CN106434745A - Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos - Google Patents
Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos Download PDFInfo
- Publication number
- CN106434745A CN106434745A CN201610831281.4A CN201610831281A CN106434745A CN 106434745 A CN106434745 A CN 106434745A CN 201610831281 A CN201610831281 A CN 201610831281A CN 106434745 A CN106434745 A CN 106434745A
- Authority
- CN
- China
- Prior art keywords
- tev
- ser
- gly
- leu
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nutrition Science (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
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
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 Na3PO4•
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 (5)
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:
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.
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..
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610831281.4A CN106434745A (en) | 2016-09-19 | 2016-09-19 | Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610831281.4A CN106434745A (en) | 2016-09-19 | 2016-09-19 | Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos |
Publications (1)
Publication Number | Publication Date |
---|---|
CN106434745A true CN106434745A (en) | 2017-02-22 |
Family
ID=58165599
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610831281.4A Pending CN106434745A (en) | 2016-09-19 | 2016-09-19 | Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106434745A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117362451A (en) * | 2023-12-04 | 2024-01-09 | 北京质肽生物医药科技有限公司 | Fusion protein containing TEV protease, preparation method and application thereof |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1533438A (en) * | 2001-06-08 | 2004-09-29 | �����Ŵ��Ƽ���˾ | Method for producing proteins in plants |
CN1905890A (en) * | 2003-11-13 | 2007-01-31 | 里卡多·阿马拉·雷默 | Pharmaceutical product comprising tissue of the male vegetal reproductive system |
CN102753700A (en) * | 2009-09-04 | 2012-10-24 | 先正达参股股份有限公司 | Stacking of translational enhancer elements to increase polypeptide expression in plants |
CA2901370A1 (en) * | 2014-08-25 | 2016-01-18 | Shengwu Ma | Production of il-37 in plants |
-
2016
- 2016-09-19 CN CN201610831281.4A patent/CN106434745A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1533438A (en) * | 2001-06-08 | 2004-09-29 | �����Ŵ��Ƽ���˾ | Method for producing proteins in plants |
CN1905890A (en) * | 2003-11-13 | 2007-01-31 | 里卡多·阿马拉·雷默 | Pharmaceutical product comprising tissue of the male vegetal reproductive system |
CN102753700A (en) * | 2009-09-04 | 2012-10-24 | 先正达参股股份有限公司 | Stacking of translational enhancer elements to increase polypeptide expression in plants |
CA2901370A1 (en) * | 2014-08-25 | 2016-01-18 | Shengwu Ma | Production of il-37 in plants |
Non-Patent Citations (5)
Title |
---|
ALLISON,R. ET AL.: ""Tobacco etch virus RNA, complete genome"", 《GENBANK》 * |
JOSE F.MARCOS ET AL.: ""Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide"", 《JOURNAL OF GENERAL VIROLOGY》 * |
PAUL G.BLOMMEL ET AL.: ""A combined approach to improving large-scale production of tobacco etch virus protease"", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
RACHEL B.KAPUST ET AL.: ""Controlled Intracellular Processing of Fusion Proteins by TEV Protease"", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
RUSSELL JOHNSON: ""Transformation of Arabidopsis thaliana using Agrobacterium tumefaciens"", 《COLBY J.RES.METH.》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117362451A (en) * | 2023-12-04 | 2024-01-09 | 北京质肽生物医药科技有限公司 | Fusion protein containing TEV protease, preparation method and application thereof |
CN117362451B (en) * | 2023-12-04 | 2024-03-08 | 北京质肽生物医药科技有限公司 | Fusion protein containing TEV protease, preparation method and application thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2015070778A1 (en) | Method for controlling pest | |
JPS63500425A (en) | molecular farming | |
WO2015070783A1 (en) | Method for controlling pest | |
WO2015070781A1 (en) | Method for controlling pests | |
WO2015067194A1 (en) | Method for controlling pests | |
WO2016138819A1 (en) | Uses of insecticidal protein | |
WO2016101612A1 (en) | Pest control method | |
CN105950541A (en) | Establishment method of hFGF21-gene knock-out human liver cell strain | |
CN108611362B (en) | Use of insecticidal proteins | |
ES2336861T3 (en) | PRODUCTION OF IL-10 IN BIORREACTOR OF GROWTH PLANT NON-FOOD. | |
DE69836075T2 (en) | PROCESS FOR CUTTING FUSION PROTEINS | |
US20110243975A1 (en) | Transformed soybean plant which accumulates vaccine, and use thereof | |
CN102653763A (en) | Meloidogyne javanica dominant-effect gene (Mj-nulg), related protein and application of Mj-nulg | |
CN108432760B (en) | Use of insecticidal proteins | |
CN106496313A (en) | Disease-resistance-related protein IbSWEET10 and its encoding gene and application | |
CN106434745A (en) | Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos | |
WO2016184397A1 (en) | Application of insecticidal protein | |
WO2016101684A1 (en) | Uses of insecticidal protein | |
KR20210047891A (en) | Composition and method for production of antibiotic-free biopharmaceuticals from lettuce chloroplasts | |
CN106349355B (en) | Resistance relevant protein IbCPK28 and its encoding gene and application | |
KR101680903B1 (en) | Transformants expressing epitope of porcine epidemic diarrhea virus and rotavirus and vaccine composition containing the same | |
CN101988058B (en) | Gene expression composition, PRRS (Porcine Reproductive and Respiratory Syndrome) oral vaccine and preparation methods thereof | |
CN100471955C (en) | A fusion gene, it expressed protein and producing process thereof | |
CN102911265B (en) | Recombination variant of human nerve growth factors and preparation method thereof | |
CN101343624B (en) | Recombined human growth hormone gene bacilliform virus, preparation and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170222 |
|
RJ01 | Rejection of invention patent application after publication |