CN114933642B

CN114933642B - Art v 1 recombinant protein and application thereof

Info

Publication number: CN114933642B
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: 2023-09-12
Anticipated expiration: 2042-06-30
Also published as: CN114933642A

Abstract

The invention relates to the technical field of protein purification, in particular to an Art v 1 recombinant protein and application thereof. The invention successfully constructs a prokaryotic in-vitro expression system for high expression of the sensitized protein Art v 1. The invention optimizes the sequence of the Art v 1 gene to obtain a nucleic acid molecule which is applicable to a prokaryotic expression system and used for encoding Art v 1 recombinant protein; and subcloning the nucleic acid molecule into a eukaryotic vector after artificial synthesis to obtain a recombinant vector, transforming a prokaryotic expression system, realizing the high-efficiency expression of the Art v 1 recombinant protein, and optimizing 200ml OD ₆₀₀ Coli at 0.6 was able to purify approximately 500 μg of protein; the preparation method of the invention has simple operation, low cost, large yield and high activity, and has wide application prospect in the fields of allergic disease diagnosis and treatment.

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 gradually over the last few decades, currently affecting 10% to 40% of the world population. Allergic rhinitis not only affects quality of life, but also can cause a huge socioeconomic burden, and has become a global health problem, but its pathogenesis is not clear. Major allergens fall into two categories, seasonal allergens mainly contain pollen and moulds, perennial allergens mainly contain dust mites, pets, pests and some moulds. Pollen is the major allergen of seasonal allergic rhinitis. Due to geographical conditions, climatic factors and human factors, the distribution of plants is obviously regional, so that regional characteristics of pollen spreading, such as artemisia are the most common allergens in the summer and autumn in northern China. Art v 1 is the major sensitizer protein for artemisia pollen, a modular glycoprotein with a defensin-like domain and a hydroxyproline-rich domain. Thus, in vitro preparation of recombinant proteins by Art v 1 is an important tool for studying the pathogenesis of seasonal allergic rhinitis caused by artemisia pollen.

At present, freeze-dried powder or crude extract of Artemisia annua is mainly used for exploring the allergic rhinitis of Artemisia annua pollen, the freeze-dried powder or crude extract of Artemisia annua is used for treating cells after being dissolved by sterile PBS buffer solution or is used for animal experiments, and the pathogenesis of the Artemisia annua allergen in the allergic rhinitis is explored, but the defect is that the freeze-dried powder or crude extract of different batches have differences, wherein the content of main sensitization proteins is different, and the experimental repeatability is poor. And the freeze-dried powder and the crude extract are complex composite components, and the pathogenic action of the protein or the components cannot be clarified in the research of pathogenic mechanism. In individual researches, natural extracts of Art v 1 are also used, and the natural proteins are separated and extracted from artemisia annua by means of liquid chromatography, mass spectrometry and the like, and have the defects of high price, low extraction quantity and the like.

With the development of molecular biology techniques, the use of genetically engineered recombinant allergens for allergic diseases has become one of the hot spots of research. Recombinant allergens are obtained by recombinant DNA technology starting from cDNA encoding the allergen, and a large number of highly purified allergens are obtained, which have good lot-to-lot consistency and can meet the quality requirements of the allergen in medicine. At present, researchers clone Art v 1 protein from Artemisia in pcDNA3.1/myc-His A vector, and express the protein by taking a mammalian HEK293F cell as a host, so that the purity of the expressed protein is higher. However, compared with the method using microorganism as host, the method has the characteristics of weak replication and regeneration capability of mammal cells, high requirement on culture conditions, large production difficulty and the like, further limits the expression of the protein, and has no case of expressing the protein in a prokaryotic model organism at present because the prokaryotic heterologous expression of the protein easily generates inclusion bodies and increases the protein purification difficulty.

Disclosure of Invention

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

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

i) Nucleic acid with a sequence shown as SEQ ID NO. 2;

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

II) nucleic acids having one or more bases modified, substituted, deleted or added to the nucleic acid as shown in I);

IV), a nucleic acid complementary or partially complementary to a nucleic acid as shown in I).

Furthermore, the Art v 1 is derived from Artemisia annua, the original nucleic acid sequence is shown as SEQ ID NO.1, the codon use frequency, the splice site of RNase and the RNA stabilizing trans-acting element are fully considered, and then the optimized nucleic acid sequence is shown as SEQ ID NO. 2. Experiments show that the Art v 1 protein encoded by the optimized sequence has high expression level, high solubility and strong protein activity.

The present invention provides a recombinant protein comprising a dissolution and/or purification 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, which may include a thrombin cleavage site (thrombin site), or may include other protease cleavage sites commonly used in molecular biology, which is not limited in this invention.

In some embodiments, the recombinant protein of the invention comprises GST-thrombin site-Art v 1-his in order from N-terminus to C-terminus; or from the N end to the C end, sequentially comprises his-GST-thiombin site-Art v 1-his; or GST-thrombin site-Art v 1-thrombin site-his from N terminal to C terminal; or GST-Art v 1-thrombin site-his is included in sequence from the N-terminal to the 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 encoding the recombinant protein, and the nucleic acid for encoding the recombinant protein is shown as SEQ ID NO. 4.

The invention provides a recombinant vector, which comprises a vector skeleton and a nucleic acid fragment, wherein the nucleic acid fragment 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 the advantages of high solubility, high yield and strong activity, and is superior to other vectors (such as pET-28 a). In some specific embodiments, the recombinant protein is expressed in pGEX-4T-1 as a subject.

Furthermore, the recombinant vector of the present invention further includes a 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 in the present invention.

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

i) Nucleic acids according to the invention;

ii) the recombinant protein of the invention;

iii) Nucleic acids encoding recombinant proteins of the invention;

iv) the recombinant vector according to the invention.

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

Still further, in some embodiments, the host of the present invention is E.coli BL21 (DE 3).

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

Further, in the present invention, 200ml of OD after 4 hours of induction at 37℃under the induction condition of 1.0mM IPTG was obtained by using LB liquid medium ₆₀₀ Coli at 0.6 was able to purify approximately 500 μg of protein.

The invention provides a preparation method of the expression system, which comprises the step of transforming or transfecting the vector of the invention into a host cell. The method for conversion comprises the following steps: chemical and electrical conversion; the transfection method comprises calcium phosphate coprecipitation, an artificial liposome method and virus transfection. In some embodiments of the invention, the preparation method of the expression system comprises transforming the recombinant vector of the invention into escherichia coli BL21 (DE 3) by a chemical transformation method.

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

Further, the product containing the Art v 1 protein according to the present invention includes fermentation broth, bacterial cells, supernatant, or active substance containing Art v 1 protein or recombinant protein of Art v 1 protein and recombinant protein thereof, which is not limited in the present invention.

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

The invention provides application of recombinant protein in preparing medicines for preventing and treating allergic diseases and/or diagnostic reagents for 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 artemisia anomala.

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

Furthermore, the diagnostic reagent of the present invention further includes ELISA reagents, western blot reagents, GST-pulldown reagents, co-IP reagents, IP reagents for detecting the expression of Art v 1 or the in vivo and in vitro protein interactions of Art v 1, and the present invention is not limited thereto.

Furthermore, the diagnostic reagent also comprises a detection paper or a detection card which is prepared from medical acceptable auxiliary materials or carriers and recombinant proteins. The detection method can be visual inspection, machine reading or a combination of visual inspection and machine reading, and the invention is not limited to this.

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

(1) The invention optimizes the sequence of the Art v 1 gene to obtain the nucleic acid molecule which is applicable to a prokaryotic expression system and codes the Art v 1 recombinant protein;

(2) The invention subclones the nucleic acid molecule into eukaryotic vector to obtain recombinant vector after artificial synthesis, and converts prokaryotic expression system to realize high-efficiency expression of Art v 1 recombinant protein, and 200ml OD after optimization ₆₀₀ Coli at 0.6 was able to purify approximately 500 μg of protein.

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

Drawings

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

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: wash buffer, 6: eluting the eluent;

FIG. 3 shows Western blot detection of artv 1 protein expression, M: marker,1: GST,2: ultrasonically crushing the supernatant, 3: GST-Art v 1;

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

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

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

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

FIG. 8 shows Art v 1 protein expression level comparison after pGEX-4T-1-Art v 1 purification, 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 refer to the content of the text and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein 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. For definitions and terms in the art, the expert may refer specifically to Current Protocols in Molecular Biology (Ausubel). The 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 present invention, the at least 80% homology means a sequence having a similarity of not less than 80% with the nucleic acid sequence, and further means a sequence having a similarity of not less than 85% with the nucleic acid sequence; further, it means a sequence having a similarity of 90% or more with the nucleic acid sequence; specifically, 80% refers to a nucleic acid sequence having a degree of similarity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the nucleic acid sequence.

In the invention, the nucleic acid sequence for encoding Art v 1 is as follows: atggctaaatgctcatatgtattttgtgcagtgttgctgatctttatcgtcgcaattggtgagatggaggccgcaggctcgaaactgtgtgagaagacctccaagacgtacagcggtaagtgcgataacaagaagtgcgacaaaaaatgcattgagtgggaaaaggcgcagcatggtgcatgtcacaaacgtgaagcgggcaaagaatcatgcttctgctatttcgattgtagcaaaagcccgccgggtgccaccccggcgccgccgggtgctgcgcctccaccggctgcggggggctctccgagcccgccggcggacggtggctctccgccaccgccggcggacggcggtagcccgccggttgatggcggttccccgccgccgccaagcacccactaa (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 SEQ ID NO. 4).

In some embodiments, the invention provides a method of making the expression system comprising the steps of:

(1) Artv 1 nucleic acid sequence optimization;

(2) Integrating the Art v 1 nucleic acid sequence with a vector backbone;

(3) The integration system is used for transforming escherichia coli, and correct transformants are screened through colony PCR verification;

(4) Inducing for 4h at 37 ℃ under the induction condition of 1.0mM IPTG by utilizing LB liquid medium;

further, the protein product after induced 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 a digestion connection mode, a PCR primer with a digestion site is designed, a target fragment with the digestion site is obtained by amplification, then, the fragment and the vector are digested, and the E.coli is transformed after being connected by T4 enzyme, so that the 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 proteins

In order to make the protein express better in colibacillus, fully consider codon frequency and splice site of RNase, RNA stabilize trans-acting element, optimize the sequence, the optimized sequence is shown in SEQ ID NO.2, we construct the prokaryotic recombinant expression vector of the protein. 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 sequence of an open reading frame (CDS) sequence of Artemisia Art v 1 is inserted between EcoRI and SalI cleavage sites after GST tag, so that GST-Art v 1 fusion protein is constructed. Meanwhile, a 6XHis fragment is inserted after the Art v 1CDS sequence to construct a pGEX-4T-1-6XHis-Art v 1 recombinant expression vector, and a plasmid map is shown in figure 1. The vectors used were all synthesized by sequencing companies.

The pGEX-4T-1-6XHis-Art v 1 recombinant expression vector which was successfully designed and constructed was transferred into E.coli competent cell BL21 (DE 3). Selecting LB liquid culture medium, and inducing expression condition is 37 deg.C 1.0mM IPTG was induced for 4h. As shown in FIG. 2, the result of coomassie brilliant blue staining of SDS-PAGE electrophoresis gel shows that GST-Art v 1 protein with higher purity is purified in lane 6, and the result shows that 200ml OD ₆₀₀ Coli at 0.6 was able to purify approximately 500 μg of protein.

The expression of Art v 1 in GST-Art v 1 fusion proteins was then detected by Western Blot experiments. Firstly, performing SDS-PAGE electrophoresis and membrane transfer on the supernatant after ultrasonic disruption of the thalli and the purified GST-Art v 1 protein, taking the purified GST protein as a control, then incubating an anti-Art v 1 antibody, detecting the expression condition of the Art v 1 protein, and simultaneously, displaying the development result while detecting the expression of the Art v 1 protein in a second lane and a third lane, wherein the experimental result is shown in a figure 3, and meanwhile, displaying fewer impurity bands in the third lane, so that the purified GST-Art v 1 has the expression of 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. ELISA results are shown in FIG. 4, OD in the negative control protein BSA group and purified GST protein group (NC group in the figure) ₄₅₀ The values were low, demonstrating that BSA and GST proteins did not bind to the Art v 1 antibody, whereas the OD was found in the GST-Art v 1 protein group (Art v 1 group in the figure) ₄₅₀ Higher values are shown, demonstrating better binding activity of purified GST-Art v 1 to anti-Art v 1 antibodies.

Comparative example 1 comparison of expression and activity of the different vector Art v 1 proteins:

cloning a gene (SEQ ID NO. 1) encoding the Art v 1 protein which is not subjected to sequence optimization into a vector Pet-28a; the gene (SEQ ID NO. 1) encoding the Artv 1 protein, which is not sequence optimized, was cloned into the vector pGEX-4T-1, and recombinant vectors pET-28a-Art v 1 and pGEX-4T-1-Art v 1 were constructed. LB liquid culture medium is selected, the induction expression condition is that 1.0mM IPTG is induced for 4 hours at 37 ℃, as shown in figure 5, pET-28a-Art v 1 is purified and then mainly expressed in the form of inclusion body, the expressed protein needs to be denatured and renatured, the experimental process is complex, the cost is improved, and meanwhile, the binding activity of the renatured protein is not as good as that of the protein which is expressed in a soluble way. The protein after pGEX-4T-1-Art v 1 purification is mainly expressed in a soluble form, the protein is directly purified in the ultrasonic disruption supernatant (as shown in figure 6),200mlOD ₆₀₀ the E.coli of 0.6 is used for protein purification, and the protein expression quantity is 100-150 mug.

Meanwhile, because pET-28a-Art v 1 is mainly expressed in an inclusion form after purification, denaturation and renaturation are needed, and the binding activity of the protein and Art v 1 antibody is reduced compared with that of the protein purified by pGEX-4T-1-Art v 1 (shown in figure 7).

Comparison of expression of the sequence of comparative example 2 pGEX-4T-1-Art v 1 before and after optimization

The non-optimized target gene (shown as SEQ ID NO. 1) is cloned in a vector pGEX-4T-1. Cloning the optimized target protein gene sequence (shown as SEQ ID NO. 2) in a vector pGEX-4T-1. The carrier was synthesized by the company. SDS-PAGE analysis of proteins before and after nickel column purification after induction of expression was performed, again using 200ml OD ₆₀₀ Coli having a protein expression level of 100 μg to 150 μg before optimization and a protein expression level of about 500 μg after sequence optimization (as shown in fig. 8) was performed for protein purification.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> daily friendship Hospital in Jilin university

<120> Art v 1 recombinant protein and uses thereof

<130> MP22014592

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 396

<212> DNA

<213> Artemisia (artemia)

<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

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

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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. Nucleic acid encoding Art v 1, the nucleic acid sequence of which is shown in SEQ ID NO. 2.

2. The recombinant protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 3.

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

4. Recombinant vector comprising a vector backbone and a nucleic acid fragment, said backbone vector being pGEX-4T-1, said nucleic acid fragment comprising the nucleic acid of claim 1 and/or the nucleic acid of claim 3.

5. An expression system comprising a host E.coli and the recombinant vector of claim 4.

6. A method for preparing the expression system according to claim 5, which comprises transforming or transfecting the recombinant vector according to claim 4 into a host E.coli.

7. The method for producing a recombinant protein according to claim 2, wherein the expression system according to claim 5 is fermented to obtain a product containing the Art v 1 protein.

8. Use of the recombinant protein of claim 2 for the preparation of a diagnostic reagent for allergic diseases caused by artemisia annua.

9. A diagnostic reagent for allergic diseases comprising the recombinant protein according to claim 2 or the recombinant protein produced by the production method according to claim 7.