgacagcagt 600
gacaggccca gagtctacac cataactgca gctgatgatt accaattctc atcacagtac 660
caaacaggtg gggtaacaat caccctgttc tcagccaaca ttgatgccat cacaagcctc 720
agcgttgggg gagagctcgt gtttaaaaca agcgtccaca gccttgtact gggcgccacc 780
atctacctta taggctttga tgggtctgcg gtaatcacta gagctgtggc cgcaaacaat 840
gggctgacga ccggcaccga caatcttatg ccattcaatc ttgtgattcc aaccaacgag 900
ataacccagc caatcacatc catcaaactg gagatagtga cctccaaaag tggtggtcag 960
gaaggggacc agatgtcatg gtcggcaagt gggagcctag cagtgacgat tcatggtggc 1020
aactatccag gggccctccg tcccgtcaca ctagtagcct acgaaagagt ggcaacagga 1080
tctgtcgtta cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca 1140
aagaacctgg ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg 1200
atactgagtg agagggaccg ccttggcatc aagacagtct ggccgacaag ggagtacacc 1260
gactttcgtg agtacttcat ggaggtggcc gacctcagct ctcccctgaa gattgcagga 1320
gcatttggct tcaaagacat aatccgggcc ataagg 1356
<210> 2
<211> 870
<212> DNA
<213>artificial sequence
<400> 2
cgtttccctc acaatccacg cgactgggac aggctcccct acctcaacct tccatacctt 60
ccacccaatg caggacgcca gtaccacctc gccatggccg catcagagtt caaggagacc 120
cctgaactcg agagcgccgt cagggccatg gaagcagcag ccagtgtaga cccactgttc 180
caatctgcac tcagtgtgtt catgtggctg gaagagaatg ggattgtgac tgacatggcc 240
aacttcgcac tcagcgaccc gaacgcccat cggatgcgaa actttcttgc aaacgcacca 300
caagcaggta gcaagtctca aagggccaaa tacgggacag caggctacgg agtggaggcc 360
cggggcccca caccagaaga agcacagagg gaaaaagaca cacggatctc aaagaagatg 420
gagaccatgg gcatctactt tgcaacacca gaatgggtag cactcaatgg gcaccgaggg 480
ccaagccccg gccagctaaa gtactggcag aacacacgag aaataccgga cccaaacgag 540
gactatctag actacgtgca tgcagagaag agccggttgg catcagaaga acaaatccta 600
agggcagcta cgtcgatcta cggggctcca ggacaggcag agccacccca agctttcata 660
gacgaagttg ccaaagtcta tgaaatcaac catggacgtg gcccaaacca agaacagatg 720
aaagatctgc tcttgactgc gatggagatg aagcatcgca atcccaggcg ggctccacca 780
aagcccaagc caagacccaa cgctccaacg cagagacccc ctggtcggct gggccgctgg 840
atcaggactg tctctgatga ggaccttgag 870
<210> 3
<211> 30
<212> DNA
<213>artificial sequence
<400> 3
ggaggaggag gatctggagg aggaggatct 30
<210> 4
<211> 752
<212> PRT
<213>artificial sequence
<400> 4
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg
1 5 10 15
Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr
20 25 30
Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr
35 40 45
Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro
50 55 60
Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr
65 70 75 80
Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr
85 90 95
Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr
100 105 110
Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr
115 120 125
Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu
130 135 140
Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val
145 150 155 160
Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly
165 170 175
Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys
180 185 190
Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile
195 200 205
Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Thr Gly Gly
210 215 220
Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu
225 230 235 240
Ser Val Gly Gly Glu Leu Val Phe Lys Thr Ser Val His Ser Leu Val
245 250 255
Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Ser Ala Val Ile
260 265 270
Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr Thr Gly Thr Asp Asn
275 280 285
Leu Met Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile Thr Gln Pro
290 295 300
Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln
305 310 315 320
Glu Gly Asp Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr
325 330 335
Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val
340 345 350
Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val
355 360 365
Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val
370 375 380
Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu
385 390 395 400
Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr
405 410 415
Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu
420 425 430
Ser Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile
435 440 445
Arg Ala Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Phe
450 455 460
Pro His Asn Pro Arg Asp Trp Asp Arg Leu Pro Tyr Leu Asn Leu Pro
465 470 475 480
Tyr Leu Pro Pro Asn Ala Gly Arg Gln Tyr His Leu Ala Met Ala Ala
485 490 495
Ser Glu Phe Lys Glu Thr Pro Glu Leu Glu Ser Ala Val Arg Ala Met
500 505 510
Glu Ala Ala Ala Ser Val Asp Pro Leu Phe Gln Ser Ala Leu Ser Val
515 520 525
Phe Met Trp Leu Glu Glu Asn Gly Ile Val Thr Asp Met Ala Asn Phe
530 535 540
Ala Leu Ser Asp Pro Asn Ala His Arg Met Arg Asn Phe Leu Ala Asn
545 550 555 560
Ala Pro Gln Ala Gly Ser Lys Ser Gln Arg Ala Lys Tyr Gly Thr Ala
565 570 575
Gly Tyr Gly Val Glu Ala Arg Gly Pro Thr Pro Glu Glu Ala Gln Arg
580 585 590
Glu Lys Asp Thr Arg Ile Ser Lys Lys Met Glu Thr Met Gly Ile Tyr
595 600 605
Phe Ala Thr Pro Glu Trp Val Ala Leu Asn Gly His Arg Gly Pro Ser
610 615 620
Pro Gly Gln Leu Lys Tyr Trp Gln Asn Thr Arg Glu Ile Pro Asp Pro
625 630 635 640
Asn Glu Asp Tyr Leu Asp Tyr Val His Ala Glu Lys Ser Arg Leu Ala
645 650 655
Ser Glu Glu Gln Ile Leu Arg Ala Ala Thr Ser Ile Tyr Gly Ala Pro
660 665 670
Gly Gln Ala Glu Pro Pro Gln Ala Phe Ile Asp Glu Val Ala Lys Val
675 680 685
Tyr Glu Ile Asn His Gly Arg Gly Pro Asn Gln Glu Gln Met Lys Asp
690 695 700
Leu Leu Leu Thr Ala Met Glu Met Lys His Arg Asn Pro Arg Arg Ala
705 710 715 720
Pro Pro Lys Pro Lys Pro Arg Pro Asn Ala Pro Thr Gln Arg Pro Pro
725 730 735
Gly Arg Leu Gly Arg Trp Ile Arg Thr Val Ser Asp Glu Asp Leu Glu
740 745 750
<210> 5
<211> 2256
<212> DNA
<213>artificial sequence
<400> 5
atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60
ccaacaaccg gaccggcgtc cattccggac gacaccctgg agaagcacac tctcaggtca 120
gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
cctggattcc ctggctcaat tgtgggtgct cactacacac tgcagagcaa tgggaactac 240
aagttcgatc agatgctcct gactgcccag aacctaccgg ccagctacaa ctactgcagg 300
ctagtgagtc ggagtctcac agtaaggtca agcacactcc ctggtggcgt ttatgcacta 360
aacggcacca taaacgccgt gaccttccaa ggaagcctga gtgaactgac agatgttagc 420
tacaatgggt tgatgtctgc aacagccaac atcaacgaca aaattgggaa cgtcctagta 480
ggggaagggg ttactgtcct cagcttaccc acatcatatg atcttgggta tgtgaggctt 540
ggtgacccca tacccgctat agggcttgac ccaaaaatgg tagcaacatg tgacagcagt 600
gacaggccca gagtctacac cataactgca gctgatgatt accaattctc atcacagtac 660
caaacaggtg gggtaacaat caccctgttc tcagccaaca ttgatgccat cacaagcctc 720
agcgttgggg gagagctcgt gtttaaaaca agcgtccaca gccttgtact gggcgccacc 780
atctacctta taggctttga tgggtctgcg gtaatcacta gagctgtggc cgcaaacaat 840
gggctgacga ccggcaccga caatcttatg ccattcaatc ttgtgattcc aaccaacgag 900
ataacccagc caatcacatc catcaaactg gagatagtga cctccaaaag tggtggtcag 960
gaaggggacc agatgtcatg gtcggcaagt gggagcctag cagtgacgat tcatggtggc 1020
aactatccag gggccctccg tcccgtcaca ctagtagcct acgaaagagt ggcaacagga 1080
tctgtcgtta cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca 1140
aagaacctgg ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg 1200
atactgagtg agagggaccg ccttggcatc aagacagtct ggccgacaag ggagtacacc 1260
gactttcgtg agtacttcat ggaggtggcc gacctcagct ctcccctgaa gattgcagga 1320
gcatttggct tcaaagacat aatccgggcc ataaggggag gaggaggatc tggaggagga 1380
ggatctcgtt tccctcacaa tccacgcgac tgggacaggc tcccctacct caaccttcca 1440
taccttccac ccaatgcagg acgccagtac cacctcgcca tggccgcatc agagttcaag 1500
gagacccctg aactcgagag cgccgtcagg gccatggaag cagcagccag tgtagaccca 1560
ctgttccaat ctgcactcag tgtgttcatg tggctggaag agaatgggat tgtgactgac 1620
atggccaact tcgcactcag cgacccgaac gcccatcgga tgcgaaactt tcttgcaaac 1680
gcaccacaag caggtagcaa gtctcaaagg gccaaatacg ggacagcagg ctacggagtg 1740
gaggcccggg gccccacacc agaagaagca cagagggaaa aagacacacg gatctcaaag 1800
aagatggaga ccatgggcat ctactttgca acaccagaat gggtagcact caatgggcac 1860
cgagggccaa gccccggcca gctaaagtac tggcagaaca cacgagaaat accggaccca 1920
aacgaggact atctagacta cgtgcatgca gagaagagcc ggttggcatc agaagaacaa 1980
atcctaaggg cagctacgtc gatctacggg gctccaggac aggcagagcc accccaagct 2040
ttcatagacg aagttgccaa agtctatgaa atcaaccatg gacgtggccc aaaccaagaa 2100
cagatgaaag atctgctctt gactgcgatg gagatgaagc atcgcaatcc caggcgggct 2160
ccaccaaagc ccaagccaag acccaacgct ccaacgcaga gaccccctgg tcggctgggc 2220
cgctggatca ggactgtctc tgatgaggac cttgag 2256
<210> 6
<211> 32
<212> DNA
<213>artificial sequence
<400> 6
cggaattcat gacaaacctg caagatcaaa cc 32
<210> 7
<211> 30
<212> DNA
<213>artificial sequence
<400> 7
cgggatccct caaggtcctc atcatagaca 30

Claims (10)

1. a kind of fusion protein of chicken infectivity bursa of Fabricius virus, which is characterized in that including VP2 section and VP3 section；
The expression of VP2 section nucleotide sequence as shown in SEQ ID NO.1, the VP3 section is as shown in SEQ ID NO.2 Nucleotide sequence expression.
2. fusion protein according to claim 1, which is characterized in that VP2 section and VP3 section put in order as VP2- VP3 is connected by Linker；
The Linker has the nucleotide sequence as shown in SEQ ID NO.3.
3. fusion protein according to claim 1 or 2, which is characterized in that the fusion protein has such as SEQ ID NO.4 Shown in amino acid sequence.
4. a kind of preparation method of the described in any item fusion proteins of claim 1-3, which is characterized in that the fusion will be encoded The gene of albumen is expressed in host.
5. the preparation method according to claim 4, which is characterized in that encoded using mammalian expression systems expression described The gene of fusion protein；
Preferably, using the gene of expressing cho cell system expression encoding said fusion protein；
Preferably, the expression vector of the gene comprising encoding said fusion protein is provided, the expression vector is imported into Chinese hamster ovary celI In, then Chinese hamster ovary celI is screened, obtains stablizing the Chinese hamster ovary celI strain for expressing the fusion protein, the Chinese hamster ovary celI strain table Reach the fusion protein；
Preferably, the screening includes pressurization screening and monoclonal screening；
It preferably, further include that will stablize the Chinese hamster ovary celI for expressing the fusion protein to be domesticated for the culture that suspends in the preparation method The step of.
6. preparation method according to claim 5, which is characterized in that sieved using glutamine synthelase screening amplification system The Chinese hamster ovary celI strain of the fusion protein is expressed in choosing；
Preferably, expression vector used in the glutamine synthelase screening amplification system is pEE6.4 or pEE12.4.
7. the protein expression system that one kind can express any one of claim 1-3 fusion protein.
8. the described in any item fusion proteins of claim 1-3 or the described in any item preparation methods of claim 4-7 are prepared into At least one of the application of the fusion protein arrived, which is characterized in that including following A)-D):
A chicken infectivity bursa of Fabricius virus vaccine) is prepared；
B the antibody of chicken infectivity bursa of Fabricius virus) is prepared；
C) the kit of preparation detection chicken infectivity bursa of Fabricius virus；
D chicken infectivity bursa of Fabricius virus diagnostic antigen) is prepared.
9. a kind of chicken infectivity bursa of Fabricius virus vaccine containing the described in any item fusion proteins of claim 1-3.
10. chicken infectivity bursa of Fabricius virus vaccine according to claim 9, which is characterized in that infections chicken cloacal bursa The concentration of fusion protein described in disease virus vaccine is 20-100 μ g/ml, preferably 30-80 μ g/ml, more preferable 50 μ g/ml；
Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes at least one in vaccine adjuvant, stabilizer and antibiotic Kind；
Preferably, the vaccine adjuvant include aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant, MF59 adjuvant or ISA201VG are, it is preferable to use ISA201VG.
CN201811104818.2A 2018-09-19 2018-09-19 Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine Pending CN109134668A (en)

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