CN106434745A

CN106434745A - Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos

Info

Publication number: CN106434745A
Application number: CN201610831281.4A
Authority: CN
Inventors: 马生武; 安托尼·迈克尔·耶尼卡; 邓绍平; 杨洪吉; 魏亮
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-09-19
Filing date: 2016-09-19
Publication date: 2017-02-22

Abstract

The invention discloses a method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos. The method comprises the following steps of: inserting coding sequences of TEV protease and enzyme cutting sites thereof between coding sequences of signal peptide and mature IL-37 proteins to form an IL-37-TEV fusion gene; according to the preference of tobacco codons, under the condition of guaranteeing no change of the amino acid sequence, optimizing the sequence of the fusion gene; connecting 5'-end upstream of the constructed IL-37-TEV fusion gene with a common promoter including other regulating areas, and connecting the 3'-end downstream with a transcription terminator; integrating a constructed recombinant expression vector onto cell chromosomes of the tobacco to culture a transgenic tobacco plant with high-efficiency stable expression of IL-37 and injecting the constructed recombinant expression vector to the tobacco leaves to instantaneously express target proteins. The method disclosed by the invention has the advantages that by construction of the plant expression vector, the mature proteins of IL-37 with high activity are expressed in the plants, and the obtained product has important application value for treatment of numerous inflammatory diseases and early detection and diagnosis of the inflammatory diseases.

Description

A kind of method of each hypotype maturation protein of utilization Nicotiana tabacum L. high efficient expression IL-37

Technical field

The present invention relates to genetic engineering and field of protein expression, and in particular to a kind of using Nicotiana tabacum L. high efficient expression IL-37 The method of each hypotype maturation protein.

Background technology

Human interleukin -37 (IL-37) is the new member for adding in cytokine IL-1 race, finds in 2000.With Other members in IL-1 race have extensive anti-inflammatory efficacy conversely, IL-37 belongs to anti-inflammatory cytokine, intrinsic scorching limiting Disease reacts (innate inflammation）With suppression acquired immunity（acquired immunity）Two aspects of reaction are all Play the effect of key.Experiment in vitro research finds, suppresses the normal expression of IL-37 in people's cell increase by IL-1, Toll Sample receptor（Toll-link receptor, TRL）The expression of the inflammatory cytokine that sends out with uric acid crystal and release, including TNF-α, IL-1 α, IL-1, IL-6 and G-CSF.Although up to the present it has not been found that corresponding with people with mice IL-37 gene, but people IL-37 is active in mice body.With the expression IL-37 transgenic mice (IL37- for building tg）Its impact to different inflammation diseases is studied, is as a result found, IL-37-tg mice is shown to inflammation relevant disease Resistance, such as endotoxemia, colitis, spinal cord injury, metabolic syndrome, myocardial ischemia etc..Additionally, injection restructuring IL- 37 protein similarly can be protected by endotoxemia to the normal mouse of non-IL-37 transgenic, and metabolic syndrome such as obesity is drawn The II patients with type Ⅰ DM for rising, acute pulmonary damage, spinal cord injury, myocardial infarction, the disease such as asthma and hepatic ischemia/reperfusion injury is made to animal The damage for becoming.Further, the order of severity and IL-37 of a lot of human inflammation's diseases expression in vivo is associated.For example, scorching There is IL-37 to express in the colon of disease property intestinal diseases patient, but do not exist in the respective organization of healthy individuals.Rheumatoid is closed The scorching IL-37 level in the patient of section is raised, and mutation, their joint of the lower extremity disease occurs in the IL-37 gene coding region of some patients The patient that sick score is no mutated relatively is low, implies this function of being mutated and enhance IL-37 anti-inflammatory response.Detection infection The peripheral blood of HIV-1 patient finds, the steady platform state of its IL-37（steady state）The relative person of being uninfected by of mRNA concentration will height 4 times.These result prompting inflammation can increase IL-37 expression in body, and the increase of IL-37 expression contributes to disease symptomses Control.It is generally believed that the molecular mechanism of IL-37 suppression inflammatory reaction is by striving unexpectedly and IL-18 receptor with IL-18（IL-18 receptor）Combination.After IL-37 is attached on IL-18R α, complex can capture orphan and catch receptor（orphan decoy）IL-1R8 is in combination, and the TLRb land of subsequent IL1R8 attracts MyD88 (myeloid Differentiation factor 88) in combination. the signal path in this mutual effect energy active cell is used In the generation of blocking inflammation, including the suppression to MAPKS and NF-KB, the suppression of mTOR signal path and to anti-inflammatory pathway The activation of STAT3, Mer and PTEN.Meanwhile, because MyD88 molecule is stranded, it participates in the journey of IL-1 and IL-18 signal path Degree is also corresponding to be reduced.IL-37 is to the suppression of antigen Specific acquired immunity mainly by the product of tolerance dendritic cell Raw.Therefore, IL-37 is not only possible provides a kind of new effective ways for treating many inflammation diseases, but also is possibly used for The early detection of inflammatory diseasess and diagnosis.Only escherichia coli expression production a small amount of recombined human IL-37 is used as reagent at present, far not Can meet as new detection, the demand of diagnosis and treatment disease means, can in the urgent need to developing another expression system Cheap bulk produces highly active restructuring IL-37 protein.

Plant is produced with the commercially valuable foreign protein of medical usage or other as a kind of bioreactor Show poly- big advantage and power is striven unexpectedly, with wide market prospect.Plant has much excellent as recombiant protein production system Point：(1) inexpensive, easy large-scale commercial production.Plant can carry out photosynthesis, it is only necessary to from soil mineral and Moisture just can obtain the transgene product needed for a large amount of people under appropriate conditions.Expand the scale of production easily, it is only necessary to very A small amount of puts into again.This forms obvious control with microorganism and animal cell expression system.Animal cell culture needs to use Expensive culture medium.Expanding the scale of production needs investment new in a large number including buying expensive new equipment（Effect tank etc. is such as sent out）；(2) Due to animal and Human virus cannot transboundary infection plant's cell, the pharmaceutical protein of plant Expression product infected by human or animal's virus Chance very little, therefore, the pharmaceutical protein ratio of zooblast production, the pharmaceutical protein Clinical practice of plant production more pacifies Entirely；(3) as higher eucaryotic cells biology, plant cell can be turned over to the protein of new synthesis as zooblast Processing modification, such as glycosylation after translating（glycosylation）, phosphorylation (phosphorylation) and subunit assembling （subunit assembly）, therefore expression product is with the immunogenicity as higher animal cells and biological activity.Carefully When bacterium is as bioreactor, it is impossible to effective post translational processing is carried out to Eukaryotic albumen, be suitable only for expression structure letter The protein of single both the non-glycated, and itself be probably human tropic pathogens.Nicotiana tabacum L. (Nicotiana tabacum) is flat as expression Platform has the advantages that other plant production system is incomparable.First, from first time in 1989 with tobacco expressed antibody [3] with Come, Nicotiana tabacum L. has become as the main platform of production recombiant protein；Secondly, Nicotiana tabacum L. is leaf vegetables yield height, and blade solubility Protein content height, can improve expression of the foreign protein in plant as recombiant protein production platform；Again, the heredity of Nicotiana tabacum L. Transformation technology is highly developed, and expression of the recombiant protein in Nicotiana tabacum L. may be selected different modes, such as cell nuclear expression (transgenic Plant), chloroplast expression and transient expression；Finally, Nicotiana tabacum L. is neither human foods, nor forage crop, therefore reduces Transgenic line contaminated food chain or the risk of feedstuff chain.

IL-37 gene is located on No. second chromosome of the mankind, and its gene expression product has five splicing isoform (isoforms a to e) .Current research is concentrated mainly on IL-37b, and the function for other hypotypes is not also known.But because of IL- 37a is similar to IL-37b with the structure of IL-37d, i.e., their coding region all includes the 4th exon, can encode β-SANYE Grass structure.This structure is considered as the characteristic structural of IL-1 family cytokine. so speculating as IL-37b, IL- 37a and IL-37d have biological function.And IL-37c and IL-37e lack the 4th exon, it is impossible to encode β-SANYE grass knot Structure, thus it is speculated that they may be without biological function or different with the function of IL-37b.IL-37 is expressed in Various Tissues, Including blood mononuclear cell, poly- phagocyte, synovial cell, tonsil B lymphocyte, plasma cell, tumor cell and skin is organized, Kidney and the epithelial cell of intestinal.The distribution of IL-37 splicing isoform also has certain tissue specificity.For example, IL-3a is main Express in the brain, IL-37b is primarily present in kidney, heartspecific expresses IL-37c, and IL-37d and IL-37e are then main Express in bone marrow and testis.Five hypotypes of IL-37 are all first synthetic proteinses precursors, produce into soft-boiled eggs after cutting off signal peptide In vain.But their signal peptide all non-classical sequences.The cutting of signal peptide is mainly by caspase-1（caspase-1）Complete. But newest research thinks that the cutting completely of IL-37 signal peptide needs other enzyme to participate.Five hypotypes in IL-37 In, IL-37b protein chain is most long, is made up of 218 aminoacid, the N-terminal signal peptide containing a 45 aminoacid composition.Although IL- 37b precursor and IL-37b maturation protein form have been found that active, but the activity of IL-37b maturation protein is than IL-37b precursor egg White activity is higher by 20-30 times.IL-37 maturation protein can also enter nucleus and be combined so as to affect genetic transcription with core DNA, And its precursor protein then can not.People IL-37 is natural with dimeric forms（dimer）Exist, but dimeric formed be not with altogether Valence link (two sulfur) mode connects.Up till now for this purpose, having not found the gene of coding caspase-1 enzyme in plant cell, Although certain plants protein sequence shows the similarity certain with animal caspases enzyme.This means when thin with plant During cellular expression IL-37 gene, the expression product of acquisition is IL-37 precursor rather than maturation protein, because of plant cell None- identified position Sheared in the N-terminal signal peptide sequence of IL-37 precursor.

At present, a kind of possible method is only the gene of encoding mature IL-37 to be expressed in plant cell, but the method It is exactly low expression amount and the low activity of destination protein with obvious shortcoming, because the albumen of new synthesis is due to lacking signal peptide Mediate transport easily causes false folding to be not only allowed to lose activity but also easily by the proteasome degradation in plant cell cytoplasm.

Alternatively possible method is that the original signal peptide sequence for replacing IL-37 gene with plant protein signal peptides makes new synthesis Destination protein molecule can realize transmembrane transport and in the process by cut away the plant protein signal peptides of grafting produce ripe The destination protein with natural N end. but this needs the signal peptide for testing many different vegetable proteins to be possible to find conjunction Suitable signal peptide, because unlike signal peptide plays the role of different to have a significant impact protein expression amount.Even if additionally, using plant Protein signal peptide is replaced, and former discovery shows the exogenous gene of thus method expression, has suitable one in protein end-product Point it is precursor protein that plant signal peptide is not removed.

Content of the invention

It is an object of the invention to overcome the shortcoming of prior art, provide a kind of using using TEV protease in vivo and External high specific and highly active double characteristic, by designing and synthesizing the IL-37 being connected in framework with TEV protease The method of each hypotype maturation protein of fusion gene Nicotiana tabacum L. high efficient expression IL-37.

The purpose of the present invention is achieved through the following technical solutions：One kind is become using each hypotype of Nicotiana tabacum L. high efficient expression IL-37 The white method of soft-boiled eggs, comprises the following steps：

S1. IL-37-TEV fusion gene is designed：

Compile including insertion TEV protease between signal peptide and each hypotype coded sequence of IL-37 maturation protein and its restriction enzyme site Code sequence is obtained between fusion gene, the natural signals peptide in each hypotype of IL-37 maturation protein and TEV protease sequence adds one section Flexible peptide linker, the IL-37-TEV fusion gene basic structure order be：Itself primary signal peptide-flexible peptide linker- The each hypotype IL-37 coded sequence of TEV protease-TEV enzyme recognition site-maturation protein；

The each hypotype of the IL-37 maturation protein is IL-37a, IL-37b, IL-37c, IL-37d and IL-37e, wherein IL- The nucleotide sequence of 37a-TEV fusion gene is as shown in SEQ ID NO.1, and the nucleotide sequence of IL-37b-TEV fusion gene is such as Shown in SEQ ID NO.2, the nucleotide sequence of IL-37c-TEV fusion gene is as shown in SEQ ID NO.3, and IL-37d-TEV melts The nucleotide sequence of gene is closed as shown in SEQ ID NO.4, the nucleotide sequence such as SEQ ID of IL-37e-TEV fusion gene Shown in NO.5；The aminoacid sequence of its IL-37a-TEV fusion gene coding is as shown in SEQ ID NO.6, and IL-37b-TEV merges The aminoacid sequence of gene code as shown in SEQ ID NO.7, IL-37c-TEV fusion gene coding aminoacid sequence such as SEQ Shown in ID NO.8, the aminoacid sequence of IL-37d-TEV fusion gene coding is as shown in SEQ ID NO.9, and IL-37e-TEV melts The aminoacid sequence of gene code is closed as shown in SEQ ID NO.10；The flexible peptide linker sequence such as SEQ ID NO.11 institute Show；

S2. recombinant expression carrier is built：

The IL-37-TEV fusion gene of step S1 design is connected to transition vector PRTL-2-Gus, after separating through enzyme action again gram The grand T-DNA to plant expression binary empty carrier, obtains the recombinant expression carrier of each hypotype of IL-37 maturation protein；

S3. express：

The recombinant expression carrier that step S2 is built is imported in Nicotiana tabacum L. is expressed.

Further, each hypotype of the IL-37 maturation protein is IL-37a, IL-37b, IL-37c, IL-37d and IL- 37e.

Further, the recombinant expression carrier is by plant composing type E35S promoter and the TEV with 5 ' end untranslateds The expression that the gene that targeting sequencing drives is carried out, using the NPTII gene of Kanamycin resistance as selection markers.

Further, the recombinant expression carrier for step S2 being built imports to the transgenic for obtaining IL-37 in tobacco cell Tobacco plant carries out each hypotype maturation protein of stable expression IL-37.

Further, the recombinant expression carrier for step S2 being built is expelled to each hypotype of transient expression IL-37 in Nicotiana tabacum L. and becomes Soft-boiled eggs are white.

Recombinant expression carrier plasmid is proceeded to by the agriculture bacillus mediated blade genetic transforming method in the present invention by commonly using In tobacco cell, induce through healing cell, differentiation and regeneration and kanamycin resistance screening obtain stably express IL-37 turn base Because of tobacco plant.

By recombinant expression carrier plasmid with containing can suppressor gene silence tomato bushy stunt virus (tomato bushy Stunt virus, TBSV) vector plasmid of gene P19 is co-injected in tobacco leaf with Agrobacterium blade injector method and realizes IL- Transient expression of 37 maturation proteins in Nicotiana tabacum L..

The present invention has advantages below：

When IL-37 is expressed by the use of plant as biological device, as plant cell does not contain caspase-1, it is unable to Recognize and shear the signal peptide sequence of IL-37 precursor and produce ripe destination protein.The invention discloses a kind of using Nicotiana tabacum L. height The method of each hypotype maturation protein of effect expression IL-37, first with the etch virus poison (TEV of Nicotiana tabacum L.）Protease is in vivo and in vitro High specific and highly active dual characteristicses, design and insert TEV egg between signal peptide and IL-37 mature protein coding sequence White enzyme and its recognition site coded sequence are allowed to form fusion gene.Thus, express the single chain polypeptide for producing to melt by plant cell Hop protein carries out IL-37 of self specificity cutting so as to produce with natural structure by the activity of TEV enzyme in the cell and becomes Soft-boiled eggs are white.In addition add one section of flexible connection peptide sequence between signal peptide and TEV enzyme to ensure the TEV as fusion unit The relatively independent folding of enzyme energy is so as to farthest keep the active function of its enzyme.Thereafter, construct containing synthesis, codon The expression vector of the fusion gene of optimization, with table of the plant composing type E35S promoters driven fusion gene in plant cell Reach；Using the mediation of pus bacillus blade method for transformation by vector introduction leaf cell, through healing cell induction, disintegrating and regeneration and card sodium Chloramphenicol resistance screening obtains transgenic tobacco plant.By recombinant expression carrier plasmid with containing can suppressor gene silence Fructus Lycopersici esculenti clump The vector plasmid of dwarf virus gene P19 is co-injected in tobacco leaf with Agrobacterium blade injector method and realizes IL-37 maturation protein Transient expression in Nicotiana tabacum L..As T-DNA inserts the randomness of plant cell chromosome DNA, and its thus to exogenesis albumen The impact that matter expression brings, the screening of the separate transgenic plant for therefore obtaining after each recombinant expression carrier transformation of tobacco is many In 25 plants.Using methods such as Western blot, Inspection and analysis are carried out to the transgenic tobacco plant for obtaining.As a result show, institute The expression vector of structure correctly expresses IL-37 maturation protein, and exists with dimeric forms, this and natural people IL-37 egg White form is completely the same.Finally, method of the IL-37 of Hisx6 mark mirror by His protein purification test kit is contained to C- end Separate from transgenic tobacco plant leaf.Hisx6 mark mirror IL-37 maturation protein and the mice renal epithelial cell culture of purification.Knot Fruit shows the secretion of inflammation that the IL-37 maturation protein of plant expression effectively can suppress to be induced by antibacterial lipopolysaccharide, such as TNF-a.Therefore the IL-37 of plant expression has significantly activity, be further with plant bioreactor large-scale production Restructuring IL-37 lays a good foundation.

For at the same implement transient expression of the IL-37 maturation protein in Nicotiana tabacum L., respectively by recombinant expression carrier plasmid with contain Have can suppressor gene silence tomato bushy stunt virus gene P19 expression vector mix by Agrobacterium blade injector method co-injection To in Ben's tobacco leaf, harvesting blade in rear 1-6d is infected.Using methods such as Western blot to the tobacco leaf that infects Carry out Inspection and analysis.As a result show, constructed expression vector equally can become soft-boiled eggs by transient expression IL-37 in tobacco leaf In vain, products obtained therefrom is to treating the early detection of many inflammation diseases and inflammation disease and diagnosing with significant application value.

Description of the drawings

Fig. 1 is IL-37-TEV fusion gene structural representation；

Fig. 2 builds flow chart for plant expression carrier plasmid；

Fig. 3 is that pBI-SP-TEV-CS-IL-37b transgene tobacco total soluble protein immunoblotting detection IL-37b becomes The white expression of soft-boiled eggs, in figure, A be using anti-IL-37 antibody；B be using anti-TEV antibody；

Fig. 4 is that pBI-SP-TEV-CS-IL-37bHis6 transgene tobacco total soluble protein immunoblotting detects IL- The expression of 37bHis6 maturation protein, in figure, A be using anti-IL-37 antibody；B be using 37 antibody of anti-TEV；

Fig. 5 is that the Nicotiana tabacum L. for injecting pBI-SP-TEV-CS-IL-37b expression vector plasmid+P19 expresses IL-37b in different time The Western blot detection of albumen, using anti-IL-37 antibody；

The purification IL-37b albumen that Fig. 6 is obtained through Ni post affinity chromatography for Western blot detection, in figure, A is use Anti-IL-37 antibody；B be using anti-TEV antibody；

Fig. 7 is the activity analysiss that tobacco expressed IL-37 maturation protein discharges inflammatory factor to suppression cell, wherein, is used Cell be mouse kidney epidermis cell；LPS containing 1ug/ml in Med phalangeal cell culture medium but without IL-37 albumen make Control is used, and IL-37 (STAND) is standard protein.

Specific embodiment

Below in conjunction with the accompanying drawings and embodiment the present invention will be further described, protection scope of the present invention be not limited to Lower described.

The invention discloses a kind of method of each hypotype maturation protein of utilization Nicotiana tabacum L. high efficient expression IL-37, is lost using Nicotiana tabacum L. The high specific of stricture of vagina virus protease (tobacco etch virus protease, TEV protease) and highly active Dual characteristicses, insertion TEV protease and its restriction enzyme site coded sequence between signal peptide and maturation IL-37 albumen coded sequence Form IL-37-TEV fusion gene；According to the Preference of Nicotiana tabacum L. codon, in the case of ensureing that aminoacid sequence is constant, right Fusion gene sequence is optimized.And entrust the Bio Basic company of Toronto to synthesize；The IL-37-TEV that will build 5' end upstream and the conventional promoter of fusion gene includes that other adjustment regions (as enhancer) are connected, and in 3' end downstream It is connected with transcription terminator；The recombinant expression carrier of structure is incorporated on tobacco cell chromosome by conversion, is cultivated efficient The transgenic tobacco plant of IL-37 is stably expressed and by injecting the recombinant expression carrier for building to tobacco leaf transient expression Destination protein.Tobacco etch virus protease (TEV protease) is the abbreviation of 27 kDa catalytic domains of Nla protease. TEV protease is height site specific cysteines protease, and the enzyme can recognize that 7 amino acid recognition site (L-Glutamic Acid-day Winter amide-Leucine-L-Tyrosine-Phe-Gln-Glycine) and cut off the peptide between glutamine and Glycine Key.Due to its high activity, the dual characteristicses of high specific, the potent agent that TEV protease is cracked for fused protein.Its upstream Pyrolysis product containing the aminoacid for increasing by 6 more, and downstream cleavage product comprises only the Glycine for increasing by 1 more.Shin et al (Protein Sci. 2005;14,936 941) fusion protein containing TEV protease of bacterial expression is reported by which Enzyme activity performance is implemented self cutting of specificity in the cell and discharges the active fusion companion with natural structure (native) (fusion partner) .Marcos and Beachy (J Gen Virol. 1997;78,1771 1778) report outer Source TEV protease has biological activity in plant cell.These initially really show to be connected with TEV protease by building Fusion gene can reach restructuring destination protein of the expression with natural structure.

Embodiment 1：Design and synthesis Sp-TEV-CS-IL-37b, Sp-TEV-CS-37a, Sp-TEV-CS-IL-37d, Sp- TEV-CS-IL-37c and Sp-TEV-CS-IL-37e fusion gene

The cDNA sequence of each protein subunit of IL-37 is obtained first in the GenBank of NCBI website, is encoded to Gen Bank accession NO.NM_014439.3 for IL37b；Gen Bank accession NO.NM_173205.1 for IL- 37a；Gen Bank accession NO.NM_173204.1 for IL-37c；Gen Bank accession NO.NM_ 173202.1 for IL-37d and Gen Bank accession NO.NM_173203.1 for IL-37e.Similarly, The nucleotide sequence of coding TEV protease is obtained from Gen Bank, is encoded to Gen Bank accession NO.NP_ 062908.1.

Design IL-37-TEV fusion gene.

As shown in figure 1, the basic structure order of fusion gene is：Itself primary signal peptide-flexible peptide linker (GGGGSx3)-TEV protease-TEV enzyme recognition site-maturation IL-37 coded sequence.

According to the Preference of Nicotiana tabacum L. codon to the TEV-IL-37 fusion gene for designing on the premise of aminoacid is not changed It is optimized, the fusion gene after optimization adds PcI1 restriction enzyme site at 5' end by synthetic during synthesis, increases at 3' end Xba I restriction enzyme site.The fusion gene of the present embodiment is synthesized and is cloned into plasmid by Toronto Bio Basics company PUC57.The nucleotide sequence of the fusion gene after synthesis such as SEQ ID NO:1st, shown in 2,3,4,5, the aminoacid of its coding Sequence such as SEQ ID NO:6th, shown in 7,8,9,10.

Embodiment 2：Recombinant expression carrier pBI-Sp-TEV-CS-IL-37b, pBI-Sp-TEV-CS-IL-37a, pBI- The structure of Sp-TEV-CS-IL-37d, pBI-Sp-TEV-CS-IL-37c and pBI-Sp-TEV-CS-IL-37e

(1) structure of recombinant expression carrier pBI-Sp-TEV-CS-IL-37b:Construction method is as shown in Fig. 2 including following Key step：

Using Nco I+ Xba I double digestion transition vector pRTL2-GUS, the carrier-pellet of 4.2 Kb for removing gus gene is reclaimed Section.Plasmid pRTL2-GUS contains E35S promoter (enhanced 35S promoter）The TEV of -5' end untranslated is leading The gus gene expression cassette of sequence-GUS- Nos3 ' terminator.

Using Pci l and pUC57-Sp-TEV-CS- IL-37b plasmid described in Xba I enzyme action, fusion gene piece is reclaimed Section is simultaneously inserted into the carrier segments that pRTL2-GUS reclaimed after Nco I+ Xba I enzyme action and obtains transition expression vector pRTL2- Sp-TEV-CS- IL-37b.

Using the transition expression vector pRTL2- Sp-TEV-CS- IL-37b described in Hind III enzyme action, reclaim to contain and melt And expression casette（E35S-Sp-TEV-CS-IL-37b-Nos3’）DNA fragmentation, and the double base being inserted into through Hind III digestion Carrier pBI101-1 obtains recombinant expression carrier pBI- Sp-TEV-CS-IL-37b.

The expression vector pBI- Sp-TEV-CS-IL- that will be built by triparental mating (tri-parental mating) 37b proceeds to Agrobacterium.

(2) structure of recombinant expression carrier pBI-Sp-TEV-CS-IL-37a:Its construction method and carrier pBI- The structure of Sp-TEV-CS-IL-37b is identical.

(3) structure of recombinant expression carrier pBI-Sp-TEV-CS-IL-37d:Its construction method and carrier pBI- The structure of Sp-TEV-CS-IL-37b is identical.

(4) structure of recombinant expression carrier pBI-Sp-TEV-CS-IL-37c:Its construction method and carrier pBI- The structure of Sp-TEV-CS-IL-37b is identical.

(5) structure of recombinant expression carrier pBI-Sp-TEV-CS-IL-37e:Its construction method and carrier pBI- The structure of Sp-TEV-CS-IL-37b is identical.

Embodiment 3：The stable conversion of Nicotiana tabacum L.

The recombinant expression carrier plasmid for building is proceeded to Agrobacterium respectively by triparental mating (tri-parental mating) LBA4404.

Nicotiana tabacum L. is cultivated.Tobacco seed（N. tabacum CV.81V9）After 70% alcohol disinfecting, one is planted in containing MS The plant tissue culture box of+8g/L agar culture medium（magenta vessel）In, incubator culture.25 ± 1 °C of temperature, wet Degree 70% or so, cultivates under illumination/8 hour dark cycle illumination in 16 hours.After 3-4 week, when seedling grows to 3-4 piece true leaf, you can Take germ-free plant leaf and be used as genetic transformation material.

Recombinant expression carrier genetic transformation Nicotiana tabacum L..Retouch with reference to (Science 1985. 227,1229-1231) such as Horsch The blade method for transformation that states is introduced into the plasmid in the present invention.By taking recombinant vector PBI-Sp-TEV-CS-IL-37 b as an example, will contain The Agrobacterium LBA4404 single bacterium colony of the plasmid be placed in 5mL liquid TYC (peptone 8g/L, yeast 4g/L, calcium chloride 6.6%)+ In 50mg/L Km+20mg/L Rif culture medium, 28 DEG C, 180rpm, shaken cultivation.Take 1 mL bacterium solution and be placed in 50 mL liquid In+50 mg/L Km+20mg/L Rif culture medium of body fluid body TYC, 28 DEG C, 180rpm, cultivate to OD600=0.5 ~ 0.6, 6000rpm, centrifugation 10min, collects thalline, the resuspended thalline of 40mL MS culture fluid, Nicotiana tabacum L. tests for sterility removes main lobe normal pulse leaf Piece edge is simultaneously cut into about 1.0cm small pieces, contaminates 5min in bacterium solution, and aseptic filter paper blots bacterium solution, is inoculated in MS+1.0mg/L In 6-BA+0.1mg/L NAA culture medium, after 3 d are co-cultured under dark condition, be forwarded to Selective agar medium (MS+ by 25 DEG C 6-BA (1 mg/L)+NAA (0.1 mg/L)+Km (100 mg/L)+Cb (500 mg/L)+sucrose (30g/L)+fine jade Fat (8g/L)) on carry out bud induction screening and culturing, cut when bud length is to about 2 cm, be inoculated in root media (MS+ Km (100 mg/L)+Cb (500 mg/L)+sucrose (30g/L)+agar (8g/L)) on carry out root induction culture.Take root Regrowth afterwards is migrated in flowerpot, is put into (25 ± 0.5) DEG C hot-house culture.

Embodiment 4：The instantaneous conversion of Nicotiana tabacum L.

Nicotiana tabacum L. is cultivated：Ben's tobacco seed (N. Benthamiana）, 4C ° of 48 h that are soaked in water, it is seeded in soil, 25 ± 1 DEG C, cultivate in the incubator of 16 h/d illumination.Can be used for testing after about 30 days when which is five leaves or six leaf phases.

PBI-Sp-TEV-CS-IL-37b, pBI-Sp-TEV-CS-IL-37a, pBI-Sp-TEV-CS- will be contained respectively Single bacterium of the LBA4404 Agrobacterium of IL-37d, pBI-Sp-TEV-CS-IL-37c and pBI-Sp-TEV-CS-IL-37e plasmid Fall to being inoculated into+100 mg/L Rif of+50 mg/L Km of TYC fluid medium, at 28 DEG C, shake 24 to 48h.Draw 100ul bacterium solution is added in 10ml TYC liquid, is further cultured for 24 h.Collects thalline, at 4 DEG C, 5000 × g is centrifuged 10 min, goes Except supernatant, thalline is stayed.Thalline is suspended in suspension (5 mg/ml d- glucoses, 500 nM MES, 20 nM Na₃PO_4• 12 H₂O, 1M acetosyringone (acetosyringone)), 6000rpm, centrifugation 10min, collects thalline, suspension is resuspended Thalline, determines OD600 value.By the suspension root containing the P19 expression of destination protein (effect be to increase) and genes of interest 1 is pressed according to titre OD=0.5 of determined Agrobacterium:1 ratio is mixed into and infects liquid.

The liquid that infects for being suspended with Agrobacterium is injected in blade by the disposable syringe for spending syringe needle from the Nicotiana tabacum L. back side.Injection After finishing, it is placed in 25 ± 1 DEG C of artificial culture rooms and cultivates.Inject after 24 h and collect injection areas tobacco leaf, once a day until 6th day.

Embodiment 5：The detection of transgene tobacco IL-37 protein expression

By taking recombinant expression carrier pBI-Sp-TEV-CS-IL-37b transgene tobacco as an example.Take fresh transgenic tobacco leaf 0.5 G, is immediately placed at liquid feeding nitrogen in mortar and grinds, and ground blade is collected in 2mL EP pipe and adds 1 mL protein extraction Liquid（200mM Tris pH8.0,100 mM NaCL, 400 mM sucrose, 10 mM EDTA, 0.1mM-coloured glaze base ethanol, 0.05% Tween-20,1 mM Phenylmethanesulfonyl fluoride（PMSF）, 0.1% aprotitin and 0.1% leupeptin）, on ice incubation 20 ~ 30min, 4 DEG C of 12000 rpm/min is centrifuged 20min.Supernatant is collected, supernatant is soluble protein extracting solution.

SDS-PAGE and Western immunoblotting (Western blot) is analyzed.Protein extract is taken, is added Sample buffer, boils sample 10min in boiling water.After protein example is separated through SDS-PAGE, using Western immunoblotting Method (Western Blot) is then detected on Protein transfer to film using antibody.Western blot is detected As a result prove, the transfer-gen plant expression IL-37b maturation protein of acquisition, and with desired dimer（dimer）Form is present, As shown in figure 3, in figure, M is molecular weight of albumen marker；WT makees negative control for non-transgenic tobacco soluble protein； Lanes b1 to b18 is partial transgenic plant；Arrow indication is tobacco expressed IL-37b maturation protein form (dimerization Body)；The numeral in figure left side is molecular weight of albumen size criteria.IL-37 is non-glycoprotein, the IL-37b maturation without signal peptide Albumen is by 173 Amino acid profiles, and molecular weight is about the dimeric molecular weight of 19 kDa. IL-37b and merges egg with IL-37-TEV The molecular weight of white second polypeptide product (i.e. signal peptide+TEV fragment) for producing after intracellular self splicing is similar, occurs two Individual protein fragments are migrated altogether as a wide band, as shown in Figure 3A.Present in the middle of band not with IL-37 antibody response Blister band is SP-TEV polypeptide.

Embodiment 6：Transgene tobacco IL-37His₆The detection of protein expression

With recombinant expression carrier pBI-Sp-TEV-CS-IL-37bHis₆As a example by transgene tobacco.Protein extracting method such as embodiment 5 method is extracted.

SDS-PAGE and Western immunoblotting (Western blot) is analyzed.As a result with described in embodiment 5 Unanimously, as shown in figure 4, in figure, M is molecular weight of albumen marker;It is negative right that WT makees for non-transgenic tobacco soluble protein According to；1 to b ' 18 of lane b ' is partial transgenic plant；Arrow indication is the IL-37bHis of expression₆Maturation protein form (dimer)；The numeral in figure left side is molecular weight of albumen size criteria.

Embodiment 7：The detection of Nicotiana tabacum L. transient expression IL-37

By taking recombinant expression carrier PBI-Sp-TEV-CS-IL-37 b injection Nicotiana tabacum L. transient expression as an example.Using egg described in embodiment 5 White matter extracting method, extracts protein the injection areas Nicotiana tabacum L. that collects from different time.

Protein using SDS-PAGE the and Western blot method Detection and Extraction described in embodiment 5.Testing result Nicotiana tabacum L. transient expression IL-37 b maturation protein is proved, and same with dimer（dimer）Form is present, as shown in figure 5, figure In, 1 to 6 day after 1-6 pointed injection expression vector（DAI）The Nicotiana tabacum L. of collection.M is molecular weight of albumen marke；IL-37+ is IL- 37 protein standard substances；WT makees negative control for non-transgenic tobacco soluble protein；Arrow indication is the IL- of Nicotiana tabacum L. transient expression 37b maturation protein (dimer), the numeral for scheming left side is molecular weight of albumen size criteria.

Embodiment 8：The purification of tobacco expressed IL-37 and detection

With from transgene tobacco purification IL-37bHis₆As a example by.Protein purification test kit using His targeting（His-Trap Kit, purchased from Cedar lane Canada）Purification is carried out with according to operation instructions.

Using the albumen of the SDS-PAGE and Western blot method detection purification described in embodiment 5, as a result as Fig. 6 Shown, wherein, M is protein molecular weight marker；TSP is transgene tobacco total soluble protein (before upper prop)；FT is flow- through；W is wash；E1 is eluted fraction 1；E2 is eluted fraction 2；Fig. 6 A arrow institute Refer to as IL-37b maturation protein (dimeric forms), when being checked using anti-TEV antibody, E1 and E2 not with antibody response, such as Fig. 6 B, does not contain SP-TEV polypeptide in the IL-37b albumen for showing purification.The numeral in figure left side is the big small tenon of molecular weight of albumen Accurate.Fig. 6 result shows the IL-37bHis of purification₆In do not contain SP-TEV protein fragments.

Embodiment 9：Tobacco expressed IL-37 maturation protein discharges the activity analysiss of inflammatory factor to suppressing cell

The aseptic B6 mouse kidney that takes gently grinds, plus HANK'S（Balanced salt solution）Liquid is rinsed, 2000 rpm, 5 min, and centrifugation is abandoned Clearly, plus 1% collagenolysises enzyme V (collagens V, Invitragen）, 37C °, 30min dissolves nephridial tissue.Plus HANK'S liquid Prevent dissolving.Centrifugation, repopulating cell to nephrocyte culture fluid K1+ /+（K1+ /+complex culture medium 50：50 DMEM and Ham ' s F12；Containing 10% calf serum（FBS）, and hormone includes the insulin of 5Ig/ml（insulin）, 34pg/ml triiodo thyroid Propylhomoserin (triiodothyronine）, 5Ig/ml transferrinss (transferrin), 1.73ng/ml sodium selenite (sodium Selenite), 8ng/ml hydrocortisone (hydrocortisone), 25ng/ml egf (epidermal Growth factor), 100u/ml penicillin (penicillin), 100u/ml streptomycin (streptomycin）In. 5% CO₂, 37C ° of certain humidified incubator culture.After 5-7 days, renal epithelial cell covers with 80%.Suspension cell is in K1+ /+liquid. Dyeed with trypan blue (Trypan blue) with 0.4%, hemocytometer（hemocytometer）Count with 0.8x10⁶Cell is dense Degree plantation is to 96 well culture plates.Culture 24hr after add 100 ul K1-/- (50/50 Mix of DMEM/Ham ' s F12), 100u/ml penicillin, the IL-37 (prIL-37) of the plant expression of 100u/ml streptomycin and variable concentrations purification, per hole most Final concentration is respectively 0,20,10,5,2,5 0.625 nM). standard IL-37 albumen makees negative control. after culture 24hr hour, Abandon supernatant, plus 100ul K1-/- culture fluid (lipopolysaccharides of lipopolysaccharide containing 1ug/ml）.After culture 24hr Supernatant is collected, uses TNF-a test kit（R&D Canada）TNF-a concentration is determined, as shown in fig. 7, as a result showing that plant is expressed IL-37 there is high bioactivity.

SEQUENCE LISTING

<110>Ma Shengwu, Peter Antonie. Michael. Ye Nika, Deng Shaoping, Yang Hongji, Wei Liang

<120>A kind of method of each hypotype maturation protein of utilization Nicotiana tabacum L. high efficient expression IL-37

<130> 2016

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1371

<212> DNA

<213>Artificial sequence

<400> 1

atgtcaggtt gtgatagaag agagacagaa actaagggta aaaacagctt caagaagcga 60

ctaagaggac caaaggttgg tggaggtgga tctggtggag gtgggtcagg agggggggga 120

tctggtgaat ctcttttcaa gggtcctaga gattacaatc caatcagtag tacaatctgt 180

cacttaacca acgaaagcga tggtcataca acaagtctct acggtatcgg atttggtcca 240

ttcattatta ccaacaagca tctcttcaga agaaacaacg gaacactcct cgttcaatca 300

ctgcatggag ttttcaaggt aaagaacaca accaccttgc aacaacattt gatagacgga 360

agagatatga ttataattag gatgccaaag gacttcccgc cttttccaca gaagttgaag 420

tttcgagagc ctcaacgtga agaacgtatt tgccttgtaa ccactaattt tcagacaaag 480

tccatgtcca gcatggtttc agatacttcc tgcacttttc cttcctctga tggaattttc 540

tggaaacact ggattcagac aaaagacggg caatgcgggt ctcctttggt ttcaactagg 600

gatgggttca tagttggcat acattcagca tcaaatttta ctaatactaa taattatttt 660

acttctgtcc ccaaaaattt tatggagctt cttactaatc aggaggctca gcagtgggtc 720

tctggctgga ggttgaatgc tgactctgtt ttgtggggcg gccacaaagt gtttatgtca 780

aaaccagaag aaccatttca acccgtgaaa gaggccactc aattaatgaa tgagcttgtg 840

tattctcaag aaaatttgta ttttcaaggc aagaacttaa acccgaagaa attcagcatt 900

catgaccagg atcacaaagt actggtcctg gactctggga atctcatagc agttccagat 960

aaaaactaca tacgcccaga gatcttcttt gcattagcct catccttgag ctcagcctct 1020

gcggagaaag gaagtccgat tctcctgggg gtctctaaag gggagttttg tctctactgt 1080

gacaaggata aaggacaaag tcatccatcc cttcagctga agaaggagaa actgatgaag 1140

ctggctgccc aaaaggaatc agcacgccgg cccttcatct tttatagggc tcaggtgggc 1200

tcctggaaca tgctggagtc ggcggctcac cccggatggt tcatctgcac ctcctgcaat 1260

tgtaatgagc ctgttggggt gacagataaa tttgagaaca ggaaacacat tgaattttca 1320

tttcaaccag tttgcaaagc tgaaatgagc cccagtgagg tcagcgattg a 1371

<210> 2

<211> 1449

<212> DNA

<213>Artificial sequence

<400> 2

atgtcctttg tgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60

gaaccccagt gctgcttaga agacccggct gtaagccccc tggaaccagg cccaagcctc 120

cccaccatga attttggtgg aggtggatct ggtggaggtg ggtcaggagg ggggggatct 180

ggtgaatctc ttttcaaggg tcctagagat tacaatccaa tcagtagtac aatctgtcac 240

ttaaccaacg aaagcgatgg tcatacaaca agtctctacg gtatcggatt tggtccattc 300

attattacca acaagcatct cttcagaaga aacaacggaa cactcctcgt tcaatcactg 360

catggagttt tcaaggtaaa gaacacaacc accttgcaac aacatttgat agacggaaga 420

gatatgatta taattaggat gccaaaggac ttcccgcctt ttccacagaa gttgaagttt 480

cgagagcctc aacgtgaaga acgtatttgc cttgtaacca ctaattttca gacaaagtcc 540

atgtccagca tggtttcaga tacttcctgc acttttcctt cctctgatgg aattttctgg 600

aaacactgga ttcagacaaa agacgggcaa tgcgggtctc ctttggtttc aactagggat 660

gggttcatag ttggcataca ttcagcatca aattttacta atactaataa ttattttact 720

tctgtcccca aaaattttat ggagcttctt actaatcagg aggctcagca gtgggtctct 780

ggctggaggt tgaatgctga ctctgttttg tggggcggcc acaaagtgtt tatgtcaaaa 840

ccagaagaac catttcaacc cgtgaaagag gccactcaat taatgaatga gcttgtgtat 900

tctcaagaaa atttgtattt tcaaggcgtt cacacaagtc caaaggtgaa gaacttaaac 960

ccgaagaaat tcagcattca tgaccaggat cacaaagtac tggtcctgga ctctgggaat 1020

ctcatagcag ttccagataa aaactacata cgcccagaga tcttctttgc attagcctca 1080

tccttgagct cagcctctgc ggagaaagga agtccgattc tcctgggggt ctctaaaggg 1140

gagttttgtc tctactgtga caaggataaa ggacaaagtc atccatccct tcagctgaag 1200

aaggagaaac tgatgaagct ggctgcccaa aaggaatcag cacgccggcc cttcatcttt 1260

tatagggctc aggtgggctc ctggaacatg ctggagtcgg cggctcaccc cggatggttc 1320

atctgcacct cctgcaattg taatgagcct gttggggtga cagataaatt tgagaacagg 1380

aaacacattg aattttcatt tcaaccagtt tgcaaagctg aaatgagccc cagtgaggtc 1440

agcgattga 1449

<210> 3

<211> 1329

<212> DNA

<213>Artificial sequence

<400> 3

atgtcctttg tgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60

gaaccccagt gctgcttaga agacccggct gtaagccccc tggaaccagg cccaagcctc 120

cccaccatga attttggtgg aggtggatct ggtggaggtg ggtcaggagg ggggggatct 180

ggtgaatctc ttttcaaggg tcctagagat tacaatccaa tcagtagtac aatctgtcac 240

ttaaccaacg aaagcgatgg tcatacaaca agtctctacg gtatcggatt tggtccattc 300

attattacca acaagcatct cttcagaaga aacaacggaa cactcctcgt tcaatcactg 360

catggagttt tcaaggtaaa gaacacaacc accttgcaac aacatttgat agacggaaga 420

gatatgatta taattaggat gccaaaggac ttcccgcctt ttccacagaa gttgaagttt 480

cgagagcctc aacgtgaaga acgtatttgc cttgtaacca ctaattttca gacaaagtcc 540

atgtccagca tggtttcaga tacttcctgc acttttcctt cctctgatgg aattttctgg 600

aaacactgga ttcagacaaa agacgggcaa tgcgggtctc ctttggtttc aactagggat 660

gggttcatag ttggcataca ttcagcatca aattttacta atactaataa ttattttact 720

tctgtcccca aaaattttat ggagcttctt actaatcagg aggctcagca gtgggtctct 780

ggctggaggt tgaatgctga ctctgttttg tggggcggcc acaaagtgtt tatgtcaaaa 840

ccagaagaac catttcaacc cgtgaaagag gccactcaat taatgaatga gcttgtgtat 900

tctcaagaaa atttgtattt tcaaggcgtt cacacagaga tcttctttgc attagcctca 960

tccttgagct cagcctctgc ggagaaagga agtccgattc tcctgggggt ctctaaaggg 1020

gagttttgtc tctactgtga caaggataaa ggacaaagtc atccatccct tcagctgaag 1080

aaggagaaac tgatgaagct ggctgcccaa aaggaatcag cacgccggcc cttcatcttt 1140

tatagggctc aggtgggctc ctggaacatg ctggagtcgg cggctcaccc cggatggttc 1200

atctgcacct cctgcaattg taatgagcct gttggggtga cagataaatt tgagaacagg 1260

aaacacattg aattttcatt tcaaccagtt tgcaaagctg aaatgagccc cagtgaggtc 1320

agcgattga 1329

<210> 4

<211> 1386

<212> DNA

<213>Artificial sequence

<400> 4

atgtcctttg tgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60

ggtggaggtg gatctggtgg aggtgggtca ggaggggggg gatctggtga atctcttttc 120

aagggtccta gagattacaa tccaatcagt agtacaatct gtcacttaac caacgaaagc 180

gatggtcata caacaagtct ctacggtatc ggatttggtc cattcattat taccaacaag 240

catctcttca gaagaaacaa cggaacactc ctcgttcaat cactgcatgg agttttcaag 300

gtaaagaaca caaccacctt gcaacaacat ttgatagacg gaagagatat gattataatt 360

aggatgccaa aggacttccc gccttttcca cagaagttga agtttcgaga gcctcaacgt 420

gaagaacgta tttgccttgt aaccactaat tttcagacaa agtccatgtc cagcatggtt 480

tcagatactt cctgcacttt tccttcctct gatggaattt tctggaaaca ctggattcag 540

acaaaagacg ggcaatgcgg gtctcctttg gtttcaacta gggatgggtt catagttggc 600

atacattcag catcaaattt tactaatact aataattatt ttacttctgt ccccaaaaat 660

tttatggagc ttcttactaa tcaggaggct cagcagtggg tctctggctg gaggttgaat 720

gctgactctg ttttgtgggg cggccacaaa gtgtttatgt caaaaccaga agaaccattt 780

caacccgtga aagaggccac tcaattaatg aatgagcttg tgtattctca agaaaatttg 840

tattttcaag gcgaacccca gtgctgctta gaaggtccaa aggtgaagaa cttaaacccg 900

aagaaattca gcattcatga ccaggatcac aaagtactgg tcctggactc tgggaatctc 960

atagcagttc cagataaaaa ctacatacgc ccagagatct tctttgcatt agcctcatcc 1020

ttgagctcag cctctgcgga gaaaggaagt ccgattctcc tgggggtctc taaaggggag 1080

ttttgtctct actgtgacaa ggataaagga caaagtcatc catcccttca gctgaagaag 1140

gagaaactga tgaagctggc tgcccaaaag gaatcagcac gccggccctt catcttttat 1200

agggctcagg tgggctcctg gaacatgctg gagtcggcgg ctcaccccgg atggttcatc 1260

tgcacctcct gcaattgtaa tgagcctgtt ggggtgacag ataaatttga gaacaggaaa 1320

cacattgaat tttcatttca accagtttgc aaagctgaaa tgagccccag tgaggtcagc 1380

gattga 1386

<210> 5

<211> 1263

<212> DNA

<213>Artificial sequence

<400> 5

atgtcctttg tgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60

ggtggaggtg gatctggtgg aggtgggtca ggaggggggg gatctggtga atctcttttc 120

aagggtccta gagattacaa tccaatcagt agtacaatct gtcacttaac caacgaaagc 180

gatggtcata caacaagtct ctacggtatc ggatttggtc cattcattat taccaacaag 240

catctcttca gaagaaacaa cggaacactc ctcgttcaat cactgcatgg agttttcaag 300

gtaaagaaca caaccacctt gcaacaacat ttgatagacg gaagagatat gattataatt 360

aggatgccaa aggacttccc gccttttcca cagaagttga agtttcgaga gcctcaacgt 420

gaagaacgta tttgccttgt aaccactaat tttcagacaa agtccatgtc cagcatggtt 480

tcagatactt cctgcacttt tccttcctct gatggaattt tctggaaaca ctggattcag 540

acaaaagacg ggcaatgcgg gtctcctttg gtttcaacta gggatgggtt catagttggc 600

atacattcag catcaaattt tactaatact aataattatt ttacttctgt ccccaaaaat 660

tttatggagc ttcttactaa tcaggaggct cagcagtggg tctctggctg gaggttgaat 720

gctgactctg ttttgtgggg cggccacaaa gtgtttatgt caaaaccaga agaaccattt 780

caacccgtga aagaggccac tcaattaatg aatgagcttg tgtattctca agaaaatttg 840

tattttcaag gcgaacccca gtgctgctta gagatcttct ttgcattagc ctcatccttg 900

agctcagcct ctgcggagaa aggaagtccg attctcctgg gggtctctaa aggggagttt 960

tgtctctact gtgacaagga taaaggacaa agtcatccat cccttcagct gaagaaggag 1020

aaactgatga agctggctgc ccaaaaggaa tcagcacgcc ggcccttcat cttttatagg 1080

gctcaggtgg gctcctggaa catgctggag tcggcggctc accccggatg gttcatctgc 1140

acctcctgca attgtaatga gcctgttggg gtgacagata aatttgagaa caggaaacac 1200

attgaatttt catttcaacc agtttgcaaa gctgaaatga gccccagtga ggtcagcgat 1260

tga 1263

<210> 6

<211> 456

<212> PRT

<213>Artificial sequence

<400> 6

Met Ser Gly Cys Asp Arg Arg Glu Thr Glu Thr Lys Gly Lys Asn Ser

1 5 10 15

Phe Lys Lys Arg Leu Arg Gly Pro Lys Val Gly Gly Gly Gly Ser Gly

20 25 30

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Ser Leu Phe Lys Gly

35 40 45

Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn

50 55 60

Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro

65 70 75 80

Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu

85 90 95

Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn Thr Thr Thr

100 105 110

Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile Ile Arg Met

115 120 125

Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro

130 135 140

Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe Gln Thr Lys

145 150 155 160

Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser

165 170 175

Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys

180 185 190

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

195 200 205

Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro

210 215 220

Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val

225 230 235 240

Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys

245 250 255

Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu Ala

260 265 270

Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln Glu Asn Leu Tyr Phe

275 280 285

Gln Gly Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp Gln Asp

290 295 300

His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val Pro Asp

305 310 315 320

Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser Ser Leu

325 330 335

Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly Val Ser

340 345 350

Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln Ser His

355 360 365

Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala Ala Gln

370 375 380

Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln Val Gly

385 390 395 400

Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe Ile Cys

405 410 415

Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys Phe Glu

420 425 430

Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys Ala Glu

435 440 445

Met Ser Pro Ser Glu Val Ser Asp

450 455

<210> 7

<211> 482

<212> PRT

<213>Artificial sequence

<400> 7

Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp

1 5 10 15

Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala Val Ser

20 25 30

Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Ser Leu

50 55 60

Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His

65 70 75 80

Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly

85 90 95

Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn

100 105 110

Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn

115 120 125

Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile

130 135 140

Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe

145 150 155 160

Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe

165 170 175

Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe

180 185 190

Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp

195 200 205

Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val

210 215 220

Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr

225 230 235 240

Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln

245 250 255

Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly

260 265 270

Gly His Lys Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gln Pro Val

275 280 285

Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln Glu Asn

290 295 300

Leu Tyr Phe Gln Gly Val His Thr Ser Pro Lys Val Lys Asn Leu Asn

305 310 315 320

Pro Lys Lys Phe Ser Ile His Asp Gln Asp His Lys Val Leu Val Leu

325 330 335

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

340 345 350

Glu Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu

355 360 365

Lys Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu

370 375 380

Tyr Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys

385 390 395 400

Lys Glu Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg

405 410 415

Pro Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu

420 425 430

Ser Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn

435 440 445

Glu Pro Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu

450 455 460

Phe Ser Phe Gln Pro Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val

465 470 475 480

Ser Asp

<210> 8

<211> 442

<212> PRT

<213>Artificial sequence

<400> 8

Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp

1 5 10 15

Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala Val Ser

20 25 30

Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Glu Ser Leu

50 55 60

Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser Thr Ile Cys His

65 70 75 80

Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly Ile Gly

85 90 95

Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe Arg Arg Asn Asn

100 105 110

Gly Thr Leu Leu Val Gln Ser Leu His Gly Val Phe Lys Val Lys Asn

115 120 125

Thr Thr Thr Leu Gln Gln His Leu Ile Asp Gly Arg Asp Met Ile Ile

130 135 140

Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gln Lys Leu Lys Phe

145 150 155 160

Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys Leu Val Thr Thr Asn Phe

165 170 175

Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe

180 185 190

Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile Gln Thr Lys Asp

195 200 205

Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe Ile Val

210 215 220

Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr

225 230 235 240

Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln

245 250 255

Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly

260 265 270

Gly His Lys Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gln Pro Val

275 280 285

Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln Glu Asn

290 295 300

Leu Tyr Phe Gln Gly Val His Thr Glu Ile Phe Phe Ala Leu Ala Ser

305 310 315 320

Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly

325 330 335

Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln

340 345 350

Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala

355 360 365

Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln

370 375 380

Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe

385 390 395 400

Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys

405 410 415

Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys

420 425 430

Ala Glu Met Ser Pro Ser Glu Val Ser Asp

435 440

<210> 9

<211> 461

<212> PRT

<213>Artificial sequence

<400> 9

Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp

1 5 10 15

Trp Glu Lys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro

35 40 45

Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr

50 55 60

Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys

65 70 75 80

His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His

85 90 95

Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile

100 105 110

Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro

115 120 125

Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile

130 135 140

Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val

145 150 155 160

Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys

165 170 175

His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser

180 185 190

Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr

195 200 205

Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu

210 215 220

Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn

225 230 235 240

Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe Met Ser Lys Pro

245 250 255

Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu

260 265 270

Leu Val Tyr Ser Gln Glu Asn Leu Tyr Phe Gln Gly Glu Pro Gln Cys

275 280 285

Cys Leu Glu Gly Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser

290 295 300

Ile His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu

305 310 315 320

Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala

325 330 335

Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile

340 345 350

Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp

355 360 365

Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met

370 375 380

Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr

385 390 395 400

Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro

405 410 415

Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val

420 425 430

Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro

435 440 445

Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp

450 455 460

<210> 10

<211> 421

<212> PRT

<213>Artificial sequence

<400> 10

Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp

1 5 10 15

Trp Glu Lys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro

35 40 45

Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr

50 55 60

Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys

65 70 75 80

His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His

85 90 95

Gly Val Phe Lys Val Lys Asn Thr Thr Thr Leu Gln Gln His Leu Ile

100 105 110

Asp Gly Arg Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro

115 120 125

Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile

130 135 140

Cys Leu Val Thr Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val

145 150 155 160

Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys

165 170 175

His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser

180 185 190

Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr

195 200 205

Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu

210 215 220

Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn

225 230 235 240

Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe Met Ser Lys Pro

245 250 255

Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu

260 265 270

Leu Val Tyr Ser Gln Glu Asn Leu Tyr Phe Gln Gly Glu Pro Gln Cys

275 280 285

Cys Leu Glu Glu Ile Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala

290 295 300

Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu

305 310 315 320

Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu

325 330 335

Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser

340 345 350

Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn

355 360 365

Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser Cys

370 375 380

Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys

385 390 395 400

His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys Ala Glu Met Ser Pro

405 410 415

Ser Glu Val Ser Asp

420

<210> 11

<211> 15

<212> PRT

<213>Artificial sequence

<400> 11

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims

1. the method for each hypotype maturation protein of a kind of utilization Nicotiana tabacum L. high efficient expression IL-37, it is characterised in that comprise the following steps：

S1. IL-37-TEV fusion gene is designed：

S2. recombinant expression carrier is built：

S3. express：

2. the method for each hypotype maturation protein of a kind of utilization Nicotiana tabacum L. high efficient expression IL-37 as claimed in claim 1, its feature exists In, each hypotype of the IL-37 maturation protein be.

3. the method for each hypotype maturation protein of a kind of utilization Nicotiana tabacum L. high efficient expression IL-37 as claimed in claim 1, its feature exists In the recombinant expression carrier is driven by plant composing type E35S promoter and the TEV targeting sequencing with 5 ' end untranslateds The expression that gene is carried out, using the NPTII gene of Kanamycin resistance as selection markers.

4. the method for each hypotype maturation protein of a kind of utilization Nicotiana tabacum L. high efficient expression IL-37 as claimed in claim 1, its feature exists In the recombinant expression carrier for building step S2 imports to the transgenic tobacco plant of acquisition IL-37 in tobacco cell and carries out surely The each hypotype maturation protein of IL-37 is expressed surely.

5. the method for each hypotype maturation protein of a kind of utilization Nicotiana tabacum L. high efficient expression IL-37 as claimed in claim 1, its feature exists In the recombinant expression carrier for building step S2 is expelled to each hypotype maturation protein of transient expression IL-37 in Nicotiana tabacum L..