CN109111508A - Avian flu virus hemagglutinin antigen and Chinese hamster ovary celI strain, preparation method and the vaccine for expressing it - Google Patents
Avian flu virus hemagglutinin antigen and Chinese hamster ovary celI strain, preparation method and the vaccine for expressing it Download PDFInfo
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- CN109111508A CN109111508A CN201811104957.5A CN201811104957A CN109111508A CN 109111508 A CN109111508 A CN 109111508A CN 201811104957 A CN201811104957 A CN 201811104957A CN 109111508 A CN109111508 A CN 109111508A
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- A—HUMAN NECESSITIES
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention provides a kind of avian flu virus hemagglutinin antigen and Chinese hamster ovary celI strain, preparation method and the vaccines of expressing it, it is related to field of biotechnology, the avian flu virus hemagglutinin antigen is the hemagglutinin expressed with the sequence as shown in SEQ ID NO.1, has the advantages that broad spectrum activity as antigen, and the hemagglutinin is the albumen of eukaryotic cell expression, avoids the epitope for losing the conformation type of the antigen.The preparation method of above-mentioned avian flu virus hemagglutinin antigen and the cell strain for expressing above-mentioned avian flu virus hemagglutinin antigen can obtain the hemagglutinin good with broad spectrum activity and antigen immunogenicity.The vaccine of above-mentioned avian flu virus hemagglutinin antigen, the production of vaccine is at low cost, and immune efficacy is high, after 3-4 week old SPF chicken is immunized, it can be detected within 14th H5 hypotype antigen measuring HI Geometric mean titers (GMT) not less than 1:64, be better than conventional vaccine.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of avian flu virus hemagglutinin antigen and express its
Chinese hamster ovary celI strain, preparation method and vaccine.
Background technique
Avian influenza virus (AIV) is sub-thread negative strand viruses, spherical in shape or Filamentous, spherical diameter 80-120nm.Gene component
For 8 segments, 10 kinds of albumen are encoded altogether, and wherein HA, NA and M are located at the cyst membrane surface of virus, are that the main protective of AIV is anti-
It is former.It is matrix protein under the viral double-deck rouge coating, it is the albumen that content is most in virion, and which constitute viruses
The frame of coating.Two kinds of glycoprotein furcellas encoded by viral gene, i.e. hemagglutinin have then been inlayed on bilayer lipid membrane
(Hemagglutinnin, HA) and neuraminidase (Neuraminidase, NA), the two be divide influenza virus sub-strain according to
According to, and its antigenicity easily makes a variation, new hypotype, easily leads to the outburst of bird flu once being formed.
HA is to constitute the prominent main component of virus envelope fibre, belongs to I type glycoprotein.HA exists in the form of tripolymer.HA
Viruses adsorption, wear mould and determine virus in terms of play a crucial role.It can be cracked into HA1 and
An important factor for HA2 two parts are viral pathogenesis power height.It can produce anti-HA antibody after AIV infection, HA antibody is main
Protection antibody plays an important role in preventing virus infection.HA albumen is the main protection antigen of bird flu influenza virus
One of.It can not only stimulate body to generate protection antibody, but also body can be induced to generate cytotoxic effect, to same
The good protection of total generation of subtype virus.
2003 year-to-date, and H5N1 subtype highly pathogenic avian influenza is in the birds in Europe, Africa, America and Asia, hair
It gave birth to pandemic and caused weight huge economic loss.The chicken embryo of inactivated avian influenza vaccine application at present or MDCK suspend carefully entirely
Born of the same parents' production, complicated using chicken embryo production vaccine art, the chicken embryo used needs to connect in venom there may be the pollution of exogenous virus
Kind chicken embryo collects allantoic fluid, inactivation, collects allantoin liquid chicken embryo waste and needs HIGH PRESSURE TREATMENT, generates more production waste,
Pressure is brought to environmental protection.The full suspension cell production avian influenza vaccine of MDCK needs to tame the process that kind of poison adapts to cell, needs
Want the period long, and sometimes up to less than expected malicious valence, toxigenic capacity and production technology are complex.Therefore, a kind of improvement
Avian influenza vaccine be existing market needs.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of avian flu virus hemagglutinin antigen, the antigen have both tool broad spectrum activity and
Good immunogenicity can produce compared with the antibody of high titre and prevent the bird flu of a variety of hypotypes after Immunizing Birds.
The second object of the present invention is to provide a kind of cell strain for expressing above-mentioned avian flu virus hemagglutinin antigen, this is thin
Born of the same parents' strain can express the influenza virus hemagglutinin antigen with broad spectrum activity.
The third object of the present invention is to provide the system of a kind of above-mentioned avian flu virus hemagglutinin antigen and above-mentioned cell strain
Preparation Method, this method is easy to operate, and the antigen immunogenicity of acquisition is good.
The fourth object of the present invention is to provide a kind of vaccine comprising above-mentioned avian flu virus hemagglutinin antigen, the vaccine
Production cost is low, and immune efficacy is high.
In order to solve the above technical problems, spy of the present invention adopts the following technical scheme that
A kind of avian flu virus hemagglutinin antigen, the avian flu virus hemagglutinin antigen are with such as SEQ ID NO.1
The hemagglutinin of shown sequence expression；The hemagglutinin is the albumen of eukaryotic cell expression.
Preferably, the eukaryocyte is Chinese hamster ovary celI.
A kind of cell strain for expressing above-mentioned avian flu virus hemagglutinin antigen；
Preferably, the cell strain is Chinese hamster ovary celI strain.
The preparation method of a kind of above-mentioned avian influenza virus antigen or above-mentioned cell strain, the preparation method include providing to include
The recombinant vector of sequence shown in SEQ ID NO.1, then the recombinant vector described in host cell expression, can be obtained avian flu
The cell strain of malicious haemagglutinin antigen and expression avian flu virus hemagglutinin antigen；
Preferably, the preparation method includes the steps that carrying out the host cell containing recombinant vector into pressurization screening.
Preferably, the screening reagent of the recombinant vector includes G418；The host cell is Chinese hamster ovary celI；The host
Cell carries out pressurization screening and includes the following steps:
A) Chinese hamster ovary celI containing recombinant vector pressurization screening 3-5 generation to cell is carried out with the culture medium containing G418 to deposit
Motility rate is 10%-50%；It is preferred that 20%-40%；More preferable 30%；
B) it is at least 90% using Chinese hamster ovary celI to the survival rate that conventional medium culture is filtered out through step a)；
C) it is primary to repeat step a) and step b)；
Preferably, the concentration of G418 is 400-1000 μ g/ml, preferably 600-900 μ g/ in the culture medium containing G418
Ml, more preferable 800 μ g/ml.
Preferably, the recombinant vector is by least one of sequence and following carrier carrier weight shown in SEQ ID NO.1
The expression vector of group: pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.
Preferably, the Chinese hamster ovary celI after pressurization screening is subjected to monoclonal screening.
Further, 36~38 DEG C will be placed in by the Chinese hamster ovary celI of screening, 5%CO2In shaking table carry out shaking flask culture 48~
72 hours, cell-seeding-density was 0.3 × 106~0.5 × 106VC/ml；
Preferably, 31~33 DEG C were cooled the temperature to the 5th day in 36~38 DEG C of cultures, pH is adjusted to 7.4~7.6；
Preferably, the Efficient Feed of 10% starting working volume, every daily test are separately added within 9th on 4th and
Concentration of glucose is surveyed, when concentration of glucose is lower than 2.5g/L, supplement glucose to 3~4g/L；
Preferably, when Cell viability is lower than 80%, harvest supernatant obtains the avian influenza virus antigen.
A kind of avian influenza vaccine, the avian influenza vaccine include above-mentioned avian flu virus hemagglutinin antigen；
Preferably, the dosage of avian flu virus hemagglutinin antigen is 1-10 μ g/ plumage part in the avian influenza vaccine, preferably
2-8 μ g/ plumage part, more preferably 3-6 μ g/ plumage part.
Preferably, the avian influenza vaccine further includes adjuvant；
Preferably, the adjuvant be selected from aluminium hydroxide gel, mineral oil, carbomer, Gel01, propolis, ISA206 or
One of ISA760VG or a variety of；
Preferably, the avian influenza vaccine includes the antigen and adjuvant that mass ratio is 1:1, preferably water-in-oil-in water.
Compared with prior art, the invention has the following beneficial effects:
Avian flu virus hemagglutinin antigen provided by the invention, the antigen are with the sequence table as shown in SEQ ID NO.1
The hemagglutinin reached；Sequence shown in SEQ ID NO.1 is that nearly 5 years popular avian influenza hemagglutinin genes is selected to be compared point
The sequence obtained after analysis, and by the sequence carry out codon optimization and modification, with reach further increase antigen broad spectrum activity and
Improve the purpose of antigen presentation amount.And the hemagglutinin is the albumen of eukaryotic cell expression, avoids and loses the antigen
The epitope of conformation type.The cell strain of the above-mentioned avian flu virus hemagglutinin antigen of expression provided by the invention, has above-mentioned fowl
The beneficial effect of influenza virus hemagglutinin antigen.
It the preparation method of above-mentioned avian flu virus hemagglutinin antigen and can obtain with broad spectrum activity and antigen immunogenicity
Good hemagglutinin, and this method is easy to operate, is suitble to large-scale production.
Vaccine provided by the invention comprising above-mentioned avian flu virus hemagglutinin antigen, the production of vaccine is at low cost, is immunized
Effect is high, after 3-4 week old SPF chicken is immunized, can be detected within 14th H5 hypotype antigen measuring HI Geometric mean titers (GMT)
Be not less than 1:64,21 days H5 hypotype antigen measuring HI Geometric mean titers (GMT) be not less than 1:256；And conventional
The immune latter 21 days HI Geometric mean titers (GMT) of inactivated vaccine are just not less than 1:64, therefore the vaccine is better than conventional vaccine.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the recombinant plasmid digestion qualification result figure that the embodiment of the present invention 3 provides.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not
Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
The present invention provides a kind of avian flu virus hemagglutinin antigen, which is with the sequence as shown in SEQ ID NO.1
The hemagglutinin of expression；Sequence shown in SEQ ID NO.1 is that nearly 5 years popular bird flu HA genes is selected to be compared
The sequence obtained afterwards, and by the optimization and modification of sequence progress codon, it further increases antigen broad spectrum activity to reach and mentions
The purpose of high antigen expression quantity.And the hemagglutinin is the albumen of eukaryotic cell expression, it is preferable to use expressing cho cell
Albumen avoids the epitope for the conformation type for losing the antigen.The present invention also provides express above-mentioned avian flu virus hemagglutinin
The cell strain of antigen, therefore the beneficial effect with above-mentioned avian flu virus hemagglutinin antigen.
Wherein, typical Chinese hamster ovary celI can be CHO-K1 cell.
The present invention also provides the preparation methods of a kind of above-mentioned avian flu virus hemagglutinin antigen and above-mentioned cell strain, including
The recombinant vector including sequence shown in SEQ ID NO.1 is provided, then the recombinant vector described in host cell expression.It is excellent at one
In the embodiment of choosing, prepare above-mentioned antigen using Chinese hamster ovary celI, by sequence shown in SEQ ID NO.1 and following carrier at least
A kind of carrier recombination: pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13；Then by recombinant vector transfect in Chinese hamster ovary celI into
Row expression, pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13 transfect recombinant vector using G418 as reporter gene
Pressurization screening is carried out using Chinese hamster ovary celI of the culture medium containing G418 to expression hemagglutinin after in Chinese hamster ovary celI, will be pressurizeed
The cell strain filtered out carries out the monoclonal screening of cell, obtains the relatively high cell strain of expression quantity by screening.Then into
One step passes through the condition of culture for optimizing the cell strain, feed supplement time, and feed supplement amount is finally obtained the cell of higher hemagglutinin
Strain, the expression quantity using cell strain expression hemagglutinin albumen are high and stable.Filter out high expression hemagglutinin
After cell strain, the cell strain is cultivated under the condition of culture of optimization, can obtain the albumen of high expression quantity, is collected albumen and is removed carefully
It can be obtained hemagglutinin after born of the same parents' fragment, the avian influenza virus antigen of vaccine can be prepared.
In one preferred embodiment, pressurization screening is carried out using following steps:
A) Chinese hamster ovary celI containing recombinant vector pressurization screening 3-5 generation to cell is carried out with the culture medium containing G418 to deposit
Motility rate is 10%-50%, such as can be, but is not limited to 10%, 20%, 30%, 40% or 50%；It is preferred that 20%-40%；More
It is preferred that 30%；
B) it is at least 90% using Chinese hamster ovary celI to the survival rate that conventional medium culture is filtered out through step a)；
C) it is primary to repeat step a) and step b)；
Red in some preferred embodiments, the concentration of G418 is 400-1000 μ in the culture medium containing G418
G/ml, such as can be, but be not limited to 400 μ g/ml, 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml
Or 1000 μ g/ml, preferably 600-900 μ g/ml, more preferable 800 μ g/ml, to achieve the purpose that optimization pressurization screening effect.
In some preferred embodiments, 36~38 DEG C will be placed in by the Chinese hamster ovary celI of screening, 5%CO2In shaking table into
Row shaking flask culture 48~72 hours, such as can be, but be not limited to 48 hours, 60 hours or 72 hours；Cell-seeding-density is
0.3×106~0.5 × 106VC/ml, such as can be, but be not limited to 0.3 × 106VC/ml、0.4×106VC/ml or 0.5 ×
106VC/ml。
Preferably, 31~33 DEG C were cooled the temperature to the 5th day in 36~38 DEG C of cultures, pH is adjusted to 7.4~7.6.
Preferably, the Efficient Feed of 10% starting working volume, every daily test are separately added within 9th on 4th and
Concentration of glucose is surveyed, when concentration of glucose is lower than 2.5g/L, supplement glucose to 3~4g/L.
Preferably, when Cell viability is lower than 80%, harvest supernatant obtains the avian influenza virus antigen.
The Chinese hamster ovary celI for stablizing high expression hemagglutinin, the expression quantity of hemagglutinin can be obtained using the above method
Up to 5mg/ml uses the Chinese hamster ovary celI filtered out production hemagglutinin with short production cycle as avian influenza hemagglutinin antigen,
Expressing quantity is high and the antigen immunogenicity that produces is good.
The present invention also provides a kind of avian influenza vaccine, the vaccine includes above-mentioned avian flu virus hemagglutinin antigen, should
The dosage of antigen is preferably 1-10 μ g/ plumage part in vaccine, such as can be, but is not limited to 1 μ g/ plumage part, 2 μ g/ plumage parts, 3 μ g/ plumages
Part, 4 μ g/ plumage parts, 5 μ g/ plumage parts, 6 μ g/ plumage parts, 7 μ g/ plumage parts, 8 μ g/ plumage parts, 9 μ g/ plumage parts or 10 μ g/ plumage parts, preferably 2-8
μ g/ plumage part, more preferably 3-6 μ g/ plumage part.
Preferably, which further includes adjuvant；Such as can be but be not limited to aluminium hydroxide gel, mineral oil,
Carbomer, Gel01, propolis, ISA206 or ISA760VG, the avian influenza vaccine are 10%-60% it is preferable to use mass fraction
ISA206VG can more effectively improve the antigen slow releasing function and immunogenicity of vaccine.
Beneficial effects of the present invention are further illustrated below with reference to preferred embodiment.
A kind of sequence for expressing hemagglutinin of embodiment 1
Current popular, nearly 5 years popular bird flu H5 hypotype HA gene orders is downloaded from Genebank to be compared point
Analysis, selects ingredient of the Dominant Epitopes as vaccine antigen, according to the inclined preferendum of Chinese hamster ovary celI codon, by avian influenza virus HA
The sequence of gene carries out the optimization and modification of codon, to improve the level of destination protein expression HA albumen.Meanwhile to make HA base
Because having the antigenicity of wide spectrum, the antigen sequence of the relatively conservative HA1 Dominant Epitopes of screening, and 3 ' in HA2 are held with flexible ammonia
Base acid form connects stromatin M2e (containing 23 amino acid), forms the base sequence of one section of artificial polypeptide, same by protein
Source modeling, searches for module in the library PDB using the homology of structure and sequence, simulates the sky of each epitope difference connection method
Between three-dimensional conformation, analyze its antigenicity, finally filter out gene sequence of the optimal combination of antigenicity as HA destination protein
Column, the albumen have the broad spectrum activity and conservative of height, design restriction enzyme site and artificial synthesized HA full length gene.
A kind of recombinant vector for expressing hemagglutinin of embodiment 2
By the amino acid sequence of the coding HA albumen of above-mentioned synthesis and C-terminal have the segment of His label by Sal I and
The insertion of Xho I site is cloned on eukaryon transfer vector pcDNA3.1.It is obtained after 16 DEG C overnight connection using T4DNA ligase
Connection product is coated in the LB plate containing ammonia section penicillin after E. coli competent DH5a conversion, and 37 DEG C were cultivated
Picking positive bacteria falls in the LB culture medium containing ammonia section penicillin and cultivates after night, extracts plasmid.
Embodiment 3 expresses the digestion identification of the recombinant vector of hemagglutinin
Plasmid DNA will be prepared, selects Sal I and Xho I site digestion with restriction enzyme, digestion products are through 1% agar
Sugared gel electrophoresis, as a result as shown in Figure 1.Wherein, swimming lane M:DNA Marker, swimming lane 1~3: plasmid after digestion, swimming lane 4: digestion
Preceding plasmid.As can be seen from the figure the purpose for having expected size occurs, and illustration purpose gene is successfully plugged into carrier.
The plasmid of positive findings is served into the raw work sequence verification in sea, final to obtain positive restructuring expression plasmid, sequence is such as
Shown in SEQ ID NO.1, wherein 1402-1431, between 1441-1476 and 1486-1512 epitope by
The flexible amino acid of CCTCCCAGC coding is connected.
The recombinant cell that embodiment 4 expresses the recombinant vector of hemagglutinin, which pressurizes, to be screened
The Chinese hamster ovary celI that the plasmid transfection to well-grown is suspended entirely carries out cell passage after 3 days, and in culture medium
The G418 of 800 μ g/ml is added, is forced into motility rate at 30% or so, stops pressurization.With conventional culture medium culture, change is newly
The culture of adhere-wall culture base 7 days, when Cell viability reaches 90% or more, repressurization screening is primary, and same Cell viability reaches
90%.
Embodiment 5 expresses the monoclonal screening of the recombinant vector cell of hemagglutinin
Positive colony is selected and is detected: in the plate after culture 7 days, the cell of adherent growth being chosen into 96 orifice plates, benefit
After adhere-wall culture base culture 7 days, 100gL suspension medium MSX expression is added culture 2 days, culture medium is miscellaneous for point in hollow plate
Detection is handed over, wherein will go to 24 orifice plates by high-expression clone, is cultivated using MSX is changed to after the culture of adhere-wall culture base 2 days, culture medium is used
It is detected in immunoblotting, high-expression clone strain is finally obtained according to experimental result.The condition of culture of all cells is 37 DEG C,
5%CO2。
Embodiment 6 expresses the culture of the recombinant vector cell of hemagglutinin
Cell is resuspended in CHO culture medium (containing 25 μM of MSX) or other appropriate medias in Chinese hamster ovary celI by screening, carefully
Born of the same parents' inoculum density is 0.3 × 106~0.5 × 106VC/ml, inoculating cell are placed in 37 ± 1 in the cell shaking flask of suitable volumes
DEG C, contain 5%CO2It is carried out shaking flask culture 48~72 hours in shaking table, continues to pass on amplification cultivation cell.
Bioreactor culture step by step is carried out as needed to ferment, and is generally carried out 5~8 times of amplifications, is amplified to designated volume
The expression of Shi Jinhang antigen cooled the temperature to 32 ± 1 DEG C to the 5th day in 37 ± 1 DEG C of cultures, and pH is adjusted to 7.5 ± 0.1, and with
Suitable revolving speed is cultivated, and the Efficient Feed of 10% starting working volume was added on the 4th and the 9th, daily to detect
Concentration of glucose, when concentration of glucose is lower than 2.5g/L, supplement glucose to 3~4g/L.When egg Cell viability is lower than 80%
When, harvest supernatant is required antigen.
By optimizing condition of culture, the feed supplement time of the cell strain, feed supplement amount is finally obtained the HA albumen of 1.5mg/ml
Expression.
A kind of avian influenza vaccine of embodiment 7
Go cell fragment by expression it is quantitative after, be prepared by mixing into vaccine with adjuvant, after 3-4 week old SPF chicken is immunized, as a result
Display can be detected H5 hypotype antigen measuring HI Geometric mean titers (GMT) on the 14th not less than 1:64, the Asia H5 on the 21st
Type antigen measuring HI Geometric mean titers (GMT) are not less than 1:256.
Comparative example 1
Kind of poison is seeded in the chick embryo allantois element liquid of 10~11 ages in days, after culture about 72 hours, collects chick embryo allantois element
Liquid inactivates, emulsification, while needing the chicken embryo of high pressure sterilization processing absorption allantoic fluid.HI antibody geometric average drop on the 21st after immune
It spends (GMT) and is not less than 1:64.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Tian Kang Biological Co., Ltd.
<120>avian flu virus hemagglutinin antigen and its Chinese hamster ovary celI strain, preparation method and vaccine are expressed
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1524
<212> DNA
<213>artificial sequence
<400> 1
atggagaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120
accgtgaccc acgcccagga catcctggag aagacccaca acggcaagct gtgcgaccac 180
gacggcgtga agcccctgat cctgagggac tgcagcgtgg ccggctggct gctgggcaac 240
cccatgtgcg acgagcccat caacgtgccc gagtggagct acatcgtgga gaaggccaac 300
cccgccaacg acctgtgcta ccccggcgac ttcaacgact acgaggagct gaagcacctg 360
ctgagcagga tcaaccactt cgagaagatc cagatcatcc ccaagaacag ctggagcagc 420
cacgagaccc ccctgggcgt gagcagcgcc tgcccctacc agggcaagag cagcttcttc 480
aggaacgtgg tgtggctgat caagaagaac aacgcctacc ccaccatcaa gaggagctac 540
aacaacacca accaggagga cctgctggtg ctgtggggca tccaccaccc caacgacgcc 600
gccgagcaga ccaggctgta ccagaacccc accacctaca tcagcgtggg caccagcacc 660
ctgaaccaga ggcctgtgcc caagatcgcc accaggagca agatcaacgg ccagagcggc 720
aggatcgact tcttctggac catcctgaag cccaacgacg ccatccactt cgagagcaac 780
ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcaccatc 840
atgaggagcg aggtggagta cggcaactgc aacaccaggt gccagacccc cgtgggcgcc 900
accaggggat ccttcggcgc catcgccggc ttcatcgagg gcggctggca gggcatggtg 960
gacggctggt acggctacca ccacagcaac gagcagggca gcggctacgc cgccgacaag 1020
gagagcaccc agaaggccat ggacggcgtg accaacaagg tgaacagcat catcgacaag 1080
atgaacaccc agttcgaggc cgtgggcagg gagttcaaca acctggagag gaggatcgag 1140
aacctgaaca agaagatgga ggacggcttc ctggacgtgt ggacctacaa cgccgagctg 1200
ctggtgctga tggagaacga gaggaccctg gacttccacg acagcaacgt gaagaacctg 1260
tacgacaagg tgaggctgca gctgaaggac aacgccaagg agctgggcaa cggctgcttc 1320
gagttctacc acaagtgcaa caacgagtgc atggagagcg tgaggaacgg cacctacgac 1380
tacccccagt accctcccag catcctgggc ttcgtgttca cgctgaccgt gcctcccagc 1440
ggccccctga aggccgagat cgcgcagagg ctggagcctc ccagcggcat cctgggcttc 1500
gtgttcaccc tccctcccag ctga 1524
Claims (10)
1. a kind of avian flu virus hemagglutinin antigen, which is characterized in that the avian flu virus hemagglutinin antigen is with such as SEQ
The hemagglutinin of the expression of sequence shown in ID NO.1；The hemagglutinin is the albumen of eukaryotic cell expression.
2. avian flu virus hemagglutinin antigen according to claim 1, which is characterized in that the eukaryocyte is that CHO is thin
Born of the same parents.
3. a kind of cell strain for expressing avian flu virus hemagglutinin antigen of any of claims 1 or 2；
Preferably, the cell strain is Chinese hamster ovary celI strain.
4. the preparation method of a kind of avian influenza virus antigen of any of claims 1 or 2 or cell strain as claimed in claim 3,
It is characterized in that it provides the recombinant vectors for including sequence shown in SEQ ID NO.1, then recombinate and carry described in host cell expression
Body can be obtained avian flu virus hemagglutinin antigen and express the cell strain of avian flu virus hemagglutinin antigen；
Preferably, the preparation method includes the steps that carrying out the host cell containing recombinant vector into pressurization screening.
5. the preparation method according to claim 4, which is characterized in that the screening reagent of the recombinant vector includes G418；
The host cell is Chinese hamster ovary celI；The host cell carries out pressurization screening and includes the following steps:
A) Chinese hamster ovary celI containing recombinant vector is subjected to pressurization screening 3-5 generation to cell survival rate with the culture medium containing G418
For 10%-50%；It is preferred that 20%-40%；More preferable 30%；
B) it is at least 90% using Chinese hamster ovary celI to the survival rate that conventional medium culture is filtered out through step a)；
C) it is primary to repeat step a) and step b)；
Preferably, in the culture medium containing G418 G418 concentration be 400-1000 μ g/ml, preferably 600-900 μ g/ml,
More preferable 800 μ g/ml.
6. preparation method according to claim 5, which is characterized in that the recombinant vector is will be shown in SEQ ID NO.1
The expression vector of at least one of sequence and following carrier carrier recombination: pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.
7. preparation method according to claim 5, which is characterized in that the Chinese hamster ovary celI after pressurization screening is carried out monoclonal
Screening.
8. preparation method according to claim 5, which is characterized in that 36~38 DEG C will be placed in by the Chinese hamster ovary celI of screening,
5%CO2It is carried out shaking flask culture 48~72 hours in shaking table, cell-seeding-density is 0.3 × 106~0.5 × 106VC/ml；
Preferably, 31~33 DEG C were cooled the temperature to the 5th day in 36~38 DEG C of cultures, pH is adjusted to 7.4~7.6；
Preferably, on 4th and it is separately added within 9th the Efficient Feed of 10% starting working volume, detects Portugal daily
Grape sugar concentration, when concentration of glucose is lower than 2.5g/L, supplement glucose to 3~4g/L；
Preferably, when Cell viability is lower than 80%, harvest supernatant obtains the avian influenza virus antigen.
9. a kind of avian influenza vaccine, which is characterized in that the vaccine includes avian influenza virus blood clotting of any of claims 1 or 2
Plain antigen；
Preferably, the dosage of avian flu virus hemagglutinin antigen is 1-10 μ g/ plumage part, preferably 2-8 μ in the avian influenza vaccine
G/ plumage part, more preferably 3-6 μ g/ plumage part.
10. avian influenza vaccine according to claim 9, which is characterized in that the avian influenza vaccine further includes adjuvant；
Preferably, the adjuvant be selected from aluminium hydroxide gel, mineral oil, carbomer, Gel01, propolis, ISA206, ISA201 or
One of ISA760VG or a variety of；
Preferably, the avian influenza vaccine includes the antigen and adjuvant that mass ratio is 1:1, preferably water-in-oil-in water.
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| CN110872358A (en) * | 2019-12-04 | 2020-03-10 | 天康生物(上海)有限公司 | HA-Fc fusion protein, preparation method thereof and vaccine |
| CN111849849A (en) * | 2020-07-29 | 2020-10-30 | 天康生物股份有限公司 | Brucella ghost strain, Brucella ghost vaccine and preparation method |
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