CN1513993A - Coding gene sequence of chinese genom cDNA library interleukin 21 and its amino acid sequence of protein - Google Patents
Coding gene sequence of chinese genom cDNA library interleukin 21 and its amino acid sequence of protein Download PDFInfo
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- CN1513993A CN1513993A CNA031230636A CN03123063A CN1513993A CN 1513993 A CN1513993 A CN 1513993A CN A031230636 A CNA031230636 A CN A031230636A CN 03123063 A CN03123063 A CN 03123063A CN 1513993 A CN1513993 A CN 1513993A
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Abstract
A coding gene sequence of interleukin 21 in Chinese genome cDNA library and the amino acid sequence of its protein are disclosed. Said coding gene can obtain recombinant expression in eucaryotic or procaryotic expression system by gene recombination. The process for preparing its expression product is also disclosed. The 51Cr release experiment is first used to prove interleukin 21 activating action to NK cells, so it can be used as genetically engineered medicine for treating tumor and viral infection.
Description
Technical field
The present invention relates to a gene and a cDNA sequence thereof by the cytokine with potential medicinal use of Chinese's genome cDNA library coding, and the chemical primary structure sequence of the interleukin II 1 that sequence determined thus, belong to the medical bioengineering technical field.
Background technology
Human interleukin is that the class that the human inner cell produces has the active proteinic general name of various biological, and wherein human interleukin 2, human interleukin 6 are as the drug use of immunomodulatory class.Human interleukin 21 is a kind of protein in its family.Human interleukin 21 has the various biological activity, mainly show the immune NK cell of activating human body, the specificity of enhancing body and non-specific immunity function, thereby bring into play effects such as antitumor, anti-virus infection and immunomodulatory, so human interleukin 21 is a kind of newtype drugs that better DEVELOPMENT PROSPECT is arranged.
Reported sequence in the world from the genomic interleukin II 1 of American.The encoding gene of separation in present Chinese's genome cDNA library of still having no way of, clones coding interleukin II 1, and express through gene recombination technology external, thereby obtain the report of the recombinant interleukin element 21 of Chinese's genome cDNA library coding, also do not have the relevant report of relevant recombinant interleukin 21 biologic activity.
Summary of the invention
The coding gene sequence that the purpose of this invention is to provide Chinese's genome cDNA library interleukin II 1.
Another object of the present invention provides the proteinic aminoacid sequence of Chinese's genome cDNA library interleukin II 1.
The 3rd purpose of the present invention provides the gene engineering expression product preparation method of Chinese's genome cDNA library interleukin II 1, uses first in the world
51The Cr release test proves the interleukin II 1 of this genetically engineered reorganization in external activation to the NK cell, and the result confirms that the interleukin II 1 of this genetically engineered reorganization has immunoregulatory activity preferably, can be used as immunomodulatory genoid engineering medicine.
For achieving the above object, the present invention is by the following technical solutions:
The coding gene sequence of Chinese's genome cDNA library interleukin II 1, this sequence is as follows:
gctgaagtgaaaacgagaccaaggtctagctctactgttggtacttatgagatccagtcctggcaacatggaga
gggttgtcatctgtctgatggtcatcttcttggggacactggtccacaaatcaagctcccaaggtcaagatcgc
cacatgattagaatgcgtcaacttatagatattgttgatcagctgcataattatgtgaatgacttggtccctga
atttctgccagctccagaagatgtagagacaaactgtgagtggtcagctttttcctgttttcagaaggcccaac
taaagtcagcaaatacaggaaacaatgaaaggataatcaatgtatcaattaaaaagctgaagaggaaaccacct
tccacaaatggagggagaagacagaaacacagactaacatgcccttcatgtgattcttatgagaaaaaaccacc
caaagaattcctagaaagattcaaatcacttctccaaaagatgattcatcagcatatctcctctagaacacacg
gaagtgaagattcctgaggatctaacttgcagttggacactatgttacatactctaatatagtagtgaaagtca
tttctttgtattccaagtggaggagccctattaaattatataaagaaata。
The aminoacid sequence of Chinese's genome cDNA library interleukin II 1, this sequence is as follows:
MRSSPGNMERVVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLHNYVNDLVPEFLPAPEDVETNCEWS
AFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNGGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMI
HQHISSRTHGSEDS。
The derived sequence of the coding gene sequence of Chinese's genome cDNA library interleukin II 1.
Described derived sequence be meant comprise new isolating have the gene order of same basic structure and use external DNA operative technique by base displacement or insert or the various allosteric bodies of this gene order that disappearance obtains.
The allosteric body of described Chinese's genome cDNA library interleukin II 1.
Described allosteric body is included on the aminoacid sequence basis of respective egg white matter through single or multiple amino acid whose displacements or inserts or various allosteric bodies that disappearance etc. forms.
The application that described Chinese's genome cDNA library interleukin II 1 is used to prepare immunomodulatory genoid engineering medicine.
The application that the allosteric body of described Chinese's genome cDNA library interleukin II 1 is used to prepare immunomodulatory genoid engineering medicine.
The invention has the beneficial effects as follows, according to the cDNA sequence of Chinese's genome cDNA of the present invention library interleukin II 1 encoding gene, can be from human CD
3 +Clone humans in the cDNA library of the cell encoding gene of interleukin II 1, this encoding gene passes through gene recombination technology, can in eukaryotic expression system (as yeast cell or zooblast) or prokaryotic expression system (as intestinal bacteria), obtain recombinant expressed, the invention provides the gene engineering expression product preparation method of Chinese's genome cDNA library interleukin II 1, use first in the world
51The Cr release test proves the interleukin II 1 of this genetically engineered reorganization in external activation to the NK cell, and the result confirms that the interleukin II 1 of this genetically engineered reorganization has immunoregulatory activity preferably, can be used as immunomodulatory genoid engineering medicine.Recombinant interleukin element 21 has the various biological activity in human body, can bring into play effects such as antitumor, anti-virus infection and immunomodulatory, is new genetically engineered drug.
Description of drawings
Fig. 1 is a techniqueflow chart of the present invention.
Embodiment
DNA (mRNA) sequence of Chinese's genome cDNA library interleukin II 1 encoding gene and the acquisition of aminoacid sequence
As shown in Figure 1, this operation comprises the steps:
(1) separation of CD3+ cell and activation: a Chinese man's of health tonsilla is got in operation, put soaking disinfection in 75% ethanol, in plate, clean blood with Hanks liquid, in the plate that fills the 5mlRPMI-1640 cell culture fluid, shred the tonsilla tissue and make cell suspension, by 200 purpose metal cells sieve, isolated mononuclear cell suspension.The individual cells suspension is changed in the test tube, carefully add 2ml lymphocyte separation medium (biochemical two factories in Shanghai produce) on the upper strata, behind the static 3min of room temperature, take out with the cellular layer of suction pipe with the intersection of cellular layer and lymphocyte separation medium.After washing repeatedly with Hanks liquid, it is 1 * 10 that counting makes cell suspension
7/ ml, the PHA of adding 10% places 37 ℃, 5%CO
2Harvested cell behind the 24h is hatched in cultivation.
(2) the total RNA of activatory CD3+ cell extracts: the above-mentioned cell suspension 3000rpm that obtains is centrifugal, and precipitation adds 1ml Trizol liquid, fully blows and beats to cell to dissolve room temperature placement 5-10 minute fully.Add the 0.2ml chloroform, ice bath is placed 2min.Centrifugal 15 minutes of 12000rpm changes the upper strata water in the one clean pipe over to, adds equivalent Virahol mixing, and the centrifugal 10min of 12000rpm abandons supernatant, and drying at room temperature 10min does not have the pure water dissolving of RNA enzyme with 50 μ l.
(3) encoding gene of RT-PCR amplification state human interleukin-12 1: use the ReverseTranscription System (Cat.No.A3500) of Promega company, with Olig (dT)
6For primer carries out the synthetic cDNA of reverse transcription to total RNA.Clone primer sequence according to the sequences Design of having reported in the world: (upstream P1:5`GCTGAAGTGAAAACGAG 3` from the genomic interleukin II 1 of American; Downstream: 5`TGGAATACA AAGAAATGAC3`).PCR reaction totally is 200 μ l, each 1 μ mol/L of upstream and downstream primer wherein, 4 kinds of each 0.2 μ mol/L of dNTPs, 10 * buffer, 20 μ l, 1.5mol/L Mgcl2, Taq archaeal dna polymerase 10U, cDNA template 10 μ l, fully the rearmounted DNA cloning instrument of mixing reacts.Reaction conditions is 94 ℃ of pre-sex change 4 minutes, then 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ were carried out 35 circulations in 30 seconds, last 72 ℃ were extended 5 minutes.Pcr amplification goes out interleukin II 1 encoding gene of 642 bp.
(4) make up recombinant cloning vector among the pGEM-T Easy Vector System I (Cat.No.A1360) that PCR product insertion Promega company is produced and change in the e. coli jm109, cut by restriction enzyme EcoRI enzyme and identify positive recombinant clone.Positive recombinant clone is carried out determined dna sequence (using ABIDNA automatic sequence determinator) with the T7 universal primer, obtains DNA (mRNA) sequence of Chinese's genome cDNA of the present invention library interleukin II 1 encoding gene:
gctgaagtgaaaacgagaccaaggtctagctctactgttggtacttatgagatccagtcctggcaacatggaga
gggttgtcatctgtctgatggtcatcttcttggggacactggtccacaaatcaagctcccaaggtcaagatcgc
cacatgattagaatgcgtcaacttatagatattgttgatcagctgcataattatgtgaatgacttggtccctga
atttctgccagctccagaagatgtagagacaaactgtgagtggtcagctttttcctgttttcagaaggcccaac
taaagtcagcaaatacaggaaacaatgaaaggataatcaatgtatcaattaaaaagctgaagaggaaaccacc+
tccacaaatggagggagaagacagaaacacagactaacatgcccttcatgtgattcttatgagaaaaaaccacc
caaagaattcctagaaagattcaaatcacttctccaaaagatgattcatcagcatatctcctctagaacacacg
gaagtgaagattcctgaggatctaacttgcagttggacactatgttacatactctaatatagtagtgaaagtca
tttctttgtattccaagtggaggagccctattaaattatataaagaaata
By comparing, this gene order and the coding gene sequence of having reported in the world from American's genome cDNA library interleukin II 1 have the difference of 5 bases, and its base variation and site are as shown in table 1:
The gene order of the interleukin II 1 of table 1, people's gene group relatively
The sequence of having reported in the world from the genomic interleukin II 1 of American | The coding gene sequence of Chinese's genome cDNA of the present invention library interleukin II 1 | |
The 196th bit base | ????A | ????C |
The 198th bit base | ????A | ????T |
The 392nd bit base | ????C | ????G |
The 500th bit base | ????C | ????A |
The 502nd bit base | ????G | ????C |
(5) use DNASATR software,, draw the proteinic chemical primary structure (aminoacid sequence) of human interleukin 21 according to DNA (mRNA) sequence of this Chinese's genome cDNA library interleukin II 1 encoding gene
MRSSPGNMERVVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLHNYVNDLVPEFLPAPEDVETNCEWS
AFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNGGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMI
HQHISSRTHGSEDS。
This aminoacid sequence has 3 amino acid whose variations with the amino acid whose primary sequence that the coding gene sequence of having reported in the world from American's genome cDNA library interleukin II 1 determines, its amino acid variation and site are as shown in table 2:
The amino acid whose primary sequence of the sequence decision of table 2, interleukin II 1 relatively
The amino acid whose primary sequence of the sequence decision of the genomic interleukin II 1 of American | The amino acid whose primary sequence of the coding gene sequence decision of Chinese's genome cDNA library interleukin II 1 | |
The 50th amino acids | ????Lys | ????His |
The 112nd amino acids | ????Ala | ????Gly |
The 152nd amino acids | ????Leu | ????Ile |
(6) foundation of the prokaryotic expression system of interleukin II 1:
After the pcr amplification product of interleukin II 1 and prokaryotic expression plasmid pGEX4T-2 (Promega company) cut 4h with restriction endonuclease BamHI and 37 ℃ of water-bath enzymes of EcoRI (Promega company), under the effect of T4 ligase enzyme (Promega company), connect and be built into recombinant plasmid.The pGEX4T-2 recombinant plasmid transformed host bacterium e. coli bl21 that inserts interleukin II 1 is made up prokaryotic expression system.
(7) prokaryotic expression of interleukin II 1 and preliminary purification:
The host bacterium BL21 that picking contains the pGEX4T-2 recombinant plasmid that inserts interleukin II 1 is inoculated in the 500mlLB nutrient solution and (contains 100 μ g/ml penbritins), and 37 ℃ of shaking culture are to OD
600Be about 0.2.Add inductor IPTG to final concentration 1mmol/L, 37 ℃ are continued to cultivate 4h.Collect the host bacterium of abduction delivering, add the resuspended bacterial plaque of 10ml aseptic double-distilled water after the centrifugal 5min of 6000rpm collects bacterial plaque, in ice bath, carry out the ultrasonication bacterium with the ultrasonic cell disintegration instrument, frequency is 15kHz, and each time is 1min, repeats 4 times, the centrifugal 10min of 12000rpm collects supernatant.B-PER with the production of PIERCE company
@GST Fusion Protein Purification Kit (Lot# D156592) purifying protein.The fusion rotein of gained adds thrombogen (Sigma company) cleavage of fusion proteins.
(8)
511 pair of NK cell activity of Cr release test interleukin II detects:
1), the preparation of target cell: the target cell (subculture in vitro separately cell strain K562) that the 24-48h that learns from else's experience cultivates, usefulness RPMI-1640 nutrient solution washing 1 time, the centrifugal 6min of 1000r/min.Remove supernatant,, cell count is adjusted to 4 * 10 with the resuspended back of RPMI-1640 nutrient solution counting
6/ ml.Get above-mentioned target cell 0.5ml, add 150 μ Ci
51Cr puts 37 ℃ of water-bath 90min, jolts once every 15min.With the RPMI-1640 nutrient solution washing that contains 5% foetal calf serum three times, remove free then
51Cr.Living cell counting is adjusted cell concn to 1 * 10 with the RPMI-1640 nutrient solution
5/ ml.The detection target cell
51The Cr mark rate is the 0.3cpm/ cell.
2), effector cell's preparation:, be mixed with 1 * 10 with the RPMI-1640 nutrient solution with lymphocyte layering liquid ordinary method separation of human peripheral blood mononuclear cell (PBMC)
7The cell suspension of/ml.
3), the effect of effector cell and target cell: aseptic effector cell and each 0.1ml of the target cell (E/T=100: 1) of getting, add in preceding 4 row of 24 well culture plates, 3 multiple holes of every row, in preceding 3 row, add different dilution interleukin IIs 1 respectively, every hole 5 μ l, its extent of dilution was respectively 1: 10,1: 100,1: 1000, and the 4th classifies the blank hole that does not add interleukin II 1 as, place 37 ℃, 5%CO
24h is hatched in cultivation.Set up nature to discharge control wells (0.1ml target cell+0.1ml RPMI-1640 nutrient solution) and maximum release aperture (0.1ml target cell+0.1ml 2%SDS) simultaneously, take out back each hole supernatant liquor 0.1ml of micropipet sucking-off, add in the little plastics tubing, measure the cpm value with the γ calculating instrument.
4), the result calculates: calculate according to formula
51Cr nature release rate and NK cell activity:
Detected result is:
51Cr nature release rate is 7.3%, the blank hole NK cell activity that the 4th row do not add interleukin II 1 is 34%, the NK cell activity that first row add 1: 10 interleukin II 1 is 46%, the NK cell activity that secondary series adds 1: 100 interleukin II 1 is that the NK cell activity that 42%, the three row add 1: 1000 interleukin II 1 is 37%.
Sequence table
<110〉Beijing Botaidi Biological Engineering Science ﹠. Technology Development Co., Lt
<120〉coding gene sequence and the protein thereof of Chinese's genome cDNA library interleukin II 1
Aminoacid sequence
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>642
<212>DNA
<213〉Genus Homo, and ethnic group (Homo sapiens, human)
<400>1
gctgaagtga?aaacgagacc?aaggtctagc?tctactgttg?gtacttatga?gatccagtcc??60
tggcaacatg?gagagggttg?tcatctgtct?gatggtcatc?ttcttgggga?cactggtcca??120
caaatcaagc?tcccaaggtc?aagatcgcca?catgattaga?atgcgtcaac?ttatagatat??180
tgttgatcag?ctgcataatt?atgtgaatga?cttggtccct?gaatttctgc?cagctccaga??240
agatgtagag?acaaactgtg?agtggtcagc?tttttcctgt?tttcagaagg?cccaactaaa??300
gtcagcaaat?acaggaaaca?atgaaaggat?aatcaatgta?tcaattaaaa?agctgaagag??360
gaaaccacct?tccacaaatg?gagggagaag?acagaaacac?agactaacat?gcccttcatg??420
tgattcttat?gagaaaaaac?cacccaaaga?attcctagaa?agattcaaat?cacttctcca??480
aaagatgatt?catcagcata?tctcctctag?aacacacgga?agtgaagatt?cctgaggatc??540
taacttgcag?ttggacacta?tgttacatac?tctaatatag?tagtgaaagt?catttctttg??600
tattccaagt?ggaggagccc?tattaaatta?tataaagaaa?ta?????????????????????642
<210>2
<211>162
<212>PRT
<213〉Genus Homo, and ethnic group (Homo sapiens, human)
<400>2
Met?Arg?Ser?Ser?Pro?Gly?Asn?Met?Glu?Arg?Val?Val?Ile?Cys?Leu?Met
1???????????????5???????????????????10??????????????????15
Val?Ile?Phe?Leu?Gly?Thr?Leu?Val?His?Lys?Ser?Ser?Ser?Gln?Gly?Gln
20??????????????????25??????????????????30
Asp?Arg?His?Met?Ile?Arg?Met?Arg?Gln?Leu?Ile?Asp?Ile?Val?Asp?Gln
35??????????????????40??????????????????45
Leu?His?Asn?Tyr?Val?Asn?Asp?Leu?Val?Pro?Glu?Phe?Leu?Pro?Ala?Pro
50??????????????????55??????????????????60
Glu?Asp?Val?Glu?Thr?Asn?Cys?Glu?Trp?Ser?Ala?Phe?Ser?Cys?Phe?Gln
65??????????????????70??????????????????75??????????????????80
Lys?Ala?Gln?Leu?Lys?Ser?Ala?Asn?Thr?Gly?Ash?Ash?Glu?Arg?Ile?Ile
85??????????????????90??????????????????95
Ash?Val?Ser?Ile?Lys?Lys?Leu?Lys?Arg?Lys?Pro?Pro?Ser?Thr?Asn?Gly
100?????????????????????105?????????????110
Gly?Arg?Arg?Gln?Lys?His?Arg?Leu?Thr?Cys?Pro?Ser?Cys?Asp?Ser?Tyr
115?????????????????????120?????????????125
Glu?Lys?Lys?Pro?Pro?Lys?Glu?Phe?Leu?Glu?Arg?Phe?Lys?Ser?Leu?Leu
130?????????????????????135?????????????140
Gln?Lys?Met?Ile?His?Gln?His?Ile?Ser?Ser?Arg?Thr?His?Gly?Ser?Glu
145?????????????????????150?????????????155?????????????????160
Asp?Ser
Claims (8)
1, the coding gene sequence of Chinese's genome cDNA library interleukin II 1, this sequence is as follows:
gctgaagtgaaaacgagaccaaggtctagctctactgttggtacttatgagatccagtcctggcaacatggaga
gggttgtcatctgtctgatggtcatcttcttggggacactggtccacaaatcaagctcccaaggtcaagatcgc
cacatgattagaatgcgtcaacttatagatattgttgatcagctgcataattatgtgaatgacttggtccctga
atttctgccagctccagaagatgtagagacaaactgtgagtggtcagctttttcctgttttcagaaggcccaac
taaagtcagcaaatacaggaaacaatgaaaggataatcaatgtatcaattaaaaagctgaagaggaaaccacct
tccacaaatggagggagaagacagaaacacagactaacatgcccttcatgtgattcttatgagaaaaaaccacc
caaagaattcctagaaagattcaaatcacttctccaaaagatgattcatcagcatatctcctctagaacacacg
gaagtgaagattcctgaggatctaacttgcagttggacactatgttacatactctaatatagtagtgaaagtca
tttctttgtattccaagtggaggagccctattaaattatataaagaaata。
2, the aminoacid sequence of Chinese's genome cDNA library interleukin II 1, this sequence is as follows:
MRSSPGNMERVVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLHNYVNDLV
PEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNGGRR
QKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHISSRTHGSEDS。
3, the derived sequence of the coding gene sequence of Chinese's genome cDNA according to claim 1 library interleukin II 1.
4, derived sequence according to claim 3 is characterized in that: described derived sequence be meant comprise new isolating have the gene order of same basic structure and use external DNA operative technique by base displacement or insert or the various allosteric bodies of this gene order that disappearance obtains.
5, the allosteric body of Chinese's genome cDNA according to claim 2 library interleukin II 1.
6, the allosteric body of Chinese's genome cDNA according to claim 5 library interleukin II 1 is characterized in that: described allosteric body is included on the aminoacid sequence basis of respective egg white matter through single or multiple amino acid whose displacements or inserts or various allosteric bodies that disappearance etc. forms.
7, Chinese's genome cDNA according to claim 2 library interleukin II 1 application of being used to prepare immunomodulatory genoid engineering medicine.
8, the application that is used to prepare immunomodulatory genoid engineering medicine according to the allosteric body of claim 5 or 6 described Chinese's genome cDNA library interleukin IIs 1.
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CN02159589 | 2002-12-31 | ||
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US7622561B2 (en) | 2002-12-13 | 2009-11-24 | Zymogenetics, Inc. | IL-21 production in prokaryotic hosts |
US7745176B2 (en) | 2002-12-13 | 2010-06-29 | Zymogenetics, Inc. | IL-21 production in prokaryotic hosts |
US7767199B2 (en) | 2002-12-13 | 2010-08-03 | Zymogenetics, Inc. | Compositions of IL-21 produced in prokaryotic hosts |
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US11518808B2 (en) | 2018-01-12 | 2022-12-06 | Amgen Inc. | Anti-PD-1 antibodies and methods of treatment |
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