CN105820218A - Protein CAA' for preventing and treating radiation damage and application thereof - Google Patents
Protein CAA' for preventing and treating radiation damage and application thereof Download PDFInfo
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- CN105820218A CN105820218A CN201610196102.4A CN201610196102A CN105820218A CN 105820218 A CN105820218 A CN 105820218A CN 201610196102 A CN201610196102 A CN 201610196102A CN 105820218 A CN105820218 A CN 105820218A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
The invention discloses protein CAA' for preventing and treating radiation damage and application thereof, and provides cyclization protein (named as the protein CAA').The cyclization protein is shown as follows: (a1) or (a2) or (a3) or (a4), wherein the cyclization protein shown in (a1) is composed of amino acid residues from the 11<th> site to the 305<th> site of the N-terminal of a sequence 7, the cyclization protein shown in (a2) is composed of amino acid residues from the 3<th> site to the 311<th> site of the N-terminal of the sequence 7, the cyclization protein shown in (a3) is shown in the sequence 7, and the cyclization protein shown in (a4) is obtained by conducting substitution and/or deletion and/or addition of one or several amino acid residues on the cyclization protein shown in (a1) or the cyclization protein shown in (a2) or the cyclization protein shown in (a3) and has the function of preventing and/or treating the adiation damage.The invention further discloses the application of the protein CAA' in product preparation, wherein a product has the functions of preventing and/or treating the radiation damage and/or activating a NF-kappaB signal path.The protein CAA' has the great application prospect and popularization value on prevention and/or treatment of the radiation damage.
Description
Technical field
The present invention relates to a kind of prevention and the PROTEIN C AA ' for the treatment of radiation damage and application thereof.
Background technology
The short-term lethal effect that heavy dose of ionizing radiation is caused mainly due to radiosensitive system such as hemopoietic system,
Acute radiation complication ARS (the Acute Radiation that a large amount of apoptosis of gastrointestinal system are caused
Syndrome) appearance, and the activation of NF-κ B signal path can the carrying out of effective inhibited apoptosis.The most straight
Connect and research and develop novel radiation protection medicine efficient, nontoxic for mechanism of radiation damage, it may be possible to a new effective way.
Small intestinal endotheliocyte to radiation extreme sensitivity expresses receptor 5 (i.e. TLR5) and the bacterial flagellin of Toll-like
In conjunction with, can be with effective activation NF-κ B signal path.Salmonella flagellin is as be currently known uniquely can be with
The specific agonist that TLR5 combines, plays the merit of brilliance in protection mice and primate reply acute radiation
Effect, but owing to flagellin derives from antibacterial, its immunogenicity and toxicity are the strongest, Given this related researcher
The flagellin deriving from salmonella typhimurium (Salmonella enterica) is done and has studied deeper into ground.
Salmonella flagellin (also known as protein A A) total length totally 505 aminoacid, including N end (1～176aa), in
Between variable region (177～401aa) and C end (402～505), by the research respectively in these three region is found, when
In the middle of removing after variable region, the Activity and stabill of this flagellin does not all reduce, more amazing be after truncate its
Immunogenicity and toxicity are all substantially reduced (body maximum is stood dosage and brought up to 25mg/kg by 12mg/kg), and this grinds
Study carefully group by its named CBLB502 (also referred to as protein A A ').
Summary of the invention
It is an object of the invention to provide a kind of prevention and the PROTEIN C AA ' for the treatment of radiation damage and application thereof.
The invention provides a kind of cyclisation albumen (named PROTEIN C AA '), be following (a1) or (a2) or (a3)
Or (a4):
(a1) the cyclisation albumen that in sequence table, sequence 7 forms from N-terminal the 11st to 305 amino acids residue；
(a2) the cyclisation albumen that in sequence table, sequence 7 forms from N-terminal the 3rd to 311 amino acids residue；
(a3) the cyclisation albumen shown in sequence 7 in sequence table；
(a4) by (a1) or (a2) or (a3) through one or several amino acid residue replacement and/or disappearance and
/ or add and have prevention and/or the cyclisation albumen for the treatment of radiation damage function.
The described PROTEIN C AA ' PROTEIN C AA ' that concretely following arbitrary described method prepares.
The gene (named gene C AA ') of encoding said proteins CAA ' falls within protection scope of the present invention.
Described gene C AA ' is concretely following (b1) or (b2) or (b3) or (b4) or (b5):
(b1) in sequence table sequence 8 from the DNA molecular shown in 5 ' end 31-915 position nucleotide；
(b2) in sequence table sequence 8 from the DNA molecular shown in 5 ' end 7-933 position nucleotide；
(b3) DNA molecular shown in sequence 8 in sequence table；
(b4) the DNA sequence hybridization limited with (b1) or (b2) or (b3) under strict conditions and coding are described
The DNA molecular of PROTEIN C AA '；
(b5) DNA molecular limited with (b1) or (b2) or (b3) at least has more than 90% homology and volume
The DNA molecular of code encoding said proteins CAA '.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing described gene C AA ' belong to the present invention
Protection domain.
The present invention also protects a kind of albumen (named Protein Q CAA '；Protein Q CAA ' is the precursor of PROTEIN C AA ',
PROTEIN C AA ' is formed after self cleavage), include following element successively: intein-C fragment, cyclisation protein fragments, include
Peptide-N fragment；
Described intein-C fragment is following (c1) or (c2):
(c1) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 3rd to 40 amino acids residue；
(c2) (c1) through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and had phase
The polypeptide fragment of congenerous；
Described intein-N fragment is following (c3) or (c4):
(c3) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 354th to 476 amino acids residue；
(c4) (c3) through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and had phase
The polypeptide fragment of congenerous；
Described cyclisation protein fragments is following (c5) or (c6) or (c7) or (c8):
(c5) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 51st to 345 amino acids residue；
(c6) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 43rd to 351 amino acids residue；
(c7) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 41st to 353 amino acids residue；
(c8) by (c5) or (c6) or (c7) through one or several amino acid residue replacement and/or disappearance and
/ or add and have the polypeptide fragment of identical function.
Described Protein Q CAA ' is specifically as shown in the sequence 3 of sequence table.
The gene (named gene QCAA ') of encoding said proteins QCAA ' falls within protection scope of the present invention.
In described gene QCAA ', the DNA molecular of encoding intein-C fragment is following (d1) or (d2):
(d1) in sequence table sequence 4 from the DNA molecular shown in the 7th to 120 nucleotide of 5 ' end；
(d2) DNA molecular limited with (d1) at least has intein described in more than 90% homology and fgs encoder
The DNA molecular of-C fragment.
In described gene QCAA ', the DNA molecular of encoding intein-N fragment is following (d3) or (d4):
(d3) in sequence table sequence 4 from the DNA molecular shown in the 1060th to 1428 nucleotide of 5 ' end；
(d4) DNA molecular limited with (d3) at least has intein described in more than 90% homology and fgs encoder
The DNA molecular of-N fragment.
In described gene QCAA ', the DNA molecular of coding cyclisation protein fragments is following (d5) or (d6) or (d7)
Or (d8):
(d5) in sequence table sequence 4 from the DNA molecular shown in the 151st to 1035 nucleotide of 5 ' end；
(d6) in sequence table sequence 4 from the DNA molecular shown in the 127th to 1053 nucleotide of 5 ' end；
(d7) in sequence table sequence 4 from the DNA molecular shown in the 121st to 1059 nucleotide of 5 ' end；
(d8) DNA molecular limited with (d5) or (d6) or (d7) at least has more than 90% homology and volume
Code encodes the DNA molecular of described cyclisation protein fragments.
Described gene QCAA ' is specifically as shown in the sequence 4 of sequence table.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing described gene QCAA ' belong to the present invention
Protection domain.Recombinant vector containing described gene QCAA ' can be insertion exogenous DNA molecule shape in expression vector
The recombiant plasmid of the DNA molecular shown in sequence 4 with sequence table become.Described expression vector concretely carrier
pET-28a(+).Recombinant vector containing described gene QCAA ' is concretely: carrier pET-28a (+) NcoI
And insert the sequence 4 of sequence table between Hind III digestion site from the 5th to 1434 nucleotide institute of 5 ' end
The recombiant plasmid that the double chain DNA molecule shown obtains.Described recombinant vector is concretely imported Host Strains by described recombinant bacterium
The recombinant bacterium obtained.Described Host Strains concretely e. coli bl21 (DE3) pLysS.
The present invention also protects a kind of method preparing described PROTEIN C AA ', comprises the steps: to cultivate any of the above institute
State recombinant bacterium, obtain described PROTEIN C AA '.
Described method specifically includes following steps: cultivate recombinant bacterium to OD600nm=0.5, it is subsequently adding IPTG and lures
Lead, then collect bacterial sediment and carry out ultrasonication, collecting precipitation.
Described method specifically includes following steps:
(1) recombinant bacterium is seeded to LB culture medium, 37 DEG C, 220rpm shaken cultivation to OD600nm=0.5, add IPTG
(making its concentration in cultivating system is 0.5mmol/L), then 30 DEG C, 200rpm shaken cultivation 4 hours；
(2) take into the cultivating system of step (1), 4 DEG C, 12000rpm be centrifuged 5 minutes, collect bacterial sediment；
(3) bacterial sediment is carried out ultrasonication (supersonic frequency 30%；Ultrasonic 5 seconds, stop 5 seconds, totally 50 minutes),
Then 4 DEG C, 12000rpm be centrifuged 20 minutes, collect precipitation.
Described method also comprises the steps (4): precipitation 2M aqueous solution of urea washing step (3) obtained,
Being subsequently adding 2M aqueous solution of urea and room temperature is placed 30 minutes, then 12000rpm is centrifuged 30 minutes, collects supernatant
Liquid also carries out anion-exchange chromatography.The design parameter of anion-exchange chromatography: chromatographic column is GE HiTrap Q FF 1ml
(GE company, article No.: 17-5053-01)；Column diameter/post is high: 7mm/25mm；A liquid: the Tris-HCl of pH7.5,50mM
Buffer；B liquid: the Tris-HCl buffer of pH7.5,50mM containing 1M NaCl.After loading, first with 10 times
Column volume A liquid carries out eluting；Then carrying out the gradient elution of 10 column volumes, during gradient elution, A liquid exists
Volume ratio in eluent is linearly decreased to 0% by 100%, corresponding B liquid volume in eluent by 0% linear on
Being raised to 100%, flow velocity is 1ml/min, co-elute 10 minutes, after collecting the mistake post of 3-5 milliliter in this elution process
Solution.
Described method also comprises the steps (5): after the post excessively step (4) obtained, solution carries out the affine layer of Ni post
Analysis.The design parameter of Ni post affinity chromatograph: chromatographic column is GE HisTrap FF 5ml (GE company, article No.
17-5319-01)；In conjunction with buffer: containing the phosphate buffer of pH7.4,20mM of 0.5M NaCl, 20mM imidazoles；
Elution buffer: containing the phosphate buffer of pH7.4,20mM of 0.5M NaCl, 500mM imidazoles.After loading, first
Eluting is carried out with the combination buffer of 10 times of column volumes；Then carrying out eluting with elution buffer, flow velocity is 2ml/min,
Collect solution after the mistake post of 2-3 column volume in this step.
Described method also comprises the steps (6): after the post excessively step (5) obtained, solution is carried out
Strep-Tag/Affinity chromatograph.Strep-Tag/The design parameter of affinity chromatograph:
Chromatographic column is to be IBA company1ml, article No. is 2-1207-001；Buffer
W: containing 1mM EDTA, the Tris-HCl buffer of pH8.0,100mM of 150mM NaCl；Buffer E: containing 1mM
The Tris-HCl buffer of pH8.0,100mM of EDTA, 150mM NaCl, 2.5mM desthiobiotin.After loading,
First carry out eluting with the Buffer W of 5 times of column volumes；Then eluting is carried out with Buffer E, in this step every 0.5
After the post excessively of column volume, solution collects a pipe, collects and merges the 2nd to 5 pipe and crosses solution after post.
Described method also comprises the steps (7): after the post excessively step (6) obtained, solution carries out sieve chromatography.
The design parameter of sieve chromatography: chromatographic column is GE Superdex 75prep grade 25ml (GE company, goods
Number: 17-1044-10).After loading, the PBS of pH7.4,0.02M containing 0.5M NaCl is used to wash
De-, flow velocity is 0.35ml/min, and that collects 1.5-2.5 column volume crosses solution (being CAA ' protein liquid) after post.
The present invention also protects the described PROTEIN C AA ' application in preparing product；The function of described product be (f1) and/
Or (f2): (f1) prevents and/or treats radiation damage；(f2) activation NF-κ B signal path.The spoke of described radiation
Penetrate source concretely60Co gamma-rays.
The present invention also protects a kind of product, and its active component is described PROTEIN C AA '；The function of described product is (f1)
And/or (f2): (f1) prevents and/or treats radiation damage；(f2) activation NF-κ B signal path.Described radiation
Radiation source concretely60Co gamma-rays.
The present invention has great application prospect and promotional value for prevention and/or the treatment of radiation damage.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE figure during using recombiant plasmid first to carry out the step 2 of embodiment 2.
Fig. 2 is the SDS-PAGE figure during using recombiant plasmid second to carry out the step 2 of embodiment 2.
Fig. 3 is the SDS-PAGE figure of CAA ' protein liquid and Tag-AA ' protein liquid.
Fig. 4 is the result by dual-luciferase reporter system detection protein active.
Fig. 5 be irradiate after the statistical of mouse survival rate.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions,
It is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats in fact
Test, results averaged.
Protein A A ' (is made up of 296 amino acid residues) as shown in the sequence 1 of sequence table, its encoding gene (gene
AA ') as shown in the sequence 2 of sequence table.
Carrier pET-28a (+): Novagen company, catalog number (Cat.No.): 69864-3.E. coli bl21 (DE3) pLysS:
TIANGEN Biotech (Beijing) Co., Ltd., catalog number (Cat.No.): CB106-01.Carrier pSFBAD09:addgene company,
Catalog number (Cat.No.) is 11963.Carrier pJJDuet30:addgene company, catalog number (Cat.No.) is 11962.Carrier pNF-κ B-Luc:
Anjelen Sci. & Tech. Inc, catalog number (Cat.No.) is 219078.Carrier pRL-SV40: Pu Luomaige company of the U.S., catalog number (Cat.No.)
For E2231.HEK293 cell: ATCC company of the U.S., catalog number (Cat.No.) is CRL-1573.DMEM culture medium: U.S. Hyclone
Company, article No. SH30243.01B.
Embodiment 1, the discovery of PROTEIN C AA '
On the basis of protein A A ' sequence and great many of experiments, select to carry out Ssp DnaE intein with protein A A '
Merge, and on the basis of carrying out great many of experiments further, design obtains Protein Q CAA '.
Protein Q CAA ' is as shown in the sequence 3 of sequence table, and its encoding gene (gene QCAA ') is such as the sequence of sequence table
Shown in 4.
Protein Q CAA ' from N-terminal the 1st amino acids residue be start codon coding methionine, the 3rd to 40
Amino acids residue is the C-terminal fragment (intein-C fragment) of intein, the 41st to the 42nd amino acids residue
The amino acid residue (HM) encoded for the recognition sequence of restricted enzyme NdeI, the 43rd to 50 amino acids residue
For affinity purification label Strep-TagII (WSHPQFEK), the 51st to 345 amino acids residue is for removing first
Protein A A ' (being made up of 295 amino acid residues) after methionine, the 346th to 351 amino acids residue is
His-Tag label (HHHHHH), the 352nd to 353 amino acids residue is the identification sequence of restricted enzyme BamHI
The amino acid residue (GS) of row coding, the 354th to 476 amino acids residue is that the N-terminal fragment of intein (includes
Peptide-N fragment).Protein Q CAA ' is precursor forms, in thalline, intein-C fragment and intein-N fragment is situated between
Leading self-splicing, thus be cyclized, (PROTEIN C AA ' is cyclisation albumen, its aminoacid sequence to form PROTEIN C AA '
As shown in the sequence 7 of sequence table).
Gene QCAA ' is start codon from the 1st to 3 nucleotide of 5 ' end, and the 7th to 120 nucleotide is intein
The coded sequence of-C fragment, 121-126 position nucleotide is the recognition sequence (CATATG) of restricted enzyme NdeI,
127th to 150 nucleotide is the coded sequence of affinity purification label Strep-TagII, the 151st to 1035 nucleotide
For the coded sequence of the protein A A ' after first methionine of removal, the 1036th to 1053 nucleotide is His-Tag
The coded sequence of label, the 1054th to 1059 nucleotide is the recognition sequence (ggatcc) of restricted enzyme BamHI,
1060th to 1428 nucleotide is the coded sequence of intein-N fragment, and the 1429th to 1434 nucleotide is two ends
Only codon.
Embodiment 2, the activity of albumen
One, the structure of recombiant plasmid
1, recombiant plasmid pET/Ssp DnaE intein-28a (+) structure
(1) with carrier pSFBAD09 as template, with the primer of CF and CR composition to carrying out PCR amplification, PCR amplification is obtained
Product.
CF:5 '-TAACCATGGGCgttaaagttatcggtc-3’；
CR:5 '-TAA CATATGATTGAAACAATTTGCAGC-3’；
In CF, underscore mark Nco I restriction endonuclease recognition sequence.In CR, square frame mark is that one section of Linker district (has
Kpn I restriction endonuclease recognition sequence, BamH I restriction endonuclease recognition sequence), underscore mark Nde I restriction endonuclease recognition sequence.
(2) with restricted enzyme Nco I and the pcr amplification product of BamHI double digestion step (1), reclaim enzyme action and produce
Thing.
(3) with restricted enzyme Nco I and BamHI double digestion carrier pET-28a (+), reclaim carrier framework (about
5300bp)。
(4) digestion products of step (2) and the carrier framework of step (3) are connected, obtain recombiant plasmid.
(5) with carrier pJJDuet30 as template, with the primer of NF and NR composition to carrying out PCR amplification, obtain PCR and expand
Volume increase thing.
NF:5 '-AAAggatcctgcttaagtttcggtact-3’；
NR:5 '-ATGAAGCTTTCATTATTTGATAGTACCAGCGTCC-3’。
In NF, underscore mark BamH I restriction endonuclease recognition sequence.In NR, underscore mark Hind III digestion identification sequence
Row.
(6) with restricted enzyme BamH I and the pcr amplification product of Hind III double digestion step (5), enzyme is reclaimed
Cut product.
(7) recombiant plasmid obtained with restricted enzyme BamH I and Hind III double digestion step (4), reclaims
Carrier framework (about 5420bp).
(8) digestion products of step (6) and the carrier framework of step (7) are connected, obtain recombiant plasmid pET/Ssp
DnaE intein-28a(+)。
2, the structure of recombiant plasmid first
(1) double chain DNA molecule shown in sequence 2 of composition sequence table.
(2) with step 1 synthesis double chain DNA molecule as template, with F1 and R1 composition primer to carrying out PCR amplification,
Obtain pcr amplification product.
F1:5 '-AAACATATG gcacaagtcattaatacaaacAG-3’；
R1:5 '-AAAGGATCC ACGCAGTAAAGAGAGGACGT-3’。
In F1, underscore mark Nde I restriction endonuclease recognition sequence, the volume of square frame mark affinity purification label Strep-TagII
Code sequence.In R1, underscore mark BamHI restriction endonuclease recognition sequence, the coded sequence of square frame mark His-Tag label.
(3) with restricted enzyme Nde I and the pcr amplification product of BamHI double digestion step (2), reclaim enzyme action and produce
Thing.
(4) with restricted enzyme Nde I and BamHI double digestion recombiant plasmid pET/Ssp DnaE intein-28a (+),
Reclaim carrier framework (about 5800bp).
(5) digestion products of step (3) and the carrier framework of step (4) are connected, obtain recombiant plasmid first.Root
According to sequencing result, recombiant plasmid first is carried out structure and is described as follows: carrier pET-28a (+) Nco I and Hind III
The sequence 4 inserting sequence table between restriction enzyme site is divided from the double-stranded DNA shown in the 5th to 1434 nucleotide of 5 ' end
Son.In recombiant plasmid first, the DNA molecular of insertion merges with the partial nucleotide on carrier framework, the sequence of formation sequence table
Open reading frame (gene QCAA ') shown in row 4, the protein (Protein Q CAA ') shown in sequence 3 of expressed sequence table,
The cyclisation albumen (PROTEIN C AA ') shown in sequence 7 of formation sequence table after self cleavage.
3, the structure of recombiant plasmid second (control plasmid)
(1) double chain DNA molecule shown in sequence 2 of composition sequence table.
(2) with step 1 synthesis double chain DNA molecule as template, with F1 and R2 composition primer to carrying out PCR amplification,
Obtain pcr amplification product.
F1:5 ' AAACATATG GCACAAGTCATTAATACAAACAG-3’；
R2:5 ' AAAGGATCCTCATTAACGCAGTAAAGAGAGGACGT-3’。
(3) with restricted enzyme Nde I and the pcr amplification product of BamHI double digestion step (2), reclaim enzyme action and produce
Thing.
(4) with restricted enzyme Nde I and BamHI double digestion carrier pET-28a (+), reclaim carrier framework (about
5300bp)。
(5) digestion products of step (3) and the carrier framework of step (4) are connected, obtain recombiant plasmid second.Root
According to sequencing result, recombiant plasmid second is carried out structure and is described as follows: carrier pET-28a (+) Nde I and BamHI enzyme
Cut the sequence 6 inserting sequence table between site from the double chain DNA molecule shown in 5 ' the 4th to 936 nucleotide of end.Weight
In group plasmid second, the DNA molecular of insertion merges with the partial nucleotide on carrier framework, sequence 6 institute of formation sequence table
The open reading frame (gene Tag-AA ') shown, the albumen (albumen Tag-AA ') shown in sequence 5 of expressed sequence table.
Two, the expression of albumen
Recombiant plasmid second that recombiant plasmid first, the step one step one built builds and carrier pET-28a (+) enter respectively
The following operation repetitive of row:
1, by plasmid transformation escherichia coli BL21 (DE3) pLysS, recombinant bacterium is obtained.
2, recombinant bacterium is seeded to LB culture medium, 37 DEG C, 220rpm shaken cultivation to OD600nm=0.5, add IPTG
(making its concentration in cultivating system is 0.5mmol/L), then 30 DEG C, 200rpm shaken cultivation 4 hours.
3, take into the cultivating system of step 2,4 DEG C, 12000rpm be centrifuged 5 minutes, collect bacterial sediment.
4, bacterial sediment is carried out ultrasonication (supersonic frequency 30%；Ultrasonic 5 seconds, stop 5 seconds, totally 50 minutes),
Then 4 DEG C, 12000rpm be centrifuged 20 minutes, collect upper cleer and peaceful precipitation respectively.
The SDS-PAGE that employing recombiant plasmid first is carried out during above step is shown in Fig. 1.In Fig. 1, swimming lane M is molecule
Amount labelling, swimming lane 1 is the cultivating system supernatant before using IPTG induction, and swimming lane 2 is the bacterium after using IPTG induction
Body precipitates, and swimming lane 3 is the supernatant after ultrasonication, and swimming lane 4 is the precipitation after ultrasonication.Result shows, gene
After QCAA ' expresses in thalline, it is cyclized under the effect of intein, forms PROTEIN C AA ' (about 33.73kDa).
The SDS-PAGE that employing recombiant plasmid second is carried out during above step is shown in Fig. 2.In Fig. 2, swimming lane M is molecule
Amount labelling, swimming lane 1 is the cultivating system supernatant before using IPTG induction, and swimming lane 2 is the bacterium after using IPTG induction
Body precipitates, and swimming lane 3 is the supernatant after ultrasonication, and swimming lane 4 is the precipitation after ultrasonication.Result shows, gene
After Tag-AA ' expresses in thalline, form albumen Tag-AA '.
Integrated comparative Fig. 1 and Fig. 2, the molecular weight that PROTEIN C AA ' shows in electrophoresis is less than albumen Tag-AA ' on the contrary,
This is the tightst so what mobility speed faster caused due to cyclisation protein structure exactly.
Three, the purification of albumen
Precipitation and employing recombiant plasmid second after carrying out, by using recombiant plasmid first, the ultrasonication that step 2 obtains walk
Precipitation after rapid two ultrasonications obtained carries out following operation repetitive respectively:
1, the 2M aqueous solution of urea of the precipitation after ultrasonication is washed, be subsequently adding 2M aqueous solution of urea and room temperature is put
Putting 30 minutes, then 12000rpm is centrifuged 30 minutes, collects supernatant.
2, supernatant step 1 obtained carries out anion-exchange chromatography.
The chromatographic column of anion-exchange chromatography is GE HiTrap Q FF 1ml (GE company, article No.: 17-5053-01)；
Column diameter/post is high: 7mm/25mm；
A liquid: the Tris-HCl buffer of pH7.5,50mM；
B liquid: the Tris-HCl buffer of pH7.5,50mM containing 1M NaCl.
After loading, first carry out eluting, to remove unconjugated foreign protein with 10 times of column volume A liquid；Then 10 are carried out
The gradient elution of individual column volume, during gradient elution, A liquid volume ratio in eluent is by 100% linear decline
To 0%, corresponding B liquid volume in eluent is by 0% linear rise to 100%, and flow velocity is 1ml/min, co-elute
10 minutes, collect solution after the mistake post of 3-5 milliliter in this elution process.
3, after the post excessively step 2 obtained, solution carries out Ni post affinity chromatograph.
The chromatographic column of Ni post affinity chromatograph is GE HisTrap FF 5ml (GE company, article No. 17-5319-01)；
In conjunction with buffer: containing the phosphate buffer of pH7.4,20mM of 0.5M NaCl, 20mM imidazoles；
Elution buffer: containing the phosphate buffer of pH7.4,20mM of 0.5M NaCl, 500mM imidazoles.
After loading, first carry out eluting, to remove foreign protein with the combination buffer of 10 times of column volumes；Then delay with eluting
Rushing liquid and carry out eluting, flow velocity is 2ml/min, collects solution after the mistake post of 2-3 column volume in this step.
4, after the post excessively step 3 obtained, solution carries out Strep-Tag/(streptavidin) is affine
Chromatography.
The chromatographic column used is to be IBA company1ml, article No. is
2-1207-001；
Buffer W: containing 1mM EDTA, the Tris-HCl buffer of pH8.0,100mM of 150mM NaCl；
Buffer E: containing 1mM EDTA, 150mM NaCl, the Tris-HCl of pH8.0,100mM of 2.5mM desthiobiotin
Buffer.
After loading, first carry out eluting with the Buffer W of 5 times of column volumes；Then eluting is carried out with Buffer E, this
In step, after the post excessively of every 0.5 column volume, solution collects a pipe, collects and merges the 2nd to 5 pipe and crosses solution after post.
5, after the post excessively step 4 obtained, solution carries out sieve chromatography.
The chromatographic column used is GE Superdex 75 prep grade 25ml (GE company, article No.: 17-1044-10).
After loading, using the PBS of pH7.4,0.02M containing 0.5M NaCl to carry out eluting, flow velocity is
0.35ml/min, that collects 1.5-2.5 column volume crosses solution after post.
After the post excessively that step 5 is collected, solution is target protein liquid.The ultrasonication that recombiant plasmid first step 2 is obtained
After precipitation carry out the target protein liquid named CAA ' protein liquid that above-mentioned steps obtains.By recombiant plasmid second step 2
Precipitation after the ultrasonication obtained carries out the target protein liquid named Tag-AA ' protein liquid that above-mentioned steps obtains.
Fig. 3 is shown in by the SDS-PAGE photo of CAA ' protein liquid and Tag-AA ' protein liquid.In Fig. 3, M1 and M2 is respectively
For different Protein Marker, swimming lane 1 for use carrier pET-28a (+) carry out not adding IPTG during step 2
Full bacterium comparison before induction, swimming lane 2 is the thalline using recombiant plasmid second to carry out after using IPTG to induce during step 2,
Swimming lane 3 is Tag-AA ' protein liquid, and swimming lane 4 is CAA ' protein liquid.
Four, the Activity determination (test cell line) of albumen
Detected by dual-luciferase reporter system, specifically comprise the following steps that
1, in Tissue Culture Plate, HEK293 cell, then cotransfection carrier pNF-κ B-Luc and carrier are added
PRL-SV40 (every 105Individual cell transfecting 100ng carrier pNF-κ B-Luc and 2ng carrier pRL-SV40), the most quiet
Put and hatch 6 hours.
2, after completing step 1, take described Tissue Culture Plate, inhale and abandon culture supernatant, add the DMEM containing testing protein
Culture medium, then stationary incubation 30 hours.
3, after completing step 1, taking described Tissue Culture Plate, add testing protein, then stationary incubation is carried out for 6 hours
Activity determination.Testing protein is that (addition form is CAA ' to the PROTEIN C AA ' for preparing of step 3 or albumen Tag-AA '
Protein liquid or Tag-AA ' protein liquid), three concentration for the treatment of (10 are respectively set-1μmol/L、10-3μm ol/L or
10-5μm ol/L), each concentration for the treatment of arranges 4 repetitions.Arrange and replace testing protein liquid with equal-volume PBS
Control treatment.
The uciferase activity of control treatment is changed multiple and is set to 1, under the different disposal concentration of different testing proteins
Relative value see Fig. 4.The activity of PROTEIN C AA ' and albumen Tag-AA ' all shows preferable ladder under variable concentrations
Degree, and activity equal 1.Result shows, PROTEIN C AA ' and albumen Tag-AA ' all can effective activation NF-κ B signal
Path, and the activity of PROTEIN C AA ' is noticeably greater than albumen Tag-AA '.
Five, the Activity determination (animal experiment) of albumen
Experimental animal: C57BL/6 mice, male, 68 week old, body weight 18 22g.
72 experimental animals are randomly divided into six groups (often groups 12), process as follows respectively:
First group: test the 1st day, (buffer with PBS to experimental animal subcutaneous injection 200 microlitre CAA ' protein liquid
Liquid adjusts protein concentration, and dosage is 0.2mg albumen/kg body weight), complete subcutaneous injection and use after 30 minutes60Co
Gamma-rays irradiation source carries out total irradiation (exposure dose is 9Gy) to experimental animal；
Second group: replace CAA ' protein liquid with Tag-AA ' protein liquid, other are with first group；
3rd group: replace CAA ' protein liquid with PBS, other are with first group；
4th group: exposure dose is 14Gy, other are with first group；
5th group: exposure dose is 14Gy, other are with second group；
6th group: exposure dose is 14Gy, other are with the 3rd group.
Testing the 1-30 days, every day adds up the survival rate of each group of experimental animal.
Result is shown in Fig. 5.Testing the 10th day, the survival rate of the 3rd group of experimental animal is 0%, depositing of second group of experimental animal
Motility rate is 90%, and the survival rate of first group of experimental animal is 100%；Test the 17th day, the survival of second group of experimental animal
Rate is 70%, and the survival rate of first group of experimental animal is 90%.Testing the 9th day, the survival rate of the 6th group of experimental animal is
0%, the survival rate of the 5th group of experimental animal is 0%, and the survival rate of the 4th group of experimental animal is 30%.Result shows, egg
White CAA ' is significantly better than albumen Tag-AA ' to the prophylactic treatment effect of radiation damage.
Claims (10)
1. a cyclisation albumen, is following (a1) or (a2) or (a3) or (a4):
(a1) the cyclisation albumen that in sequence table, sequence 7 forms from N-terminal the 11st to 305 amino acids residue；
(a2) the cyclisation albumen that in sequence table, sequence 7 forms from N-terminal the 3rd to 311 amino acids residue；
(a3) the cyclisation albumen shown in sequence 7 in sequence table；
(a4) by (a1) or (a2) or (a3) through one or several amino acid residue replacement and/or disappearance and
/ or add and have prevention and/or the cyclisation albumen for the treatment of radiation damage function.
2. described in coding claim 1, it is cyclized the gene of albumen.
3. gene as claimed in claim 2, it is characterised in that: described gene is following (b1) or (b2) or (b3)
Or (b4) or (b5):
(b1) in sequence table sequence 8 from the DNA molecular shown in 5 ' end 31-915 position nucleotide；
(b2) in sequence table sequence 8 from the DNA molecular shown in 5 ' end 7-933 position nucleotide；
(b3) DNA molecular shown in sequence 8 in sequence table；
(b4) the DNA sequence hybridization limited with (b1) or (b2) or (b3) under strict conditions and coding are described
The DNA molecular of cyclisation albumen；
(b5) DNA molecular limited with (b1) or (b2) or (b3) at least has more than 90% homology and volume
Code encodes the DNA molecular of described cyclisation albumen.
4. an albumen, includes following element successively: intein-C fragment, cyclisation protein fragments, intein-N sheet
Section；
Described intein-C fragment is following (c1) or (c2):
(c1) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 3rd to 40 amino acids residue；
(c2) (c1) through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and had phase
The polypeptide fragment of congenerous；
Described intein-N fragment is following (c3) or (c4):
(c3) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 354th to 476 amino acids residue；
(c4) (c3) through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and had phase
The polypeptide fragment of congenerous；
Described cyclisation protein fragments is following (c5) or (c6) or (c7) or (c8):
(c5) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 51st to 345 amino acids residue；
(c6) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 43rd to 351 amino acids residue；
(c7) polypeptide fragment that in sequence table, sequence 3 forms from N-terminal the 41st to 353 amino acids residue；
(c8) by (c5) or (c6) or (c7) through one or several amino acid residue replacement and/or disappearance and
/ or add and have the polypeptide fragment of identical function.
5. the gene of albumen described in coding claim 4.
6. contain the recombinant vector of gene, expression cassette, transgenic cell line or restructuring described in Claims 2 or 3 or 5
Bacterium.
7. recombinant vector as claimed in claim 6, it is characterised in that: described recombinant vector is to insert in expression vector
Enter the recombiant plasmid of the DNA molecular shown in sequence 4 with sequence table that exogenous DNA molecule is formed.
8. prepare a method of protein described in claim 1, comprise the steps: to cultivate recombinant bacterium, weighed
Profit requires protein described in 1；Described recombinant bacterium imports recombinant vector described in claim 7 in Host Strains and obtains
Recombinant bacterium.
9. the application in preparing product of the protein described in claim 1；The function of described product be (f1) and/or
(f2):
(f1) prevent and/or treat radiation damage；
(f2) activation NF-κ B signal path.
10. a product, its active component is protein described in claim 1；The function of described product is (f1)
And/or (f2):
(f1) prevent and/or treat radiation damage；
(f2) activation NF-κ B signal path.
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| Application Number | Priority Date | Filing Date | Title |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113993538A (en) * | 2019-03-28 | 2022-01-28 | 加图立大学校产学协力团 | Composition for preventing or treating graft-versus-host disease comprising flagellin-derived TLR5 agonist as active ingredient |
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2016
- 2016-03-31 CN CN201610196102.4A patent/CN105820218A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 叶丙雨: "CBLB502衍生物的构建及活性研究", 《万方学位论文全文数据库》 * |
| 赵仲麟等: "内含肽介导的蛋白环化研究进展", 《中国农业科技导报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113993538A (en) * | 2019-03-28 | 2022-01-28 | 加图立大学校产学协力团 | Composition for preventing or treating graft-versus-host disease comprising flagellin-derived TLR5 agonist as active ingredient |
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