CN108265064A - A kind of recombination 3 allergoid albumen of artemisia annua and its application - Google Patents

Abstract

The present invention relates to a kind of main allergic protein Art a3 of artemisia annua of mammalian cell expression, encoding gene, expression way, purifying process and recombinant protein detection method and application.Artemisia annua allergic protein belongs to vegetable protein in itself, and certain difficulty has been expressed in existing common protokaryon or eukaryotic expression system, also and has no that pertinent literature discloses；And the problems such as traditional extracting mode is commonly present complicated component, quality is difficult to control, be vulnerable to exogenous noxious material, the pollution of pathogen microorganism.For this purpose, the present invention provides such as SEQ ID NO：The encoding gene of recombination Art a3 shown in 1, and aim at expression, purifying, recombinant protein detection method and the application of this design, compared with prior art, the recombination Art a3 of the present invention have higher expression quantity and activity, while also establish basis for cell strain exploitation, process optimization, preclinical and clinical research and industrialized production during the drug development.

Description

A kind of recombination 3 allergoid albumen of artemisia annua and its application

Technical field

The invention belongs to biopharmaceutical technologies, and in particular to a kind of recombination 3 allergoid albumen of artemisia annua and its volume Code gene and expression and purification method.

Background technology

In rising year by year, European incidence is risen the incidence of anaphylaxis pollinosis by the 1% of early 20th century in recent years To 20%, and it is expected that it can reach 35% within 20 years futures.China pollinosis patient has exceeded 10,000,000 people, city dweller's morbidity Rate is 0.9% or so, and Endemic Area is up to 5%, and as city trees flowers and plants kind is more and more, pollen hypersensitivity symptom patient has increase Trend.

Pollen is the male sex-cell of higher plant, under the action of wind or worm, is propagated in air, some people are exhaling After inhaling people's pollen during inhaling, allergic reaction will be generated.Pollen is drifted with apparent seasonal.The anemophilous pollen in spring Mostly from trees；The anemophilous pollen of late spring and early summer are mostly from herbage；Anemophilous pollen at the beginning of late summer and autumn is mostly from miscellaneous Grass.It is currently known to the pollen of mankind's allergy is caused mainly to have artemisia pollen (Artemisia), Ambrosia pollen (Ambrosia), Ricinus pollen (Ricinus), Humulus pollen (Hummlus), Chenopodium pollen (Chenlpldum), Amaranthus Pollen (Amaranthus), Casuarina pollen (Casuarina), Betula pollen (Betula), Pinus pollen (Pinus), Picea pollen (Picea), Cryptomeria pollen (Cryptomeria), plane pollen (Platanus) etc..These plants Pollen is because its pathogenicity is strong, and sensitization rate is high, and quantity is big, and it is wide to disseminate range；And the range of plant itself distribution is wide, quantity is also rich Richness, the influence to pollen contamination is big, thus these plants become more important pollen contamination source plant.Since pollen contamination source is planted The influence of the factors such as distribution climate, soil, biology and the landform of object, thus different regions, main pollen contamination source are planted Object also differs.In China, because most regional (such as Beijing, Xinjiang, Shanxi, Shandong, Wuhan, Shenyang, Guangzhou, Ningxia etc.) Main pollen is Artemisia Plant Pollen, therefore the pollen contamination source plant in China is the most serious with sagebruss.

At present, clinically mainly using allergic rhinitis and allergy caused by artemisia annua crude extract treatment Artemisia pollen-induced Property the anaphylactias such as asthma desensitization treatment, such as the artemisia annua powder drops of my actor playing a martial role in Chinese operas's object of Zhejiang in June, 2016 approval is For artemisia annua extracting solution.Since the composition of natural allergen extracting solution is extremely complex, it is extremely difficult to define its component, extracting method Time-consuming, and process is cumbersome, and cost is higher, and is easily polluted by exogenous noxious material, pathogen microorganism.In addition, it adopts It is diagnosed with crude extract, the extent of reaction of the patient to allergen each component can not be specified, easily cause mistaken diagnosis.

And on the other hand, artemisia annua allergen protein belongs to vegetable protein in itself, in existing common protokaryon or eukaryon table Certain difficulty has been expressed up in system, also and has seen that pertinent literature discloses.

Invention content

In order to overcome disadvantage mentioned above, inventor is according to the published Artemisia vulgaris clone1Art of NCBI The mRNA sequence of v3allergen precursor (Art v3) from NCBI EST retrieval obtain with Art v3 high homologies but The cDNA sequence of the Artemisia annua of unknown gene function is named as 3 allergoid (hereinafter referred to as rArt of artemisia annua A3 it), and by the gene is optimized in mammalian cell CHO expression systems, raising is added in MOLECULE DESIGN The effect original paper of rArt a3 expression, inventor surprisingly have found that rArt a3 have compared with prior art after gene optimization There are higher expression quantity, and the biological activity for having height similar compared with native protein, in being treated available for pollen hypersensitivity.

It is an object of the present invention to provide a kind of DNA sequence dna for encoding rArt a3 albumen, base sequence such as SEQ ID NO:Shown in 1.The sequence has carried out codon optimization for mammalian cell CHO expression systems, is more conducive to rArta3 and is feeding It is expressed in newborn zooblast CHO.

It is a further object to provide a kind of rArt a3 albumen, amino acid sequence such as SEQ ID NO:3 institutes Show.

It is a further object to provide a kind of carrier containing rArt a3 genes after above-mentioned code optimization, preferably , the carrier is pcDNa3.1, pcDNa3.3-TOPO, pOptiVECTM-TOPO, pCHO1.0.

It is a further object to provide a kind of mammalian cell Chinese hamster ovary celI strain comprising carrier described above, Preferably, the mammalian cell Chinese hamster ovary celI strain is CHO-K1, CHO-DHFR, DG44, CHO-S.

Carrier described above is preferably pCHO1.0 or pOptiVECTM-TOPO, more preferably pCHO1.0.

Mammalian cell Chinese hamster ovary celI strain described above is preferably DG44 or CHO-S cell strains, and more preferably CHO-S is thin Born of the same parents' strain.

It is a further object to provide a kind of expression of rArt a3 albumen, the method includes following steps Suddenly：

A. carrier of the structure containing above-mentioned coding rArt a3 genes；

B. it will be transferred to after the vector linearization of step A in mammalian cell Chinese hamster ovary celI strain, and train under suitable conditions It supports；

C. recovery purifying protein.

It is a further object to provide a kind of rArt a3 method for purifying proteins, the purification process is as follows：

A. rArta3 Fed batch fementation liquid low-temperature and high-speeds are collected by centrifugation supernatant, 20mM phosphate buffer ultrafiltration, 0.45 μm of membrane filtration.

B. cation-exchange chromatography, with equilibration buffer chromatographic column, then with fully-automatic intelligent protein purification system Q valves carry out equilibration buffer and elution buffer automatic in system (AKTA avant150, purchased from GE healthcare companies) Configuration, by the Fed batch fementation liquid pre-processed in step A by detaching filler, then with elution buffer gradient elution, Collect eluting peak, equilibration buffer and elution buffer pH=6.0.

The rArta3 that the present invention expresses in mammalian cell not only has higher yield, but also compared with nArta3 With closely similar biological activity.Also, preparation and purification are simple for process, and recombination allergic effect can be obtained through step purifying It is former.It is had the advantage that compared with crude extract：(1) recombinant allergen has higher purity, and non-change is free of compared with crude extract Answer ultimate constituent, enzyme, enzyme inhibitor and toxic protein etc.；(2) recombinant protein has preferable specific, and ingredient is answered in crude extract Miscellaneous, patient may only react with which part ingredient, poor specificity, and recombinant allergen ingredient is single, and specificity is good；(3) weight The group alternative natural extracting solution of allergenic activity reduces the epitope that IgE is combined compared with natural extracting solution, effectively reduces The allergic reaction of IgE mediations, while retain structural domain necessary to allergic effect pro T lymphocyte identifies, there is preferable immunogenicity, subtract The danger of few immunization therapy improves the effect of desensitization treatment.

Description of the drawings

Fig. 1 shows rArt a3 gene order comparison diagrams are recombinated before and after optimization.

It is natural rArt a3 gene nucleotide series that wherein optimization presequence is corresponding；Sequence is corresponding for this after optimization The gene nucleotide series of the recombination rArta3 of invention, i.e. sequence after codon optimization.

Fig. 2-a, Fig. 2-b are CAI index of the rArt a3 genes in mammalian cell CHO expression systems before and after optimization.

Wherein, Fig. 2-a represent natural rArt a3 gene nucleotide series CAI in mammalian cell CHO expression systems Index is calculated as 0.72 through program；Fig. 2-b represent the rArt a3 codons of the present invention after optimization in mammalian cell CHO CAI indexes are calculated as 0.96 through program in expression system.

Fig. 3-a, 3-b are optimal close in mammalian cell CHO expressive hosts for rArt a3 genes before and after codon optimization Numeral frequency distribution administrative division map.

Wherein Fig. 3-a represent that rArt a3 natural gene nucleotides sequences are listed in optimal password in mammalian cell CHO systems Sub- frequency distribution administrative division map, as can be seen from the figure：The poor efficiency codon of rArt a3 natural gene nucleotide sequences occurs Percentage is 13%；Fig. 3-b represent the rArt a3 codons of the invention after optimization in mammalian cell CHO systems most Excellent codon frequency distributed areas figure, the rArt a3 Codon sequences poor efficiency codons of the present invention after optimization are 0。

Fig. 4-a, 4-b are rArt a3 genes average GC in mammalian cell CHO expression systems before and after codon optimization Base contents distributed areas figure.

Wherein, it is flat to represent that rArt a3 natural gene nucleotides sequences are listed in mammalian cell CHO expression systems by Fig. 4-a Equal GC base contents are：48.20%；Fig. 4-b represent the rArt a3 codons of the present invention after optimization in mammalian cell Average GC base contents are in CHO expression systems：56.87%.

Fig. 5 is the agarose gel electrophoresis figure of rArt a3 gene PCR products.

Wherein, swimming lane 1 is 500bp DNA Ladder；Swimming lane 2 contains AvrII and BstZ17I restriction enzyme sites for both ends RArt a3 gene PCR products.

Fig. 6 is rArta3 expression plasmid pCHO1.0-rArt a3 building process figures.

Fig. 7-a, 7-b are fed-batch fed-batch cultivation expression identification figure of the rArt a3 genes in mammalian cell strain.

Wherein, Fig. 7-a are fed-batch fed-batch cultivation liquid supernatant SDS- of the rArt a3 genes in mammalian cell strain PAGE gel electrophoresis figures.Wherein swimming lane 1 is mammal ghost fed-batch fed-batch cultivation supernatant culture solution, swimming lane after 10 days 2-3 is fed-batch fed-batch cultivation the 3-4 days supernatant culture solution of the rArt a3 genes in mammalian cell strain, and swimming lane 4 is Pre-dyed albumen the loading Marker, swimming lane 5-10 of 10-250KD ranges are respectively rArt a3 genes in mammalian cell strain The 5-10 days supernatant culture solutions of fed-batch fed-batch cultivation.

Fig. 7-b exempt from for fed-batch fed-batch cultivation liquid liquid supernatant protein of the rArt a3 genes in mammalian cell strain Epidemic disease trace figure.Wherein swimming lane 1 is natural A rt a3 protein samples, and swimming lane 2 is the pre-dyed albumen loading of 10-250KD ranges Marker；Swimming lane 3-10 is respectively fed-batch fed-batch cultivation of the rArt a3 genes in mammalian cell strain the 3-10 days Supernatant culture solution.

Fig. 8-a, when 8-b is pH=5.5, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying chromatograms And gel electrophoresis figure.

Wherein, when Fig. 8-a are pH=5.5, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying chromatographies Figure；

When Fig. 8-b are pH=5.5, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying gel electrophoresis figures. Swimming lane 4 is the non-pre-dyed albumen Marker of 11-100KD, other each swimming lanes are collected for each elution to be in charge of.

Fig. 9-a, when 9-b is pH=6.0, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying chromatograms And gel electrophoresis figure.

Wherein, when Fig. 9-a are pH=6.0, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying chromatographies Figure；

When Fig. 9-b are pH=6.0, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying gel electrophoresis figures. Swimming lane 6 is the non-pre-dyed albumen Marker of 11-100KD, other each swimming lanes are collected for each elution to be in charge of.

Figure 10-a, when 10-b is pH=6.5, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying chromatographies Figure and gel electrophoresis figure.

Wherein, when Figure 10-a are pH=6.5, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying chromatographies Figure；

When Figure 10-b are pH=6.5, rArt a3 fed-batch fed-batch cultivation supernatant cation chromatographic purifying gel electrophoresises Figure.Swimming lane 7 is the non-pre-dyed albumen Marker of 11-100KD, other each swimming lanes are collected for each elution to be in charge of.

Specific embodiment

With reference to specific embodiment, the present invention is further explained, it should be appreciated that reference embodiment is merely to illustrate the present invention Rather than it limits the scope of the invention.

Embodiment 1rArt a3 codon optimizations

Inventor is according to the published Artemisia vulgaris clone1Art v3allergen of NCBI MRNA sequence (the GenBank accession number of precursor (Art v3)：EU564846.1 it) is obtained and Art from NCBI EST retrievals CDNA sequence (the NCBI accession number of v3 sequences high homology but the Artemisia annua of unknown gene function： GW332468.1), sequence such as SEQ ID No：Shown in 2, the rArt a3 of the present invention are obtained after codon optimization is carried out to the gene Gene, nucleotide sequence such as SEQ ID No：Shown in 1, amino acid sequence such as SEQ ID No：Shown in 3.Here is to rArt a3 Each parameter comparison is as follows before and after codon optimization：

1. codon adaptation indexI (CAI)

By Fig. 2-a it is found that codon does not have before optimizing, rArt a3 original genes are in mammalian cell CHO expression systems Middle codon adaptation indexI (CAI) is 0.72.By Fig. 2-b it is found that by codon optimization, rArt a3 genes are in mammal CAI indexes are 0.96 in cell CHO expression systems.It is most preferable in the expression system to be considered the gene during usual CAI=1 High efficient expression state, CAI indexes are lower to show that gene expression in the host is poorer, it can be seen that have passed through The gene order obtained after codon optimization can improve table of the rArt a3 genes in mammalian cell CHO expression systems Up to level.

2. optimal codon frequency of use (FOP)

By Fig. 3-a it is found that based on mammalian cell CHO expression vectors, before codon does not optimize, rArt a3 genes It is 13% that percentage, which occurs, in the poor efficiency codon (utilization rate is less than 40% codon) of sequence.What this was not optimized Gene may be decreased translation efficiency or even can be dismissed translation assemblage using series connection rare codon, these codons.By scheming For 3-b it is found that after by codon optimization, there is poor efficiency codon in mammalian cell CHO systems in rArt a3 genes Frequency be 0.

3.GC base contents (GC curve)

G/C content ideal distribution region is 30%-70%, and any peak of appearance outside this region all can be to some extent Influence transcription and translation efficiency.It can by the GC base average contents distributed areas figure comparison of Fig. 4-a, the rArt a3 genes of Fig. 4-b Know, by showing that rArt a3 gene GC bases average content is 48.20% in Fig. 4-a, by being removed after optimization is shown in Fig. 4-b The G/C content peak value occurred outside 30%-70% regions, the GC base average contents for finally obtaining rArt a3 after optimizing are 56.87%.

Embodiment 2：Expression plasmid structure containing rArt a3 genes

RArt a3 genes after codon optimization are introduced into AvrII restriction enzyme site sequences at 5 ' ends, are introduced at 3 ' ends BstZ17I restriction enzyme site sequences, and full genome synthesis is carried out, by the genetic fragment of synthesis, pUC57 plasmids are building up to (by Nanjing Jin Sirui Science and Technology Ltd.s provide) in, a kind of long-term preservation plasmid is obtained, is denoted as pUC57-rArt a3 plasmids.

Using pUC57-rArt a3 plasmids as template, PCR amplification is carried out, the primer sequence is as follows：

Sense primer (SEQ ID No：4)：

M13F:TGT AAA ACG ACG GCC AGT

Downstream primer (SEQ ID No：5)：

M13R:CAG GAA ACA GCT ATG AC

50 μ L of total volume are reacted, wherein a concentration of 10 μm of ol/L primers respectively add 2.5 μ L, the dNTP of a concentration of 10mmol/L to add 1 μ L, archaeal dna polymerase used are Q5 (#M0491L, purchased from New England BioLabs companies), and 2U/ μ L add 0.5 μ L.Reaction Condition for 98 DEG C 5 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 25 cycle after, product is analyzed through 1.0% agarose gel electrophoresis, as a result Show that primer size and expected size (420bp) are consistent (the results are shown in Figure 5).

With AvrII (#R0174S, purchased from New England BioLabs companies) and BstZ17I (#R0594S, purchased from New England BioLabs companies) after double digestion, 1% agarose electrophoresis, obtained gene outcome DNA gel QIAquick Gel Extraction Kit (DP214, purchased from Beijing Tiangeng biochemical technology Co., Ltd) is purified.With T4 ligases (#M0202S, purchased from New England BioLabs it) is connected in pCHO1.0 plasmids (purchased from Life companies), is transformed into Top10 competent cells (CB104, purchased from north Capital Tiangeng biochemical technology Co., Ltd) in, in the LB solid mediums containing kanamycins (0408, purchased from Amresco companies) In 37 DEG C of overnight incubations.

Picking positive colony bacterium carries out PCR identifications, and positive findings are carried out sequencing comparison within second day, complete with expected sequence Entirely unanimously to get to the expression plasmid after rArt a3 codon optimizations, it is denoted as pCHO-rArt a3 (plasmid construction such as Fig. 6 institutes Show).

Embodiment 3：Stablize expression mammalian cell strain containing rArt a3 genes to prepare

Puromycin is aminoglycosides antibiotics, by the albumen for interfering ribosomes function blocking mammalian cell Matter synthesizes, and the pac genes from streptomycete have the function of to release Puromycin toxicity.PCHO1.0 carriers contain pac genes, Therefore using Puromycin as the screening antibiotic that pCHO1.0 is expression vector.MTX is antifol, in the cell It can inhibit the activity of DHFR after conversion, inhibit nucleic acid synthesis, cause cytotoxicity.PCHO1.0 contains DHFR genes, can profit By the use of MTX as screening reagent.Contain Puromycin and MTX resistant genes using the cell after transfection, be continuously increased screening reagent Concentration increase in positive cell target gene copy number so as to improve expression quantity.

Correct pCHO-rArt a3 plasmids NruI limitation restriction endonucleases (#R0192S, purchased from New England will be sequenced BioLabs companies) linearisation, in electrotransfection to CHO-S host cells after, be separately added into the Puromycin of low concentration (A1113802, purchased from Life companies) and MTX (M8407, purchased from Sigma-Aldrich companies) are placed in carbon dioxide incubator 37 DEG C, 8%CO₂Carry out pressurization screening.Cell viability is calculated after 10 days, when Cell viability is transferred to shaking table more than more than 30% In, 37 DEG C, 8%CO₂, 130rpm, which suspends, to be cultivated, and the pressurization screening of Puromycin and MTX concentration is continuously improved and improves purpose base Because of copy number so as to improve expression quantity.

Embodiment 4：Stablize expression mammal strain fed-batch fed-batch cultivation containing rArt a3 genes

High expression rArt a3 stabilizations expression mammal strain will be contained and be inoculated in Dynamis culture mediums (A2617501, purchase From life companies) in, 37 DEG C, 8%CO₂, cultivate in 130rpm shaking tables.

It 3rd day, samples and calculates cell density, when cell density reaches 5 × 10⁶After/mL, glucose is added in final concentration 4g/L and 10%CD EfficientFeed^TMIn C (A2503104, purchased from Life companies) to culture medium.

Cell density is calculated daily, adds within the 5th, 7 day glucose to final concentration of 4g/L and 10%CD EfficientFeed^TMIn C to culture medium.

It collects supernatant culture solution within 10th day or detects the motility rate and density of cell, received when the motility rate of cell is less than 70% Collect supernatant culture solution.With glucose and CD EfficientFeed^TMC batch feeding streams add, different time rArt a3 supernatants Culture solution SDS-PAGE and western blot figure are shown in Fig. 7-a and Fig. 7-b, the results show that rArt a3 have in mammalian cell compared with High expression quantity, can reach 20mg/L.

Embodiment 5：The purifying process of rArt a3 albumen tentatively optimizes

It is mainly pure using cation exchange to rArt a3 culture solutions by the rArt a3 sequence analyses built to this patent Change method, and by being screened to cationic buffer liquid and pH, almost step purifying rArt a3 purity just reaches more than 90%, base Meet in sheet to rArt a3 albumen external biological activity ratings.Chromatographic stuffing selected as HiTrap SP HP (17-1152-01, Purchased from GE healthcare companies), it is as follows：

1. culture solution removal of impurities pretreatment

RArt a3 host cell strain fed-batch culture liquid, 12000rpm are obtained by embodiment 4,15min low-temperature centrifugations are received Collect supernatant, 20mM phosphate buffer ultrafiltration, 0.45 μm of membrane filtration can carry out chromatographing pure up to culture solution supernatant after processing Change.

2. cation-exchange chromatography method optimizes

Buffer solution mother liquor, wherein buffer solution Q1 are configured first：0.2M Na₂HPO₄, buffer solution Q2：0.2M NaH₂PO₄, delay Fliud flushing Q3：ddH₂O, buffer solution Q4：4M NaCl, and 0.45 μm of membrane filtration is crossed respectively.Fully-automatic intelligent protein purification system is used again Q valves carry out equilibration buffer and elution buffer automatic in system (AKTA avant150, purchased from GE healthcare companies) Be configured pH=5.5,6.0,6.5, by previous step pre-process pH=5.5,6.0,6.5 fed-batch culture liquid are secondary in three batches to be flowed through HiTrap SP HP cation-exchange chromatography posts, the above-mentioned corresponding pH value culture solution of secondary purifying in three batches, according to 25%, 50%, 100% isocratic elution, sample peak are concentrated mainly on 25% eluting peak.

When Fig. 8-a are equilibration buffer and elution buffer is pH=5.5, rArt a3 ion-exchange purification chromatograms, figure When 8-b is equilibration buffer and elution buffer is pH=5.5, SDS-PAGE analysis charts after rArt a3 ion-exchange chromatographies；Figure When 9-a is equilibration buffer and elution buffer is pH=6.0, rArt a3 ion-exchange purification chromatograms, Fig. 9-b are balance When buffer solution and elution buffer are pH=6.0, SDS-PAGE analysis charts after rArt a3 ion-exchange chromatographies；Figure 10-a are flat When weighing apparatus buffer solution and elution buffer are pH=6.5, rArt a3 ion-exchange purification chromatograms, Figure 10-b are equilibration buffer During with elution buffer for pH=6.5, SDS-PAGE analysis charts after rArt a3 ion-exchange chromatographies, the results show that equalizing and buffering Liquid and elution buffer are pH=6.0, and rArt a3 purity and the rate of recovery are all significantly improved compared with other pH.

Embodiment 6：The analysis of rArt a3 protein actives

Obtained rArt a3 albumen BCA determination of protein concentration kits (Cat No will be purified:23225, it is purchased from Pierce companies) measure protein concentration, respectively with compared with natural A rt a3 (hereinafter referred to as nArt a3) and pollen hypersensitivity disease Human serum reacts；Table 1 represents that rArt a3 and nArt a3 react OD with 24 pollen hypersensitivity patients serums₄₅₀Absorption value, as a result table Bright rArt a3 react compared with nArt a3 with pollen patients serum, OD₄₅₀Absorption value is basically identical, illustrates thin in mammal The rArt a3 expressed in born of the same parents have very similar biological activity compared with nArt a3, available for pollen desensitization treatment.

Table 1：RArt a3 react OD with nArt a3 with pollen patients serum₄₅₀Absorption value

Sequence table

<110>Co., Ltd of Changzhou Jing Sen Laboratorios Biologicos Farmaceuticos (LABIOFAM) of Jiangsu Zhonghong Biopharma Institute Inc.

<120>A kind of recombination 3 allergoid albumen of artemisia annua and its application

<130>A kind of recombination 3 allergoid albumen of artemisia annua and its application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 354

<212> DNA

<213> Artemisia annua

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atggcttcta tgactatgcg tgttttatgt gtcattgtag cctgcatggt ggttgtcgca 60

ccttatgcgg aagctctctc atgtagtgaa gtaacgagca agttggcgcc atgctttaac 120

tacctaaagt ctggtggtaa ggtgccacca gcatgttgcg acggagtcaa gggactaaac 180

tccgctgcta aaacgacccc tgatagaaag acggcatgca cttgcatgaa gagtgcttat 240

aaatcataca atggcatcaa cgctgataat gctgctggcc ttcctggcaa gtgtggtgtt 300

aatattccct acaagatcag ccttagcacc gactgcaaca aggtcaagtg ataa 354

<210> 2

<211> 354

<212> DNA

<213> Artemisia annua

<400> 2

atggcctcta tgaccatgag ggtgctgtgc gtgatcgtgg cctgtatggt ggtggtggct 60

ccttacgccg aggctctgtc ctgcagcgag gtgacatcca agctggcccc atgtttcaat 120

tatctgaaga gcggaggcaa ggtgccacct gcttgctgtg acggcgtgaa gggcctgaac 180

tccgccgcta agaccacacc cgacaggaag accgcctgca catgtatgaa gtccgcctac 240

aagtcttata acggcatcaa tgctgacaac gctgctggac tgcctggcaa gtgcggagtg 300

aatatcccct acaagatctc tctgtccacc gattgtaaca aggtgaagtg atga 354

<210> 3

<211> 116

<212> PRT

<213> Artemisia annua

<400> 3

Met Ala Ser Met Thr Met Arg Val Leu Cys Val Ile Val Ala Cys Met

1 5 10 15

Val Val Val Ala Pro Tyr Ala Glu Ala Leu Ser Cys Ser Glu Val Thr

20 25 30

Ser Lys Leu Ala Pro Cys Phe Asn Tyr Leu Lys Ser Gly Gly Lys Val

35 40 45

Pro Pro Ala Cys Cys Asp Gly Val Lys Gly Leu Asn Ser Ala Ala Lys

50 55 60

Thr Thr Pro Asp Arg Lys Thr Ala Cys Thr Cys Met Lys Ser Ala Tyr

65 70 75 80

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

85 90 95

Lys Cys Gly Val Asn Ile Pro Tyr Lys Ile Ser Leu Ser Thr Asp Cys

100 105 110

Asn Lys Val Lys

115

<210> 4

<211> 18

<212> DNA

<213>Artificial primer ()

<400> 4

tgtaaaacga cggccagt 18

<210> 5

<211> 17

<212> DNA

<213>Artificial primer ()

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caggaaacag ctatgac 17

Claims (10)

1. A kind of 1. encoding gene for recombinating 3 allergoid albumen of artemisia annua, which is characterized in that its base sequence such as SEQ ID NO： Shown in 1.
2. 2. a kind of carrier containing encoding gene as described in claim 1, the carrier is pcDNA3.1, pcDNA3.3- TOPO、pOptiVEC^TM- TOPO or pCHO1.0.
3. 3. a kind of carrier containing encoding gene as claimed in claim 2, the carrier is pCHO1.0.
4. 4. a kind of Chinese hamster ovary celI strain for including carrier as claimed in claim 3, the cell strain is CHO-S cell strains.
5. 5. a kind of expression for recombinating 3 allergoid albumen of artemisia annua, the method include following step：

   A. structure contains carrier as claimed in claim 3；

   B. it will be transferred to after the vector linearization of step A in CHO host cells as claimed in claim 4, and in suitable condition It is lower to expand culture；

   C. recovery purifying protein.
6. 6. a kind of recombination 3 allergoid protein purification technique of artemisia annua, the purifying process are：To being collected into such as claim Culture medium described in 5 carries out that supernatant is collected by centrifugation, and is obtained after ultrafiltration with one step purified pool of cation-exchange chromatography.
7. 7. a kind of rArt a3 albumen is preparing the application in treating artemisia annua pollen hypersensitivity disease drug, the rArt a3 albumen Amino acid sequence such as SEQ ID NO:Shown in 3.
8. 8. the use as claimed in claim 7, the encoding gene such as SEQ ID NO of the r Art a3 albumen:Shown in 1.
9. 9. the use as claimed in claim 7, the r Art a3 albumen is recombinantly expressed to obtain by Chinese hamster ovary celI.
10. 10. the use as claimed in claim 7, the r Art a3 albumen is expressed to obtain by following methods：

    A, encoding gene as claimed in claim 8 is building up on pCHO1.0 carriers；

    B, CHO-S cell strains will be transferred to after above-mentioned vector linearization, and expand culture under suitable conditions；

    C. recovery purifying protein.