CN107022557A - New death receptor 5 antibody fusion protein and its application - Google Patents
New death receptor 5 antibody fusion protein and its application Download PDFInfo
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Abstract
The present invention relates to a kind of amino acid sequence such as SEQ ID NO：SDR5 Fc recombinant proteins (ZJ501 5) and its genes of SEQ ID NO shown in 2：1, and disclose the effect of the sDR5 Fc recombinant proteins and its gene in the medicine for preparing treatment immunological liver diseases.And the bioactivity of the sDR5 Fc products of the biological activity ratio R＆D systems companies of sDR5 Fc albumen of the present invention in vitro is high 3 times, the ratio of N-terminal shearing variant only has 1% or so, protein drug stability is high, sDR5 Fc albumen of the present invention can significantly improve the survival rate of Con A inducing acute oneself immunity hepatitis mouse, have potential application value to treatment human autoimmune's property hepatopathy.
Description
Technical field
The present invention relates to recombinant protein, more particularly to a kind of new death receptor 5 antibody fusion protein and its application.
Background technology
Counted according to the World Health Organization, the ratio of global hepatitis and healthy population is about 1/12 at this stage, i.e., every 12
There may be a hepatitis among personal, this ratio is nearly ten times of HIV infection crowd.At present, countries in the world
The hepatitis such as hepatitis B, hepatitis account for the 1/12 of population in the world close to 500,000,000.China is the most country of hepatitis patient, is often close on 30
Therefore ten thousand people die from liver related disease, such as hepatic sclerosis, liver cancer.It is global that having more than 500,000 people dies from primary carcinoma of liver every year,
And wherein up to 80% primary carcinoma of liver is as caused by chronic hepatitis.In 400,000,000 chronic hepatitis patients, there is 75% to live in
Asia, and China is global hepatitis and liver cancer burden most heavy country, China reaches for the costly for the treatment of hepatitis B every year
More than 100000000000.
The illness performance that hepatitis and cardiovascular and cerebrovascular disease develop is not quite similar but has common feature, returns thorough investigation
Bottom, its committed step is directed to the rapid apoptosis of liver cell, cardiac muscle cell or brain cell.Apoptosis is due to that cell receives born of the same parents
Cell active death process caused by Apoptosis mechanism in outer dead signal, active cell, to maintain human internal environment stable
Institute is required, with important biological action.But when cardiovascular and cerebrovascular disease or hepatitis occur, multiple stressor is such as
The active cell apoptotic signal such as cell factor, oxidative stress and DNA damage, causes substantial amounts of cardiac muscle cell, brain cell or liver thin
Born of the same parents are dead.Apoptosis is the major way that myocardial infarction, cerebral infarction and hepatitis cause cell death, and is considered as these diseases
The fundamental mechanism that disease develops, its correlation molecule also turns into the crucial target spot for the treatment of myocardial infarction, cerebral infarction and hepatitis.Suppress
Apoptosis can effectively prevent the development of hepatitis or cardiovascular and cerebrovascular disease.
Death receptor 5 (Death receptor, DR5) is the member of TNF (TNF) receptor family, and it is swollen
The spy of tumor necrosis factor apoptosis induction ligand related (TNF-related apoptosis-inducing ligand, TRAIL)
The opposite sex, high-affinity receptor, in normal cell surface low expression, the high expression in inflammation, ischemic or the tissue of canceration.Total length
DR5 contains 411 amino acid, belongs to I type transmembrane glycoprotein.DR5 can effectively activate intracellular signal transduction way after being combined with TRAIL
Footpath, induced apoptosis reaction.It is thin that Apoptosis (Fig. 1) and myocardial infarction, cerebral infarction and the hepatitis of DR5 mediations are induced
Born of the same parents' death is closely related, and it can be used as cardiovascular and cerebrovascular disease and the co-drug target spot of hepatitis
SDR5 (soluble death receptor 5, soluble DR5, abbreviation sDR5) is a kind of soluble protein, mainly by DR5
Film outskirt composition, size about 26kD.Human soluble Death receptor 5 Protein is by the DR5 competition binding TRAIL with cell membrane surface, so as to block
Or the signal path that closing TRAIL is combined with DR5, functioning cell apoptosis is reduced, mitigates functioning cell damage, to reach preventer
Official's exhaustion and the effect of patient death.In addition, sDR5 is due to being human body oneself protein, with small toxicity, non-immunogenicity it is excellent
Point, the potential quality of great " wide spectrum " medicine as diseases such as myocardial infarction, cerebral infarction and hepatitis.
In the prior art, the recombined human DR5-Fc fusion proteins (research reagents) that R＆D systems companies are sold are in connection
Part is how several different from the amino acid of the other fusion protein medicines listed, and activity is substantially low；Stability is also poor, finds
The N-terminal for having different proportion shears the presence of variant.These bring fiber crops to the process exploitation in later stage and the formulation of quality standard
It is tired, it is not suitable for declaring as biological new drug.
At present, listing is at home and abroad showed no as medicine sDR5-Fc albumen, belongs to a kind new medicine.Do not have also in the world
Any a company can GMP volume productions sDR5-Fc albumen, only Sigma (article No.s on a large scale：D9563, has stopped production), Enzo life
Sciences (article No.s：ALX-522-005), EXBIO antibodies (article No.s：EXB0007), Abcam (article No.s：AB83547)
With R＆D systems (article No.s：631-T2) totally 5 production sDR5-Fc albumen are as biochemical reagents, the DR5 used in this 5 kinds of commodity
Gene order is not quite similar, and engineering cell species used is also variant, suppresses the Apoptosis ability ratio of trail protein induction
It is poor, and stability is poor, and the N-terminal for having different proportion shears the presence of variant, and these shortcomings give the process exploitation in later stage
Formulation with quality standard makes troubles.
The content of the invention
Based on this, the present invention provides a kind of new DR5-Fc recombinant proteins, and the DR5-Fc recombinant proteins bioactivity is high, surely
It is qualitative good.
The present invention provides a kind of nucleotide sequence of encoding D R5-Fc recombinant proteins, and it has：
a)SEQ ID NO：Base sequence described in 1；Or
B) with SEQ ID NO：1 complementary pairing sequence；Or
C) with a or b nucleotide sequence coded mutually homotactic protein, but because of the degeneracy of genetic code with a or b
The different sequence of nucleotide sequence.
The present invention also provides one kind by above-mentioned nucleotide sequence (SEQ ID NO：1) the DR5-Fc recombinant proteins of coding is more
Peptide, it has SEQ ID NO：Amino acid sequence shown in 2, or amino acid sequence such as SEQ ID NO：Shown in 2, and amino acid has
One or more substitutions, but the immovable DR5-Fc recombinant proteins of bioactivity.
It is a further object of the present invention to provide the application of above-mentioned DR5-Fc recombinant proteins and its coding gene sequence.
Concrete technical scheme is as follows.
Above-mentioned DR5-Fc recombinant proteins are applied in the medicine for preparing treatment oneself immunity hepatitis.
Above-mentioned nucleotides sequence is listed in the medicine for preparing treatment oneself immunity hepatitis and applied.
The present invention is also provided and removed and SEQ ID NO：2 belong to the DR5-Fc recombinant proteins of the different base compositions of same sequence
And its application.
Concrete technical scheme is as follows.
SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5 or SEQ ID NO：Amino acid sequence shown in 6
DR5-Fc recombinant proteins.
Above-mentioned DR5-Fc recombinant proteins are applied in the medicine for preparing treatment oneself immunity hepatitis.
Research of the present invention by inventor for a long time, reselects protein gene sequence, and realize that sDR5-Fc albumen is (special
It is not ZJ501-5) high yield (primary cell colony screening yield has reached 0.5g~1g/L) in Chinese hamster ovary celI, and the present invention
The sDR5- of the biological activity ratio R＆D systems companies of described sDR5-Fc recombinant proteins (particularly ZJ501-5) in vitro
The bioactivity of Fc products is high 3 times, and the ratio of N-terminal shearing variant only has less than 1%, and protein drug stability is high, the present invention
Described sDR5-Fc recombinant proteins (particularly ZJ501-5) can significantly improve Con A inducing acute oneself immunity hepatitis mouse
Survival rate, to treatment human autoimmune's property hepatopathy there is potential application value.
Brief description of the drawings
Fig. 1 is sDR5-Fc gene agarose gel electrophoresis figure.
Fig. 2 is that DR5-Fc recombinant proteins (ZJ501-5) DotBlot detects figure.
Fig. 3 is the non-reduced electrophoretograms of 8%SDS-PAGE.
Fig. 4 is the SEC-HPLC analysis charts of ZJ501-5 after purification.
Fig. 5 is the TIC figures of ZJ501-5 after carrying out reduction.
Fig. 6 is ZJ501-5 mass spectrogram of the data after Biopharmalynx software processings.
Fig. 7 is commercialization Trail killing activity examination criteria curves.
Fig. 8 is commercialization DR5-Fc fusion protein biological detection standard curves.
Fig. 9 is ZJ501-5 bioactivity examination criteria curves.
Figure 10 is mouse liver injury model experiment ALT, AST liver function test result schematic diagram.
Figure 11 is mouse liver injury model experiment ALT, AST liver function test result schematic diagram.
Figure 12 is mouse liver injury model experiment HE stain control group result schematic diagrams.
Figure 13 is that mouse liver injury model experiment HE dyes sDR5-Fc result schematic diagrams.
Figure 14 is hepatic tissue section TUNEL coloration result schematic diagrames.Wherein A is ConA model group murine liver tissues TUNEL
Coloration result；B is DAPI coloration results；C is that 9mg/kg sDR5-Fc (501-5) treat murine liver tissue TUNEL coloration results,
D is DAPI coloration results.
Figure 15 is hepatic tissue section TRAIL immunohistochemical staining result schematic diagrams.Wherein, A figures are ConA model group mouse
Hepatic tissue TRAIL immunohistochemical staining results, show that the positive lymphocytes of a large amount of TRAIL are raised and arrive liver organization, so that
Trigger the hepatocellular apoptosis of TRAIL-DR5 mediations；B figures are that 9mg/kg sDR5-Fc (501-5) treatments murine liver tissue TRAIL exempts from
Epidemic disease histochemical staining result, is shown to be raised due to lymphocyte positive few TRAIL and arrives liver organization, therefore the liver triggered is thin
Born of the same parents' apoptosis is also few.
Figure 16 is the testing result schematic diagram of serum alt level.
Figure 17 is the testing result schematic diagram of AST levels in serum.
Figure 18 is the testing result schematic diagram of Human Serum Albumin level.
Figure 19 is the survivorship curve schematic diagram of mouse.
Embodiment
For the ease of understanding the present invention, the present invention will be described more fully below.The present invention can be with many not
With form realize, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is made to this
The understanding of the disclosure of invention is more thorough comprehensive.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook
People, molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) in institute
The condition stated, or according to the condition proposed by manufacturer.Used various conventional chemical reagent, are commercially available in embodiment
Product.
Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention
The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose of example is applied, the limitation present invention is not used in.Term "and/or" used in the present invention includes one or more related listed
The arbitrary and all combination of project.
The design and restructuring of one restructuring DR5-Fc expressed sequences
Inventor passes through long-term experience accumulation, and people DR5 is carried out merging for various ways by construction of fusion protein with Fc,
Mass spectrometry results show that destination protein there occurs the shearing of 11 amino acid (ITQQDLAPQQR) of N-terminal mostly, in order to give expression to
N-terminal more homogeneous destination protein, finally screens fusion protein convectional signals peptide basis and gets on to fall middle catenation sequence and N-terminal
18 amino acid, are named as sDR5-Fc (ZJ501-5).Plasmid is transiently transfected, expression supernatant carries out mass spectrum point after purification
Analysis and activity analysis.
ZJ501-5 DNA sequence dna：
ATGGGTGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTCCTGTTTCCAAGCATGGCGAGCATGtc
cagcccctcagagggattgtgtccacctggacaccatatctcagaagacggtagagattgcatctcctgcaaatatg
gacaggactatagcactcactggaatgacctccttttctgcttgcgctgcaccaggtgtgattcaggtgaagtggag
ctaagtccctgcaccacgaccagaaacacagtgtgtcagtgcgaagaaggcaccttccgggaagaagattctcctga
gatgtgccggaagtgccgcacagggtgtcccagagggatggtcaaggtcggtgattgtacaccctggagtgacatcg
aatgtgtccacaaagaagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctg
gggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatg
cgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatg
ccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggac
tggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaa
agccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtca
gcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaac
aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagag
caggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcc
tctccctgtctccgggtaaatga(SEQ ID NO.1)
ZJ501-5 amino acid sequence
MGVLLTQRTLLSLVLALLFPSMASMSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVE
LSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO.2)
SDR5-Fc (ZJ501-5) gene order is connected to by pL101 by conventional technical means such as digestion, connections true
Nuclear expression carrier is simultaneously identified with Hind3 and EcoR1 double digestions.The visible 1300bp of agarose gel electrophoresis or so purpose fragment
And 9kb or so pL101 carrier segments, double digestion the result after vector construction, as a result see Fig. 1.
The expression of two DR5-Fc recombinant proteins and the detection of physicochemical property
SDR5-Fc/pL101 plasmids are carried out with Lipofectamine2000 to transiently transfect CHO.K1 cells, after 48 hours
Collect supernatant and carry out DotBlot detections, be as a result the positive, see Fig. 2.
Recovery CHO.K1-S cells, by 5 × 105After/ml 10ml are resuspended, FreeStyle is usedTM CHO
ExpressionMedium serum free mediums, and final concentration 8mmol/L Glutamine cultures are added, it is placed in 37 DEG C 8%
CO2 shaken cultivation casees 120rpm is cultivated.Work as cell number>1×106Simultaneously liquid feeding, to 30ml, maintains cell number 2- for passage during/ml
5×105/ ml, subsequently passes on and maintains density to 2-5 × 10 every time5/ ml, CHO.K1-S need to be passed on more than three times.
The day before transfection, shaking flask adjusts cell density to 5-6 × 10 after counting5/ ml 100ml, are placed in 37 DEG C of 8%CO2 and shake
Swing incubator 120rpm to be cultivated, next day can be transfected.
Transfection same day CHO.K1-S is counted and is calculated motility rate, and density should be in 1.2-1.5 × 106/ ml, motility rate>95%.Adjustment
Density is 1 × 106It is standby in/ml, 30ml/ parts of addition 100ml shaking flasks.
Gently overturn and mix FreeStyleTMMAX Reagent tetra- times, take 37.5ul FreeStyleTM MAX
Reagent adds 0.6ml OPTIproTMIn SFM dilutions, while taking 37.5ug plasmids to add 0.6ml OPTIproTM SFM
In dilution, both are well mixed, 10-20min is stored at room temperature.
Mixture after incubation is added in above-mentioned ready CHO.K1-S cells shaking flask, 37 DEG C of 8%CO2 shaken cultivations
Case 120rpm is cultivated 7 days, and product carries out SDS-PAGE, SEC-HPLC and mass spectral analysis after purification through affinity column.
Mass spectrometry procedure：
1. first by sample displacement buffer solution to 50mMNH4FA (pH6.6), and detect its concentration.
2. each sample carries out digestion with Ides；
3. add DTT Reduction of Disulfide
4. mass spectrum sample introduction is analyzed
Referring to SDS-PAGE (Fig. 3), ZJ501-5 SEC-HPLC (Fig. 4) (purity of protein reaches 99.47%) after purification and
Mass spectrum (figure of ZJ501-5 TIC (Fig. 5) after carrying out reduction and the ZJ501-5 data after Biopharmalynx software processings
6).According to Fig. 5 and Fig. 6, N-terminal spliced body ratio is analyzed, referring to following table, wherein, M (O) represents corresponding albumen n end and has more one
Methionine is simultaneously oxidized, and S-S represents to form disulfide bond.O represents first amino acid and is oxidized.And Fig. 5, Fig. 6 import data
Storehouse post analysis draw the result of a N-terminal amino acid that subtracts one and N-terminal an amino acid that subtracts two.
N-terminal spliced body ratio such as following table
As a result show, ZJ501-5 purity reaches 99%, and only micro N-terminal shears an amino acid and two amino
The variant of acid occurs, and ratio is 1.11%, is deposited wherein only about 0.11% or so N-terminal shears two Amino acid variants
.
ZJ501-5 is the ZJ501-2 in ZJ501-1, ZJ501-3, on the basis of ZJ501-4 albumen, removes N-terminal unstable
Sequence, the N-terminal finally obtained more homogeneous destination protein.ZJ501-1, ZJ501-2, ZJ501-3, ZJ501-4, ZJ501-5
The N-terminal spliced body ratio of albumen see the table below.
| Sample ID | Temperature | N- ends spliced body % |
| ZJ501-1 | 37℃ | 31.35 |
| ZJ501-2 | 37℃ | 34.72 |
| ZJ501-3 | 37℃ | 48.69 |
| ZJ501-4 | 37℃ | 51.66 |
| ZJ501-5 | 37℃ | 1.11 |
From the foregoing, it will be observed that ZJ501-5 protein drug stability highests.
The sequence of ZJ501-1, ZJ501-2, ZJ501-3, ZJ501-4 albumen is as follows：
ZJ501-1
MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPS
EGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCR
KCRTGCPRGMVKVGDCTPWSDIECVHKEGSSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.3)
ZJ501-2
MEQRGQNAPAASGARKRHGPGPREARGARPGPRVPKTLVLVVAAVLLLVSAESALITQQDLAPQQRAAPQQKRSSPS
EGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCR
KCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.4)
ZJ501-3
MGVLLTQRTLLSLVLALLFPSMASMITQQDLAPQQRAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHW
NDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO.5)
ZJ501-4 amino acid sequences (signal peptide-aim sequence)
MGVLLTQRTLLSLVLALLFPSMASMAAPQQKRSSPSEGLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTR
CDSGEVELSPCTTTRNTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKEEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ
IDNO.6)。
Three ZJ501-5 Biological Activity Identification
1. set up commercialization Trail killing activity detection methods
The Jurkat cell of exponential phase is collected, cell is resuspended with 10%FCS RPMI-1640/DMEM after counting, is adjusted
Whole cell density 8*104/ ml, is added in 96 porocyte culture plates by 100ul/ holes, is placed in 37 DEG C of 8% CO2gas incubator
Middle culture 20-24 hours.
Commercialization Trail is resuspended in the 0.03ug/ml's of final concentration containing actinomycin D by final concentration 500-1000ng/ml
In above-mentioned complete culture solution, with the multiple proportions commercialization Trail of complete culture solution 2 containing actinomycin D, common 15-20 concentration gradient.
Sample after dilution is added in 96 porocyte culture plates by 100ul/ holes, is placed in training in 37 DEG C of 8% CO2gas incubator
Support 18-22 hours.Add the 20 of Fresh:The MTS/PMS chromophoric solution 20ul/ holes of 1 mixing, 37 DEG C of 8% carbon dioxide training
Support and continue to be incubated culture 3-4 hours (not needing lid lid) in case, its A490-A630 value is detected with MS ELIASAs.
Using M5 analysis software fit standard curves：Abscissa is the concentration of sample, and ordinate is A490-A630, from 4
Parametric equation regression model, curve is reversed S-shape.
1.1 commercialization Trail killing activity testing results
Using M5 analysis software fit standard curves：Abscissa is the concentration of sample, and ordinate is A490-A630, from 4
Parametric equation regression model, curve is reversed S-shape.Software calculates EC50 for 8.382ng/ml automatically, and EC90 is 27.65ng/ml.
(referring to Fig. 7).
2. set up commercialization DR5-Fc fusion protein bioactivity detection methods
The Jurkat cell of exponential phase is collected, cell is resuspended with 10%FCS RPMI-1640/DMEM after counting, is adjusted
Whole cell density 8*104/ ml, is added in 96 porocyte culture plates by 100ul/ holes, is placed in 37 DEG C of 8% CO2gas incubator
Middle culture 20-24 hours.
Calculate commercialization Trail killing activities EC90, with the 0.03ug/ml of final concentration containing actinomycin D it is above-mentioned completely
Final concentration of EC90 Trail concentration is added in nutrient solution, with the above-mentioned Trail complete culture solutions containing actinomycin D and EC90
Serial multiple proportions commercialization DR5-Fc (R＆D systems companies), 10ng/ml 2 multiple proportions, 15 concentration.Sample after dilution is pressed
100ul/ holes are added in 96 porocyte culture plates, are placed in cultivating 18-22 hours in 37 DEG C of 8% carbon dioxide culture medium.
Add the 20 of Fresh:The MTS/PMS chromophoric solution 20ul/ holes of 1 mixing, 37 DEG C of 8% CO2gas incubator
It is middle to continue to be incubated culture 3-4 hours (not needing lid lid).Its A490-A630 value is detected with MS ELIASAs.
Using M5 analysis software fit standard curves：Abscissa is the concentration of sample, and ordinate is A490-A630, from 4
Parametric equation regression model, curve is positive serpentine.Three different experiments are carried out, compare EC50 variation situation.
2.1 commercialization DR5-Fc fusion proteins (R＆D systems companies) bioactivity testing result analyzes soft using M5
Part fit standard curve：Abscissa is the concentration of sample, and ordinate is A490-A630, from 4 parametric equation regression models, bent
Line is positive serpentine.Three different experiments are carried out, compare EC50 variation situation.EC50 is 71.82ng/ml.Referring to Fig. 8.
3ZJ501-5 bioactivity and relative activity detection method
Method is with 2, and sample is ZJ501-5 (SEQ ID NO.2), and reference product is that (R＆D systems are public by commercialization DR5-Fc
Department).Test and carry out three experiments respectively for two different operating persons, calculate experiment ZJ501-5 Relative biological activities (%) every time
=commercialization DR5-Fc EC50/ sample DR5-Fc EC50*100.The variation situation for comparing different operating between person and experimentai batches.
3.1 ZJ501-5 bioactivity detections of the present invention and relative activity result
ZJ501-5EC50 average out to 20.25ng/ml, commercialization DR5-Fc EC50 are 71.82ng/ml, and ZJ501-5 is relative
Bioactivity is 355%.(referring to Fig. 9).
Four sDR5-Fc treatments oneself immunity hepatitis, virus hepatitis.
The mouse liver injury model of Con-A inductions, its pathologic process and the pathology of a variety of acute and chronic hepatitis known today
Process is similar, particularly its pathological characteristic that specific hepatic lesion is induced by activated T lymphocytes, can be very good simulation
The pathogenic course of human autoimmune's property hepatopathy, virus hepatitis.
1. 36 male C57BL/6 mouse, 6~8 weeks, are randomly divided into 6 groups, every group 6.Model control group, sDR5-Fc
Treatment group (27mg/kg, 9mg/kg, 3mg/kg and 1mg/kg), positive controls (silibinin27mg/kg).
2. PBS, sDR5-Fc (ZJ501-5 of the present invention) 27mg/kg, sDR5-Fc9mg/ is injected intraperitoneally in every mouse respectively
Kg, sDR5-Fc 3mg/kg, sDR5-Fc 1mg/kg, silibinin 27mg/kg, volume injected is 20ml/kg
3. after 1 hour, the equal tail vein injection Con A 17mg/kg of every mouse, volume injected is 10ml/kg, in ConA
8h, 24h distinguish tail vein blood after injection, mouse and Culling heart blood are put to death in 48h CO2, after collection liver is fixed through 4%PFA
FFPE, section, HE dyeing.Use DeadEnd Fluorometric TUNEL System (Promega, G3250) reagent
Box, carries out hepatic tissue section TUNEL dyeing.Hepatic tissue section is carried out using TRAIL antibody (Santa Cruz, sc-6079) to exempt from
Epidemic disease histochemical staining.Mouse blood room temperature places 1h, 3000rpm, 4 degree of centrifugation 10min, separates upper serum, is built up using Nanjing
Biotech firm's ALT, AST detection kit carries out ALT, AST Liver function grade.
Referring to Figure 10-13.Figure 10 and Figure 11 show that intraperitoneal injection 9mg/kg sDR5-Fc can treat the mouse of ConA inductions
Oxyhepatitis, is mainly shown as that sDR5-Fc can be remarkably decreased serum aminotransferase levels at commencement.
After Figure 12 and Figure 14 (above arranging 2 figures) displays, mouse injection ConA 48h, it is dead that large area liver cell occurs in liver organization
Die.Figure 13 and Figure 14 (2 figures of lower row) display, mouse peritoneal injection 9mg/kg sDR5-Fc, then receive after ConA inductions 48h, absolutely
Most of liver cell survival, apoptosis liver cell is seldom, hepatic tissue structural integrity.After Figure 15 display mouse mainlines ConA, greatly
Measure the positive lymphocytes of TRAIL to be raised to liver organization, so as to trigger a large amount of apoptosis of liver cell that TRAIL-DR5 is mediated；
And after 9mg/kg sDR5-Fc (501-5) treatment mouse, because sDR5-Fc blocks TRAIL to be combined with the DR5 of surface of hepatocytes,
Lymphocyte positive few TRAIL, which is raised, arrives liver organization, therefore the hepatocellular apoptosis triggered is also seldom.
Experiment conclusion：SDR5-Fc of the present invention can substantially suppress the rise of serum transaminase in oxyhepatitis mouse model, suppression
The pathological change of liver processed, plays obvious liver protective effect.In Dose-response experiment, 9mg/kg sDR5-Fc is injected intraperitoneally
For optimal dosage.
4. 56 male C57BL/6 mouse, 6~8 weeks, are randomly divided into 7 groups, every group 8.Blank control group, model comparison
Group, model administration group (give 1.~5. number gene order sDR5-Fc albumen, are followed successively by SEQ IDNO.3-SEQ ID respectively
NO.6, SEQ ID NO.2,10mg/kg).
5. every mouse difference tail vein injection PBS, sDR5-Fc 1.~5. 10mg/kg, volume injected is 10ml/
kg。
6. after 1 hour, the equal tail vein injection ConA 15mg/kg of every mouse, volume injected is 10ml/kg, in ConA
Venous blood collection under the equal jaw of every mouse of 8h after injection, mouse blood room temperature is placed in 1h, 3000rpm, 4 degree of centrifugation 10min, separation
Layer serum, builds up biotech firm ALT, AST, albumin detection reagent box using Nanjing and carries out serum alt, AST and albumin
The detection of level.Referring to Figure 16, Figure 17 and Figure 18.
Experiment conclusion：The sDR5-Fc albumen energy of the relatively other gene orders of sDR5-Fc (ZJ501-5) of the present invention
Become apparent from reducing the transaminase level (such as Figure 16 and 17) of ConA inducing mouse hepatitis, under same dose, ZJ501-5 sequences
SDR5-Fc albumen has more preferable curative effect, shows as liver function more preferably, can synthesize more seralbumins and be secreted into blood
(such as Figure 18).
7. 21 male C57BL/6 mouse, 6~8 weeks, are randomly divided into 3 groups, every group 7.Model control group, 2. number gene
Sequence sDR5-Fc (ZJ501-2) treatment group 32.2mg/kg, 5. number gene order sDR5-Fc (ZJ501-5) treatment group 21.6mg/
kg.Every mouse difference tail vein injection PBS, sDR5-Fc 2. 32.2mg/kg, sDR5-Fc 5. 21.6mg/kg, volume injected are equal
For 20ml/kg.After 1 hour, the equal tail vein injection ConA 15mg/kg of every mouse, volume injected is 10ml/kg.In Con A
The survival condition of mouse is observed after injection in 48h, survivorship curve is drawn.As a result referring to Figure 19.
Experiment conclusion：SDR5-Fc (ZJ501-5) of the present invention can significantly improve ConA inducing acute LADAs
The survival rate of hepatitis mice, and ZJ501-5 gene orders sDR5-Fc albumen under 21.6mg/kg dosage, curative effect is better than
Dosage is the sDR5-Fc albumen of 32.2mg/kg ZJ501-2 gene orders.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (6)
1. a kind of nucleotide sequence of encoding D R5-Fc recombinant proteins, it is characterised in that it has：
a)SEQ ID NO：Base sequence described in 1；Or
B) with SEQ ID NO：1 complementary pairing sequence；Or
C) with a or b nucleotide sequence coded mutually homotactic protein, but because of the degeneracy of genetic code with a or b core
The different sequence of nucleotide sequence.
2.SEQ ID NO：The DR5-Fc recombinant proteins of amino acid sequence shown in 2, or amino acid sequence such as SEQ ID NO：2 institutes
Show, and amino acid has one or more substitutions, but the immovable DR5-Fc recombinant proteins of bioactivity.
3. the DR5-Fc recombinant proteins described in claim 2 are applied in the medicine for preparing treatment oneself immunity hepatitis.
4. the nucleotides sequence described in claim 1 is listed in the medicine for preparing treatment oneself immunity hepatitis and applied.
5.SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5 or SEQ ID NO：The DR5- of amino acid sequence shown in 6
Fc recombinant proteins.
6. the DR5-Fc recombinant proteins described in claim 5 are applied in the medicine for preparing treatment oneself immunity hepatitis.
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| CN109954136A (en) * | 2017-12-14 | 2019-07-02 | 深圳市中科艾深医药有限公司 | Application of the people sDR5-Fc recombination fusion protein as Treatment of Cerebral Stroke drug |
| CN109954131A (en) * | 2017-12-14 | 2019-07-02 | 深圳市中科艾深医药有限公司 | A kind of application of tumor necrosin relative death inducing ligand antagonist as septicopyemia therapeutic agent |
| CN111450232A (en) * | 2019-01-21 | 2020-07-28 | 中国科学院深圳先进技术研究院 | Application of fusion protein in preparation of medicine for treating hepatitis C |
| CN112294945A (en) * | 2019-08-01 | 2021-02-02 | 深圳市中科艾深医药有限公司 | Application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating myocardial infarction and ischemia-reperfusion injury |
| CN112386686A (en) * | 2019-08-14 | 2021-02-23 | 深圳市中科艾深医药有限公司 | Application of human sDR5-Fc recombinant fusion protein in preparation of acute kidney injury prevention and treatment drugs |
| CN112439052A (en) * | 2019-08-27 | 2021-03-05 | 深圳市中科艾深医药有限公司 | Application of human sDR5-Fc recombinant fusion protein in preparation of medicine for preventing and treating liver ischemia-reperfusion injury |
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|---|---|---|---|---|
| CN109954136A (en) * | 2017-12-14 | 2019-07-02 | 深圳市中科艾深医药有限公司 | Application of the people sDR5-Fc recombination fusion protein as Treatment of Cerebral Stroke drug |
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| CN111450232A (en) * | 2019-01-21 | 2020-07-28 | 中国科学院深圳先进技术研究院 | Application of fusion protein in preparation of medicine for treating hepatitis C |
| CN111450232B (en) * | 2019-01-21 | 2023-08-01 | 中国科学院深圳先进技术研究院 | Application of fusion protein in preparation of medicine for treating hepatitis C |
| CN112294945A (en) * | 2019-08-01 | 2021-02-02 | 深圳市中科艾深医药有限公司 | Application of human sDR5-Fc recombinant fusion protein in preparation of drugs for preventing and treating myocardial infarction and ischemia-reperfusion injury |
| CN112386686A (en) * | 2019-08-14 | 2021-02-23 | 深圳市中科艾深医药有限公司 | Application of human sDR5-Fc recombinant fusion protein in preparation of acute kidney injury prevention and treatment drugs |
| CN112439052A (en) * | 2019-08-27 | 2021-03-05 | 深圳市中科艾深医药有限公司 | Application of human sDR5-Fc recombinant fusion protein in preparation of medicine for preventing and treating liver ischemia-reperfusion injury |
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