CN114933642A

CN114933642A - Art v 1 recombinant protein and application thereof

Info

Publication number: CN114933642A
Application number: CN202210761944.5A
Authority: CN
Inventors: 孙立薇; 朱冬冬; 沙骥超; 孟粹达; 赵晨; 郎金琦; 崔娜
Original assignee: China-Japan Union Hospital of Jilin University
Current assignee: China-Japan Union Hospital of Jilin University
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2022-08-23
Anticipated expiration: 2042-06-30
Also published as: CN114933642B

Abstract

The invention relates to the technical field of protein purification, and particularly relates to an Art v 1 recombinant protein and application thereof. The invention successfully constructs a prokaryotic in-vitro expression system with high expression of the allergenic protein Art v 1. The sequence of the Art v 1 gene is optimized to obtain a nucleic acid molecule which is suitable for a prokaryotic expression system and encodes the Art v 1 recombinant protein; the nucleic acid molecule is artificially synthesized and then is subcloned to a eukaryotic vector to obtain a recombinant vector, and a prokaryotic expression system is transformed, so that the high-efficiency expression of the Art v 1 recombinant protein is realized, and the advantages are goodAfter digestion 200ml OD ₆₀₀ Coli with a 0.6 size can purify about 500 μ g protein; the preparation method disclosed by the invention is simple to operate, low in cost, large in yield, high in activity and wide in application prospect in the field of diagnosis and treatment of allergic diseases.

Description

Art v 1 recombinant protein and application thereof

Technical Field

The invention relates to the technical field of protein expression, in particular to an Art v 1 recombinant protein and application thereof.

Background

Allergic rhinitis refers to a non-infectious chronic inflammatory reaction that occurs after exposure of the nasal mucosa to an allergen. Epidemiological surveys have shown that the incidence of AR has increased over the past decades, currently affecting 10% to 40% of the world population. Allergic rhinitis not only affects the quality of life, but also causes a huge social and economic burden, and has become a global health problem, but the pathogenesis of the allergic rhinitis is still unclear. The major allergens are divided into two categories, seasonal allergens mainly comprising pollen and moulds, perennial allergens mainly comprising dust mites, pets, pests and some moulds. Pollen is the major allergen in seasonal allergic rhinitis. Due to geographical conditions, climatic factors and human factors, the distribution of plants has obvious regionalism, so that the regional characteristics of pollen spreading are caused, for example, wormwood is the most common allergen in summer and autumn in northern areas of China. Art v 1 is the major allergenic protein of wormwood pollen, a modular glycoprotein with a defensin-like domain and a hydroxyproline-rich domain. Therefore, the in vitro preparation of the recombinant protein Art v 1 is an important means for researching the pathogenesis of seasonal allergic rhinitis caused by artemisia apiacea pollen.

At present, freeze-dried powder or crude extract of artemisia apiacea is mainly used for researching the artemisia apiacea pollen in the allergic rhinitis, the freeze-dried powder or crude extract obtained after the artemisia apiacea pollen is dissolved by sterile PBS buffer solution is used for treating cells or is used for animal experiments, and the pathogenesis of the artemisia apiacea allergen in the allergic rhinitis is researched, but the method has the defect that the freeze-dried powder or crude extract in different batches has difference, wherein the content of main sensitization protein is different, and the experiment repeatability is poor. Furthermore, freeze-dried powder and crude extract are complex composite components, and the pathogenic effect of the protein or the components cannot be clarified in the research of pathogenic mechanism. In some studies, natural extracts of Art v 1 have been used, and the natural proteins are isolated and extracted from Artemisia annua by liquid chromatography, mass spectrometry, or the like, which is expensive and low in extraction yield.

With the development of molecular biology technology, the use of recombinant allergens by genetic engineering for allergic diseases has become one of the hot spots for research. Recombinant allergens are highly purified in large quantities by recombinant DNA technology starting from cDNA encoding allergens, which have good lot-to-lot consistency and can meet medical quality requirements for allergens. At present, researchers clone Art v 1 protein from wormwood into pcDNA3.1/myc-His A vector, and express the Art v 1 protein by taking a mammalian cell HEK293F cell as a host, so that the purity of the expressed protein is high. However, compared with the method using microorganisms as hosts, the expression of the protein is further limited by the characteristics of weak replication and regeneration capacity of mammalian cells, high requirement on culture conditions, high difficulty in expanding production and the like, and the protein is easy to generate inclusion bodies due to prokaryotic heterologous expression, so that the difficulty in protein purification is increased, and the case that the protein is expressed in prokaryotic model organisms does not exist at present.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an Art v 1 recombinant protein and applications thereof.

The invention provides a nucleic acid encoding Art v 1, comprising any one of the following I) to IV:

I) the sequence is shown as SEQ ID NO. 2;

II) having at least 80% homology to the nucleic acid shown in I) and encoding a protein having the same or similar function as the nucleic acid shown in I);

II) the nucleic acid shown in the I) is modified, substituted, deleted or added with one or more bases;

IV) nucleic acids complementary or partially complementary to the nucleic acids indicated under I).

Furthermore, Art v 1 is derived from wormwood, the original nucleic acid sequence of the Art v 1 is shown as SEQ ID No.1, the Art v is optimized after the codon usage frequency, the splicing sites of RNase and the RNA stable trans-acting element are fully considered, and the optimized nucleic acid sequence is shown as SEQ ID No. 2. Experiments show that the optimal sequence coded Art v 1 protein has high expression quantity, high solubility and strong protein activity.

The present invention provides a recombinant protein comprising a solubilizing and/or purifying element, and Art v 1. Further, the solubilizing and/or purifying elements are GST and his.

Furthermore, the recombinant protein of the present invention further includes a protease cleavage site, where the protease cleavage site may include a thrombin cleavage site (thrombin site) or other protease cleavage sites commonly used in molecular biology, which is not limited in this disclosure.

In some embodiments, the recombinant protein of the invention comprises GST-thrombin site-Art v 1-his in sequence from N-terminal to C-terminal; or comprises his-GST-thrombin site-Art v 1-his from the N end to the C end in sequence; or GST-thrombin site-Art v 1-thrombin site-his is sequentially included from the N end to the C end; or GST-Art v 1-symbol site-his is included from N terminal to C terminal, which is not limited in the present invention. In some specific embodiments, the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3.

Furthermore, the invention also provides a nucleic acid for coding the recombinant protein, and the nucleic acid for coding the recombinant protein is shown as SEQ ID NO. 4.

The invention provides a recombinant vector, which comprises a vector framework and a nucleic acid segment, wherein the nucleic acid segment comprises the nucleic acid disclosed by the invention and/or the nucleic acid for encoding the recombinant protein disclosed by the invention. In the invention, pGEX-4T-1 is used as a vector for expression, and the purified recombinant protein has high solubility, high yield and strong activity and is superior to other vectors (such as pET-28 a). In some embodiments, the recombinant protein is expressed from pGEX-4T-1 as a subject.

Furthermore, the recombinant vector of the present invention further includes the recombinant vector formed by the nucleic acid or the nucleic acid encoding the recombinant protein in a single or repeated tandem form, which is not limited by the present invention.

The invention provides an expression system, which comprises a host and at least one of the following i) to iv):

i) the nucleic acid of the present invention;

ii) a recombinant protein according to the invention;

iii) a nucleic acid encoding a recombinant protein according to the invention;

iv) the recombinant vector of the invention.

Furthermore, the expression system is a prokaryotic expression system, and the host is escherichia coli.

Further, in some embodiments, the host of the present invention is escherichia coli BL21(DE 3).

In the present invention, the expression system also includes an expression system formed by other types of prokaryotic hosts transformed or transfected with the recombinant vector, and the present invention is not limited herein.

Further, in the present invention, 200ml of OD was obtained after 4 hours of induction at 37 ℃ and 1.0mM IPTG using LB liquid medium ₆₀₀ Coli at 0.6 can purify about 500. mu.g of protein.

The invention provides a method for preparing the expression system, which comprises transforming or transfecting a host cell with the vector. The method for transformation comprises the following steps: chemical conversion and electrical conversion; the transfection method comprises calcium phosphate coprecipitation, an artificial liposome method and virus transfection. In some embodiments of the present invention, the expression system is prepared by transforming the recombinant vector of the present invention into E.coli BL21(DE3) by chemical transformation.

The invention provides a preparation method of an Art v 1 protein, which obtains a product containing the Art v 1 protein by fermenting the expression system.

Further, the product containing the Art v 1 protein of the present invention includes fermentation broth, bacteria, supernatant of the Art v 1 protein and recombinant protein thereof, or active substances containing the Art v 1 protein or recombinant protein, which is not limited in the present invention.

Furthermore, the invention also provides a preparation containing the product, and the preparation can be bacterial powder, granules or bacterial liquid, which is not limited in the invention.

The invention provides an application of recombinant protein in preparing a medicament for preventing and treating allergic diseases and/or a diagnostic reagent for the allergic diseases. The allergic diseases comprise skin allergy and respiratory allergy. The respiratory tract allergy comprises allergic rhinitis, allergic asthma and the like caused by wormwood.

Furthermore, the invention also provides a medicament for preventing and treating allergic diseases and/or a diagnostic reagent for the allergic diseases, which comprises the recombinant protein and/or the recombinant protein produced by the preparation method.

Furthermore, the diagnostic reagent of the present invention further comprises an ELISA reagent, a western blot reagent, a GST-pulldown reagent, a co-IP reagent and an IP reagent for detecting Art v 1 expression or in vivo and in vitro protein interaction of Art v 1, which is not limited in the present invention.

Furthermore, the diagnostic reagent of the invention also comprises detection paper or a detection card which is made of medically acceptable auxiliary materials or carriers and recombinant proteins. The detection method may be visual observation, machine reading, or a combination of visual observation and machine reading, which is not limited in the present invention.

The invention obtains at least one of the following beneficial effects:

(1) the sequence of the Art v 1 gene is optimized to obtain a nucleic acid molecule which is suitable for a prokaryotic expression system and codes the Art v 1 recombinant protein;

(2) the invention artificially synthesizes the nucleic acid molecule, then subclones the nucleic acid molecule to a eukaryotic vector to obtain a recombinant vector, transforms a prokaryotic expression system, realizes the high-efficiency expression of the Art v 1 recombinant protein, and optimizes the expression to 200ml OD ₆₀₀ Coli at 0.6 can purify about 500. mu.g of protein.

(3) The preparation method of the invention has the advantages of simple operation, low cost, high yield, high activity of the recombinant protein, no influence of the label and wide application prospect in the field of diagnosis and treatment of allergic diseases.

Drawings

FIG. 1 shows a map of pGEX-4T-1-6XHis-Art v 1 plasmid;

FIG. 2 shows the expression purification of GST-Art v 1 protein, M: marker, 1: before induction, 2: after induction, 3: effluent, 4: binding buffer, 5: washing buffer, 6: eluting the solution;

FIG. 3 shows Western blot for detecting Art v 1 protein expression, M: marker, 1: GST, 2: ultrasonication of the supernatant, 3: GST-Art v 1;

FIG. 4 shows ELISA detection of Art v 1 protein binding activity;

FIG. 5 shows the expression pattern of the Art v 1 protein after purification of pET-28a-Art v 1, M: marker, 1: before induction, 2: after induction, 3: crushing supernatant, 4: crushing and precipitating;

FIG. 6 shows the expression pattern of the Art v 1 protein after purification of pGEX-4T-1-Art v 1, M: marker, 1: before induction, 2: after induction, 3: crushing supernatant, 4: crushing and precipitating;

FIG. 7 shows ELISA detection of Art v 1 protein binding activity;

FIG. 8 shows comparison of the expression level of Art v 1 protein after purification of pGEX-4T-1-Art v 1, M: marker, 1: before optimization, 2: and (5) after optimization.

Detailed Description

The invention provides the Art v 1 recombinant protein and application thereof, and a person skilled in the Art can realize the Art v 1 recombinant protein by appropriately improving process parameters by referring to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

In the invention, the homology of at least 80% refers to a sequence with the similarity of more than or equal to 80% with the nucleic acid sequence, and further refers to a sequence with the similarity of more than or equal to 85% with the nucleic acid sequence; furthermore, the sequence has a similarity of more than or equal to 90% with the nucleic acid sequence; specifically, the 80% refers to the nucleic acid sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of similarity with the nucleic acid sequence.

In the invention, the nucleic acid sequence coding for Art v 1 is: atggctaaatgctcatatgtattttgtgcagtgttgctgatctttatcgtcgcaattggtgagatggaggccgcaggctcgaaactgtgtgagaagacctccaagacgtacagcggtaagtgcgataacaagaagtgcgacaaaaaatgcattgagtgggaaaaggcgcagcatggtgcatgtcacaaacgtgaagcgggcaaagaatcatgcttctgctatttcgattgtagcaaaagcccgccgggtgccaccccggcgccgccgggtgctgcgcctccaccggctgcggggggctctccgagcccgccggcggacggtggctctccgccaccgccggcggacggcggtagcccgccggttgatggcggttccccgccgccgccaagcacccactaa (shown in SEQ ID NO. 2).

In the invention, the amino acid sequence of the recombinant protein is MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPEFMAKCSYVFCAVLLIFIVAIGEMEAAGSKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHKREAGKESCFCYFDCSKSPPGATPAPPGAAPPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPSTHVDHHHHHH (shown as SEQ ID NO. 3).

In the invention, the nucleic acid sequence of the coding recombinant protein is atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctggttccgcgtggatccccggaattcatggctaaatgctcatatgtattttgtgcagtgttgctgatctttatcgtcgcaattggtgagatggaggccgcaggctcgaaactgtgtgagaagacctccaagacgtacagcggtaagtgcgataacaagaagtgcgacaaaaaatgcattgagtgggaaaaggcgcagcatggtgcatgtcacaaacgtgaagcgggcaaagaatcatgcttctgctatttcgattgtagcaaaagcccgccgggtgccaccccggcgccgccgggtgctgcgcctccaccggctgcggggggctctccgagcccgccggcggacggtggctctccgccaccgccggcggacggcggtagcccgccggttgatggcggttccccgccgccgccaagcacccacgtcgaccaccaccaccaccaccac (shown as SEQID NO. 4).

In some embodiments, the present invention provides a method for preparing the expression system, comprising the steps of:

(1) optimizing an Art v 1 nucleic acid sequence;

(2) the Art v 1 nucleic acid sequence is integrated with the vector framework;

(3) transforming the integration system into escherichia coli, and screening correct transformants through colony PCR verification;

(4) inducing for 4h by using LB liquid culture medium under the induction condition of 1.0mMIPTG at 37 ℃;

further, the protein product after induction expression is purified by a nickel column to obtain the Art v 1 recombinant protein.

In the invention, the recombinant vector is constructed by utilizing an enzyme digestion connection mode, a PCR primer with an enzyme digestion site is designed, a target fragment with the enzyme digestion site is obtained through amplification, then the fragment and the vector are subjected to enzyme digestion, and escherichia coli is transformed after the fragment and the vector are connected by using T4 enzyme, so that a correct recombinant vector is obtained.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1 in vitro preparation and use of Art v 1 recombinant protein

In order to make the protein better expressed in Escherichia coli, the codon usage frequency, splicing sites of RNase and RNA stable trans-acting elements are fully considered, sequence optimization is carried out, the optimized sequence is shown as SEQ ID NO.2, and a prokaryotic recombinant expression vector of the protein is constructed. pGEX-4T-1 is selected as a vector template, the vector is a prokaryotic expression vector and is provided with a GST tag, and a sequence-optimized reading frame region (CDS) sequence of wormwood Art v 1 is inserted between EcoRI and SalI enzyme cutting sites behind the GST tag to construct GST-Art v 1 fusion protein. Meanwhile, a 6XHis fragment is inserted into the CDS sequence of the Art v 1, and a pGEX-4T-1-6XHis-Art v 1 recombinant expression vector is constructed, wherein a plasmid map is shown in figure 1. All vectors used were synthesized by sequencing.

The pGEX-4T-1-6XHis-Art v 1 recombinant expression vector successfully designed and constructed is transferred into Escherichia coli competent cell BL21(DE 3). LB liquid culture medium is selected, and induction expression condition is 1.0mMIPTG induction at 37 ℃ for 4 h. The Coomassie blue staining of SDS-PAGE is shown in FIG. 2, and GST-Art v 1 protein with higher purity is purified in lane 6, which shows that 200ml OD ₆₀₀ Coli at 0.6 can purify about 500. mu.g of protein.

Expression of Art v 1 in the GST-Art v 1 fusion protein was subsequently detected by Western Blot assay. Firstly, SDS-PAGE electrophoresis and membrane transfer are carried out on supernatant obtained after the thalli are subjected to ultrasonication and purified GST-Art v 1 protein, the purified GST protein is used as a control, then an anti-Art v 1 antibody is incubated, and the expression condition of the Art v 1 protein is detected, the experimental result is shown in figure 3, the expression of the Art v 1 protein is detected in a second lane and a third lane, and meanwhile, the development result shows that the impurity band in the third lane is less, which indicates that the purified GST-Art v 1 has the expression of the Art v 1 and has higher purity compared with the purified GST-Art v 1.

The purified GST-Art v 1 protein was then assayed for Art v 1 binding activity. The results of ELISA experiments are shown in FIG. 4, in which OD was measured in the BSA group as the negative control protein and in the purified GST protein group (NC group in the figure) ₄₅₀ The values were all lower, demonstrating that BSA and GST proteins had no binding activity to the Art v 1 antibody, whereas in the GST-Art v 1 protein group (Art v 1 group in the figure) OD ₄₅₀ Shows a higher value, which proves that the purified GST-Art v 1 has better binding activity with the anti-Art v 1 antibody.

Comparative example 1 comparison of protein expression and activity of different vectors Art v 1:

cloning a gene (SEQ ID NO.1) which is not subjected to sequence optimization and codes the Art v 1 protein into a vector Pet-28 a; the gene (SEQ ID NO.1) which is not optimized in sequence and codes the Artv 1 protein is cloned in a vector pGEX-4T-1, and recombinant vectors pET-28a-Art v 1 and pGEX-4T-1-Art v 1 are constructed. An LB liquid culture medium is selected, induction expression conditions are 1.0mMIPTG induction at 37 ℃ for 4h, as shown in figure 5, pET-28a-Art v 1 is mainly expressed in an inclusion body form after being purified, expressed protein needs to be denatured and renatured, the experimental process is complex, the cost is improved, and meanwhile, the protein binding activity after renaturation is not as good as that of soluble expressed protein. pGEX-4T-1-Art v 1 purified protein is mainly expressed in a soluble form, and the protein is directly purified in ultrasonic disruption supernatant (as shown in figure 6), and 200ml OD ₆₀₀ 0.6 of Escherichia coli is subjected to protein purification, and the protein expression amount is 100-150 mug.

Meanwhile, since pET-28a-Art v 1 is mainly expressed in the form of inclusion body after purification, denaturation and renaturation are required, and the binding activity of the protein and the Art v 1 antibody is reduced compared with that of the protein after pGEX-4T-1-Art v 1 purification (as shown in FIG. 7).

Comparative example 2 pGEX-4T-1-Art v 1 sequence comparison of expression before and after optimization

The non-optimized target gene (shown as SEQ ID NO.1) is cloned into a vector pGEX-4T-1. The optimized target protein gene sequence (shown as SEQ ID NO. 2) is cloned in a vector pGEX-4T-1. The vector was synthesized by the company. After induction of expression, proteins before and after nickel column purification were subjected to SDS-PAGE analysis using 200ml OD ₆₀₀ The protein was purified in 0.6 E.coli, and the expression level of the protein before optimization was 100. mu.g to 150. mu.g, and the expression level of the protein after sequence optimization was about 500. mu.g (as shown in FIG. 8).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Zhongri friendship Hospital of Jilin university

<120> Art v 1 recombinant protein and application thereof

<130> MP22014592

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 396

<212> DNA

<213> Artemisia (Artemisia)

<400> 1

atggcaaagt gttcatatgt tttctgtgcg gttcttctga ttttcatagt tgctatcgga 60

gaaatggagg ccgctggttc aaagttgtgt gaaaagacaa gcaagacgta ttcgggtaag 120

tgcgacaaca agaaatgtga caaaaagtgt atagagtggg agaaagcgca acatggtgct 180

tgtcacaaga gagaagccgg caaagaaagt tgcttttgct actttgactg ttccaaatcg 240

cctcctggag caacaccagc gcctcctggt gcagctcctc ccccagctgc tggcggctct 300

ccgtcacctc ccgctgatgg tggctcacca cctcctccag ctgatggtgg atctcctcct 360

gtagatggtg gctctccacc tcctccgtcc actcac 396

<210> 2

<211> 396

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggctaaat gctcatatgt attttgtgca gtgttgctga tctttatcgt cgcaattggt 60

gagatggagg ccgcaggctc gaaactgtgt gagaagacct ccaagacgta cagcggtaag 120

tgcgataaca agaagtgcga caaaaaatgc attgagtggg aaaaggcgca gcatggtgca 180

tgtcacaaac gtgaagcggg caaagaatca tgcttctgct atttcgattg tagcaaaagc 240

ccgccgggtg ccaccccggc gccgccgggt gctgcgcctc caccggctgc ggggggctct 300

ccgagcccgc cggcggacgg tggctctccg ccaccgccgg cggacggcgg tagcccgccg 360

gttgatggcg gttccccgcc gccgccaagc acccac 396

<210> 3

<211> 369

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg

210 215 220

Gly Ser Pro Glu Phe Met Ala Lys Cys Ser Tyr Val Phe Cys Ala Val

225 230 235 240

Leu Leu Ile Phe Ile Val Ala Ile Gly Glu Met Glu Ala Ala Gly Ser

245 250 255

Lys Leu Cys Glu Lys Thr Ser Lys Thr Tyr Ser Gly Lys Cys Asp Asn

260 265 270

Lys Lys Cys Asp Lys Lys Cys Ile Glu Trp Glu Lys Ala Gln His Gly

275 280 285

Ala Cys His Lys Arg Glu Ala Gly Lys Glu Ser Cys Phe Cys Tyr Phe

290 295 300

Asp Cys Ser Lys Ser Pro Pro Gly Ala Thr Pro Ala Pro Pro Gly Ala

305 310 315 320

Ala Pro Pro Pro Ala Ala Gly Gly Ser Pro Ser Pro Pro Ala Asp Gly

325 330 335

Gly Ser Pro Pro Pro Pro Ala Asp Gly Gly Ser Pro Pro Val Asp Gly

340 345 350

Gly Ser Pro Pro Pro Pro Ser Thr His Val Asp His His His His His

355 360 365

His

<210> 4

<211> 1107

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctggttccgc gtggatcccc ggaattcatg gctaaatgct catatgtatt ttgtgcagtg 720

ttgctgatct ttatcgtcgc aattggtgag atggaggccg caggctcgaa actgtgtgag 780

aagacctcca agacgtacag cggtaagtgc gataacaaga agtgcgacaa aaaatgcatt 840

gagtgggaaa aggcgcagca tggtgcatgt cacaaacgtg aagcgggcaa agaatcatgc 900

ttctgctatt tcgattgtag caaaagcccg ccgggtgcca ccccggcgcc gccgggtgct 960

gcgcctccac cggctgcggg gggctctccg agcccgccgg cggacggtgg ctctccgcca 1020

ccgccggcgg acggcggtag cccgccggtt gatggcggtt ccccgccgcc gccaagcacc 1080

cacgtcgacc accaccacca ccaccac 1107

Claims

1. A nucleic acid encoding Art v 1, comprising any one of I) to IV:

I) the sequence is shown as SEQ ID NO. 2;

II) nucleic acid with one or more modified, substituted, deleted or added bases of the nucleic acid shown in I);

IV), a nucleic acid which is complementary or partially complementary to the nucleic acid shown in I).

2. A recombinant protein comprising a solubilizing element and/or a purifying element, and Art v 1; the solubilizing and/or purifying element comprises GST and/or his; the Art v 1 is encoded by the nucleic acid of claim 1.

3. The recombinant protein according to claim 2, wherein the amino acid sequence is as shown in SEQ ID No. 3.

4. A nucleic acid encoding the recombinant protein of claim 2 or 3, comprising the nucleic acid of claim 1, as set forth in SEQ ID No. 4.

5. A recombinant vector comprising a vector backbone and a nucleic acid fragment comprising the nucleic acid of claim 1, and/or the nucleic acid of claim 4.

6. Expression system, characterized in that it comprises a host and at least one of the following i) to iv):

i) the nucleic acid of claim 1;

ii) the recombinant protein of claim 2 or 3;

iii) the nucleic acid of claim 4;

iv) the recombinant vector of claim 5.

7. A method for producing the expression system according to claim 6, which comprises transforming or transfecting a host with the recombinant vector according to claim 5.

8. A process for producing a recombinant protein according to claim 2 or 3, wherein the expression system according to claim 6 is fermented to obtain a product containing the Art v 1 protein.

9. Use of the recombinant protein according to claim 2 or 3 for the preparation of a medicament for the prevention and/or treatment of allergic diseases and/or a diagnostic agent for allergic diseases.

10. A pharmaceutical and/or diagnostic agent for preventing and/or treating allergic diseases, which comprises the recombinant protein according to claim 2 or 3 or the recombinant protein produced by the production method according to claim 7.