CN1110319A - Method for preparation of human interleukin-6 and its products - Google Patents
Method for preparation of human interleukin-6 and its products Download PDFInfo
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- CN1110319A CN1110319A CN 94104006 CN94104006A CN1110319A CN 1110319 A CN1110319 A CN 1110319A CN 94104006 CN94104006 CN 94104006 CN 94104006 A CN94104006 A CN 94104006A CN 1110319 A CN1110319 A CN 1110319A
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Abstract
The present invention relates to a preparation method of human interleukin-6 and its product. Its method includes: human leukocyte separation; leukocyte total mRNA separation; artificial synthesis of primer containing restriction endonuclease nicks; synthesis of cDNA, and the whole length of its product nucleotide sequnce is 486 nucleotides, and the cDNA of the obtained IL-6 is cloned on plasmid carrier, and the promoter of the carrier pFrc is the heterozygous promoter of -35(trpB) and -10 (LacUv), and after induction and high-effective expression, the high-activity IL-6 protein can be obtained. It is 162 amino acid residues, and its expression activity is 2.8 times that of full-length maturation protein.
Description
The present invention relates to preparation method of a kind of human interleukin-6 and products thereof.
Used some medicines of many illnesss (as chemotherapeutics etc.) can make people's immune system disorder or reduce immunologic function, people's resistibility is descended, thereby easily cause infectation of bacteria, cause other illnesss, if recover or improve people's immunologic function, then just can improve people's resistance against diseases, people's the immunocyte and the growth of hematopoietic cell and differentiation are subjected to the regulation and control of many cytokines, interleukin-is exactly the cytokine of above-mentioned these emiocytosises, the growth of their induction of lymphocyte and hemopoietic stem cell and differentiation, tens kinds of interleukin-have been found at present, interleukin 6 has antitumous effect, for example can suppress human breast cancer clone's growth, interleukin-6 has hemopoietic function, for example the hemopoietic stem cell of handling with IL-6 can recover or strengthen the function of hemopoietic system, thereby aspect treatment septicemia and the thrombopenia important role is being arranged, IL-6 is for the immunologic function that improves human body, defending and fighting against diseases and carry out postoperative, the more convalescent diagnosis in back and treat significant and wide application prospect of disease.
The object of the invention provides preparation method of a kind of human interleukin-6 and products thereof, the polypeptide active height that this method obtains.
For achieving the above object, the present invention by the following technical solutions: the preparation method comprises following step:
(1) blood middle leukocytes separates;
(2) separation of the total mRNA of white corpuscle: the kit method of pressing the trace mrna purifying of Pharmacia separates;
(3) synthetic contains the primer at restriction enzyme point of contact, in the synthetic upstream primer, imported the BamHI site, imported the EcoRI site at the synthetic downstream primer, and convert the terminator codon TAG of natural IL-6 the terminator codon TAA of intestinal bacteria preference to, the sequence of primer is:
PL?5' CG?GGATCC?GC?ATG?GAA?CGA?ATT?GAC?AAA?CAA?3'
PR?5' CG?GAATTC?A?TTA?CAT?TTG?CCG?AAG?AG?3'
(4) cDNA is synthetic: contain human interleukin-6 mRNA synthetic primer in the presence of, utilize the DNA synthesis system of reversed transcriptive enzyme, by polymerase chain reaction (PCR) amplification IL-6 gene fragment, its nucleotides sequence is classified as:
ATG?GAA?CGA?ATT?GAC?AAA?CAA?ATT?CGG?TAC?ATC?CTC?GAC?GGC
10
ATC?TCA?GCC?CTG?AGA?AAG?GAG?ACA?TGT?AAC?AAG?AGT?AAC?ATG
20
TGT?GAA?AGC?AGC?AAA?GAG?GCA?CTG?GCA?GAA?AAC?AAC?CTG?AAC
30
CTT?CCA?AAG?ATG?GCT?GAA?AAA?GAT?GGA?TGC?TTC?CAA?TCT?GGA
50
TTC?AAT?GAG?GAG?ACT?TGC?CTG?GTG?AAA?ATC?ATC?ACT?GGT?CTT
60 70
60 70
TTG?GAG?TTT?GAG?GTA?TAC?CTA?GAG?TAC?CTC?CAG?AAC?AGA?TTT
80
GAG?AGT?AGT?GAG?GAA?CAA?GCC?AGA?GCT?GTG?CAG?ATG?AGT?ACA
90
AAA?GTC?CTG?ATC?CAG?TTC?CTG?CAG?AAA?AAG?GCA?AAG?AAT?CTA
100 110
GAT?GCA?ATA?ACC?ACC?CCT?GAC?CCA?ACC?ACA?AAT?GCC?AGC?CTG
120
CTG?ACG?AAG?CTG?CAG?GCA?CAG?AAC?CAG?TGG?CTG?CAG?GAC?ATG
130 140
ACA?ACT?CAT?CTC?ATT?CTG?CGC?AGC?TTT?AAG?GAG?TTC?CTG?CAG
150
TCC?AGC?CTG?AGG?GCT?CTT?CGG?CAA?ATG?TAG
160 164
(5) IL-6cDNA clone and intestinal bacteria transform:
The pTrc carrier of cutting through the EcoRI+BamHI enzyme is connected with IL-6cDNA, the promotor of this expression vector pTrc is-35(trpB) with-10(IacUV
5) hybrid promoter; The plasmid that is loaded with the IL-6 gene is through cultivating the intestinal bacteria that obtain positive colony.
(6) IL-6 purifying, mensuration.
Hydrogen base acid sequence is:
Met?Glu?Arg?Ile?Asp?Lys?Gln?Ile?Arg?Tyr?Ile?Leu?Asp?Gly
10
Ile?Ser?Ala?Leu?Arg?Lys?Glu?Thr?Cys?Asn?Lys?Ser?Asn?Met
20
Cys?Glu?Ser?Ser?Lys?Glu?Ala?Leu?Ala?Glu?Asn?Asn?Leu?Asn
30 40
Leu?Pro?Lys?Met?Ala?Glu?Lys?Asp?Gly?Cys?Phe?Gln?Ser?Gly
50
Phe?Asn?Glu?Glu?Thr?Cys?Leu?Val?Lys?Ile?Ile?Thr?Gly?Leu
60 70
Leu?Glu?Phe?Glu?Val?Tyr?Leu?Glu?Tyr?Leu?Gln?Asn?Arg?Phe
80
Glu?Ser?Ser?Glu?Glu?Gln?Ala?Arg?Ala?Val?Gln?Met?Ser?Thr
90
Lys?Val?Leu?Ile?Gln?Phe?Leu?Gln?Lys?Lys?Ala?Lys?Asn?Leu
100 110
Asp?Ala?Ile?Thr?Thr?Pro?Asp?Pro?Thr?Thr?Asn?Ala?Ser?Leu
120
Leu?Thr?Lys?Leu?Gln?Ala?Gln?Asn?Gln?Trp?Leu?Gln?Asp?Met
130 140
Thr?Thr?His?Leu?Ile?Leu?Arg?Ser?Phe?Lys?Glu?Phe?Leu?Gln
150
Ser?Ser?Leu?Arg?Ala?Leu?Arg?Gln?Met?End
160 164
Below in conjunction with embodiment the present invention is elaborated:
(1) blood middle leukocytes separates:
Get human blood, with 1: ratio 1.5(V/V) adds cellular segregation liquid, carries out gradient separations down in centrifugal 15 minutes at 1000 rev/mins, collects the white corpuscle part.
(2) separation of the total mRNA of white corpuscle:
Undertaken by the described method of Pharmacia trace mrna purification kit:
To contain 10
7The leukocyte suspension of individual cell is placed in the 1.5ml plastic centrifuge tube, and under the 1000rpm, the centrifugal supernatant liquor of abandoning adds 0.4ml and extracts damping fluid homogenate in the pipe, again with the dilution of 0.8ml elutriant, under the 10000xg centrifugal 1 minute, must contain the homogenate clear liquid of mRNA.
Other gets a 1.5ml plastic centrifuge tube, adds 1ml oligo(dt)-Mierocrystalline cellulose, under the 1000xg centrifugal 1 minute, remove stock solution.The homogenate clear liquid is joined oligo(dT)-the Mierocrystalline cellulose centrifuge tube in, mixed 3 minutes, make mRNA affinity absorption, more centrifugal 10 seconds under 16000xg, abandon supernatant liquor.
In pipe, add 1ml high-salt buffer (10mM Tris-Hcl, 1mM EDTA, 0.5MNaCL PH7.4) and wash, with Oligo(dT)-cellulose suspension, in centrifugal 10 seconds, abandon supernatant liquor.Repeat above-mentioned steps 4 times.
(0.1MNacL PH7.4) washs for 10mM Tris-Hcl, 1mM EDTA, repeated washing 3 times to add the 1ml low salt buffer at Guan Zhongzai.Oligo(dT after the washing)-Mierocrystalline cellulose adds the 0.3ml low salt buffer and suspends, and puts on the micro-column oligo(dT in post)-Mierocrystalline cellulose is centrifugal with 0.5ml low salt buffer washing three times again, removes washings.Add 0.2ml elution buffer (10mMTris-Hcl, 1mMEDTA PH7.4) centrifugal, add 10 μ l Glycogen solution, 40 μ l potassium acetate solutions in the 400 μ lmRNA solution that elute, add 1ml-20 ℃ of cold ethanol (95%) again,-20 ℃ left standstill 30 minutes at least, 4 ℃ centrifugal 5 minutes mRNA precipitation, be deposited in-80 ℃ of storages, elutriant is 0.05 in the maximum absorption of 260nm, and being equivalent to RNA concentration is 2 μ g/ml.
(3) cDNA's is synthetic:
By Boehringer company from Poly(A) the described method of the synthetic cDNA trial-production box of RNA carries out.20 μ lmRNA are put in the plastic centrifuge tube, add the synthetic damping fluid of 4 μ l, first chain in ice is molten, 1 μ lRNase inhibitor, 2 μ l dATP.dcTp.dGTP.dTTp mixed solutions, 2 μ loligo(dT)
15Primer, 2 μ lAMV reversed transcriptive enzymes and an amount of redistilled water always add 20 μ l, behind the mixing, give temperature 60 minutes at 42 ℃, and it is molten that mixed solution is put into ice again.
In mixed solution, add the synthetic component of second chain again, comprise: 40 μ l, second chain synthesizes damping fluid, 1 μ lRNaseH solution, 5 μ l E.coli archaeal dna polymerase and suitable redistilled waters, cumulative volume 100 μ l, centrifugal 10 seconds after mixing, giving temperature 60 minutes in 12 ℃ again gives temperature for 22 ℃ and gave in 60 minutes and 65 ℃ warm 10 minutes, add 4 μ l T4 archaeal dna polymerases again, gave temperature 10 minutes in 37 ℃, last, add 10 μ l EDTA solution (0.2mol/L) and 2 μ l sarkosye solution (10%W/V) termination reactions, cDNA extracts with phenol/chloroform, obtains with ethanol sedimentation.
(4) pcr gene amplification:
In the aseptic centrifuge tube of 0.5ml, add: 37 μ l redistilled waters, 5 μ l amplification buffers (0.5mol/L kcl, 0.1mol/L Trise Hcl, 15m mol/L Mgcl
20.1% gelatin, 1%TriTonx-100.PH8.3) .5 μ l dNTP mixed solution, 5 μ l primers, 1 μ l template DNA solution, behind the mixing centrifugal 10 minutes, put into 95 ℃ of water-baths again and give 10 minutes centrifugal 10 seconds of sex change, add 1 μ l(3 unit then) the Taq archaeal dna polymerase, add 50 μ l paraffin oils behind the mixing, in 72 ℃ of insulations 2 minutes, again in 93 ℃ (1 minutes)-50 ℃ (1 minute).-72 ℃ (3 minutes) circulation 35 times, the amplification sample reclaims the IL-6cDNA amplified band on sepharose electricity arteries and veins.
(5) IL-6cDNA clone and intestinal bacteria transform
Through the IL-6cDNA of PCR method amplification, reclaim through agarose electricity arteries and veins, the phenol extracting, behind the ethanol sedimentation, DNA is dissolved in the 15 μ l redistilled waters again.
The pTre carrier of cutting through EcoRI+Bam HI enzyme is connected with IL-6cDNA, should carry out adding in the pipe 4.5 μ l redistilled waters, 1.5 μ l ligation damping fluid (50mMol/L Tris-Hcl 10mMol/L Mgcl after the connection in the 0.5ml plastic centrifuge tube
2, 20mMol/L DTT.1mMOL/L ATP and 50ug BSA, PH7.8) 2 μ l carrier DNAs, 6 μ l IL-6DNA fragments and 1 μ l T4 dna ligase, behind the sample mixing, insulation is 24-72 hour in 14-16 ℃ of water-bath.
Getting 5-7 μ l connection product (being loaded with the plasmid of IL-6 gene) joins in the E.coli HB101 competent cell suspension of 100-200 μ l, mixing is placed in the ice bath 30 minutes again in 42 ℃ of thermal shocks 2 minutes, add 1ml LB nutrient solution (not containing antibiotic), 37 ℃ are incubated 1 hour, coat on the LB solid medium of proper volume (containing penbritin), cultivate liquid for 37 ℃, extracted the intestinal bacteria of positive colony.
(6) the IL-6 product is identified
Intestinal bacteria after screening are through three grades of cultivations, cellular lysate.Go inclusion body to carry out SDS-PAGE mensuration again and show that IL-6 albumen accounts for 30% of tropina, can obtain pure IL-6 albumen behind the IL-6 purifying, molecular weight of albumen is 18KD, and the SDS-PAGE collection of illustrative plates of the IL-6 of purifying is seen Fig. 1.
In the experiment promotor of expression vector pTrc be-35(trpB) with-10(lacUV
5) hybrid promoter, this promotor has very strong initial functional transcription, the Laco site is that the thing binding site is met in Lac resistance, induces and can efficiently express by inductor.Anti-Term is one section E.coli rrnB sequence, can prevent premature transcription termination, and its conformation is seen Fig. 2, and this expression vector market is on sale.Or according to preparation such as (Herman Proc.Natl Acad Sci VSA 80: 21-25 1983).
Advantage of the present invention: adopted the primer and the distinctive sequence that contain the restriction enzyme point of contact among the present invention, synthetic through cDNA, its nucleotides sequence is classified 486 nucleotides as, the cDNA of the IL-6 that obtains is cloned on the plasmid vector, the promoter of this expression vector pTrc is-35(trpB) with-10(lacUV5) hybrid promoter, and other characteristics efficiently express cDNA, the product that the method obtains (polypeptide) is 162 amino acid residues, according to work such as Brakenhoff, (J.Immunol.143: 1175-1182,1989), When 23 amino acid of the N of ripe IL-6 terminal deletion, its expression activity is 2.8 times of total length maturation protein.
Claims (2)
1, a kind of preparation method of human interleukin-6 is characterized in that it comprises following step:
(1) blood middle leukocytes separates;
(2) separation of the total mPNA of white corpuscle: the kit method of pressing the trace mrna purifying of Pharmacia separates;
(3) synthetic contains the primer at restriction enzyme point of contact, has imported the BamHI site in the synthetic upstream primer, has imported the EcoRI site at the synthetic downstream primer, and the sequence of its primer is:
PL?5′?CG?GGATCC?GC?ATG?GAA?CGA?ATT?GAC AAA?CAA?3′
PR?5′?CG?GAATTC?A?TTA?CAT?TTG?CCG?AAG?AG?3′
(4) CDAN is synthetic: contain human interleukin-6 mRNA synthetic primer in the presence of, utilize the DNA synthesis system of reversed transcriptive enzyme, by polymerase chain reaction (PCR) amplification IL-6 gene fragment, its nucleotides sequence is classified as:
ATG?GAA?CGA?ATT?GAC?AAA?CAA?ATT?CGG?TAC?ATC?CTC?GAC?GGC
10
ATC?TCA?GCC?CTG?AGA?AAG?GAG?ACA?TGT?AAC?AAG?AGT?AAC?ATG
20
TGT?GAA?AGC?AGC?AAA?GAG?GCA?CTG?GCA?GAA?AAC?AAC?CTG?AAC
30
CTT?CCA?AAG?ATG?GCT?GAA?AAA?GAT?GGA?TGC?TTC?CAA?TCT?GGA
50
TTC?AAT?GAG?GAG?ACT?TGC?CTG?GTG?AAA?ATC?ATC?ACT?GGT?CTT
60 70
TTG?GAG?TTT?GAG?GTA?TAC?CTA?GAG?TAC?CTC?CAG?AAC?AGA?TTT
80
GAG?AGT?AGT?GAG?GAA?CAA?GCC?AGA?GCT?GTG?CAG?ATG?AGT?ACA
90
AAA?GTC?CTG?ATC?CAG?TTC?CTG?CAG?AAA?AAG?GCA?AAG?AAT?CTA
100 110
GAT?GCA?ATA?ACC?ACC?CCT?GAC?CCA?ACC?ACA?AAT?GCC?AGC?CTG
120
CTG?ACG?AAG?CTG?CAG?GCA?CAG?AAC?CAG?TGG?CTG?CAG?GAC?ATG
130 140
ACA?ACT?CAT?CTC?ATT?CTG?CGC?AGC?TTT?AAG?GAG?TTC?CTG?CAG
150
TCC?AGC?CTG?AGG?GCT?CTT?CGG?CAA?ATG?TAG
160 164
(5) IL-6cDNA clone and intestinal bacteria transform:
The pTrc carrier of cutting through the EcoRI+BamHI enzyme is connected with IL-6cDNA, and the promotor of this expression vector pTrc is-35 (trpB) and-10 (lacVW
5) hybrid promoter; The plasmid that is loaded with the IL-6 gene is through cultivating the intestinal bacteria that obtain positive colony.
(6) IL-6 purifying is measured.
2, obtain product by the described preparation method of claim 1, it is characterized in that its hydrogen base acid sequence is:
Met?Glu?Arg?Ile?Asp?Lys?Gln?Ile?Arg?Tyr?Ile?Leu?Asp?Gly
10
Ile?Ser?Ala?Leu?Arg?Lys?Glu?Thr?Cys?Asn?Lys?Ser?Asn?Met
20
Cys?Glu?Ser?Ser?Lys?Glu?Ala?Leu?Ala?Glu?Asn?Asn?Leu?Asn
30 40
Leu?Pro?Lys?Met?Ala?Glu?Lys?Asp?Gly?Cys?Phe?Gln?Ser?Gly
50
Phe?Asn?Glu?Glu?Thr?Cys?Leu?Val?Lys?Ile?Ile?Thr?Gly?Leu
60 70
Leu?Glu?Phe?Glu?Val?Tyr?Leu?Glu?Tyr?Leu?Gln?Asn?Arg?Phe
80
Glu?Ser?Ser?Glu?Glu?Gln?Ala?Arg?Ala?Val?Gln?Met?Ser?Thr
90
Lys?Val?Leu?Ile?Gln?Phe?Leu?Gln?Lys?Lys?Ala?Lys?Asn?Leu
100 110
Asp?Ala?Ile?Thr?Thr?Pro?Asp?Pro?Thr?Thr?Asn?Ala?Ser?Leu
120
Leu?Thr?Lys?Leu?Gln?Ala?Gln?Asn?Gln?Trp?Leu?Gln?Asp?Met
130 140
Thr?Thr?His?Leu?Ile?Leu?Arg?Ser?Phe?Lys?Glu?Phe?Leu?Gln
150
Ser?Ser?Leu?Arg?Ala?Leu?Arg?Gln?Met?End
160 164
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94104006A CN1055312C (en) | 1994-04-13 | 1994-04-13 | Method for preparation of human interleukin-6 and its products |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94104006A CN1055312C (en) | 1994-04-13 | 1994-04-13 | Method for preparation of human interleukin-6 and its products |
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|---|---|
| CN1110319A true CN1110319A (en) | 1995-10-18 |
| CN1055312C CN1055312C (en) | 2000-08-09 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409453A (en) * | 2013-08-16 | 2013-11-27 | 四川卧龙国家级自然保护区管理局 | Preparation method of recombinant panda IL-6 immunological adjuvant |
-
1994
- 1994-04-13 CN CN94104006A patent/CN1055312C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409453A (en) * | 2013-08-16 | 2013-11-27 | 四川卧龙国家级自然保护区管理局 | Preparation method of recombinant panda IL-6 immunological adjuvant |
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| Publication number | Publication date |
|---|---|
| CN1055312C (en) | 2000-08-09 |
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