CN1055312C - 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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- CN1055312C CN1055312C CN94104006A CN94104006A CN1055312C CN 1055312 C CN1055312 C CN 1055312C CN 94104006 A CN94104006 A CN 94104006A CN 94104006 A CN94104006 A CN 94104006A CN 1055312 C CN1055312 C CN 1055312C
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Abstract
The present invention relates to a method for preparing human interleukin-6 and a product thereof, and the method comprises separating human leukocyte, separating total mRNA of leucocyte, artificially synthesizing a primer containing restriction incision enzyme nicks and synthesizing cDNA. The full length of the product nucleotide sequence is 486 ribonucleotides, cDNA of the obtained IL-6 is cloned to a plasmid carrier, and a promotor of the carrier pFrc is a hybrid promoter of-35(trpB) and-10(LacUv). The gene is induced to obtain high-activity IL-6 proteins after being expressed with high efficiency, the protein is 162 amino acid residual radicals, and the expression activity of the protein is 2.8 times of the expression activity of mature proteins in full length.
Description
The present invention relates to a kind of preparation method and product thereof of human interleukin-6.
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, and is disease-resistant, diseases prevention 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, 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) cDNA is synthetic: contain human interleukin-6 mRNA synthetic primer in the presence of, it is synthetic that the method that provides from the synthetic cDNA trial-production of Poly (A) RNA box by Boehringer company is made cDNA, 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
10ATC?TCA?GCC?CTG?AGA?AAG?GAG?ACA?TGT?AAC?AAG?AGT?AAC?ATG
20TGT?GAA?AGC?AGC?AAA?GAG?GCA?CTG?GCA?GAA?AAC?AAC?CTG?AAC
30 40CTT?CCA?AAG?ATG?GCT?GAA?AAA?GAT?GGA?TGC?TTC?CAA?TCT?GGA
50TTC?AAT?GAG?GAG?ACT?TGC?CTG?GTG?AAA?ATC?ATC?ACT?GGT?CTT
60 70TTG?GAG?TTT?GAG?GTA?TAC?CTA?GAG?TAC?CTC?CAG?AAC?AGA?TTT
80GAG?AGT?AGT?GAG?GAA?CAA?GCC?AGA?GCT?GTG?CAG?ATG?AGT?ACA
90AAA?GTC?CTG?ATC?CAG?TTC?CTG?CAG?AAA?AAG?GCA?AAG?AAT?CTA
100 110GAT?GCA?ATA?ACC?ACC?CCT?GAC?CCA?ACC?ACA?AAT?GCC?AGC?CTG
120CTG?ACG?AAG?CTG?CAG?GCA?CAG?AAC?CAG?TGG?CTG?CAG?GAC?ATG
130 140ACA?ACT?CAT?CTC?ATT?CTG?CGC?AGC?TTT?AAG?GAG?TTC?CTG?CAG
150TCC?AGC?CTG?AGG?GCT?CTT?CGG?CAA?ATG?TAG
160 164
(5) IL-6 cDNA clone and intestinal bacteria transform:
The pTrc carrier of cutting through EcoRI+BamHI enzyme is connected with IL-6cDNA, and the promotor of this expression vector pTrc is-35 (trpB) and-10 (lacUV
5) hybrid promoter; The plasmid that is loaded with the IL-6 gene obtains the intestinal bacteria of positive colony through routine conversion and resistance culture medium culturing.
(6) IL-6 purifying is measured.Aminoacid sequence is: Met Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly
10Ile?Ser?Ala?Leu?Arg?Lys?Glu?Thr?Cys?Asn?Lys?Ser?Asn?Met
20Cys?Glu?Ser?Ser?Lys?Glu?Ala?Leu?Ala?Glu?Asn?Asn?Leu?Asn
30 40Leu?Pro?Lys?Met?Ala?Glu?Lys?Asp?Gly?Cys?Phe?Gln?Ser?Gly
50Phe?Asn?Glu?Glu?Thr?Cys?Leu?Val?Lys?Ile?Ile?Thr?Gly?Leu
60 70Leu?Glu?Phe?Glu?Val?Tyr?Leu?Glu?Tyr?Leu?Gln?Asn?Arg?Phe
80Glu?Ser?Ser?Glu?Glu?Gln?Ala?Arg?Ala?Val?Gln?Met?Ser?Thr
90Lys?Val?Leu?Ile?Gln?Phe?Leu?Gln?Lys?Lys?Ala?Lys?Asn?Leu
100 110Asp?Ala?Ile?Thr?Thr?Pro?Asp?Pro?Thr?Thr?Asn?Ala?Ser?Leu
120Leu?Thr?Lys?Leu?Gln?Ala?Gln?Asn?Gln?Trp?Leu?Gln?Asp?Met
130 140Thr?Thr?His?Leu?Ile?Leu?Arg?Ser?Phe?Lys?Glu?Phe?Leu Gln
150Ser?Ser?Leu?Arg?Ala?Leu?Arg?Gln?Met?End
160 164Thr?Thr?His?Leu?Ile?Leu?Arg?Ser?Phe?Lys?Glu?Phe?Leu?Gln
150Ser?Ser?Leu?Arg?Ala?Leu?Arg?Gln?Met?End
160 164
Below in conjunction with embodiment the present invention is elaborated:
Fig. 1: the SDS-PAGE collection of illustrative plates of the IL-6 of purifying (1-6), wherein 7 is standard protein.
Fig. 2: construction of recombinant plasmid.
(1) blood middle leukocytes separates:
Get human blood, the ratio adding cellular segregation liquid with 1: 1.5 (V/V) carried out gradient separations down in centrifugal 15 minutes at 1000 rev/mins, collected the white corpuscle part.
(2) separation of the total mRNA of white corpuscle:
The described method of skill Pharmacia trace mrna purification kit is carried out:
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, removes stock solution.The homogenate clear liquid is joined in oligo (dT)-Mierocrystalline cellulose centrifuge tube, mixed 3 minutes, make the absorption of mRNA affinity, more centrifugal 10 seconds under 16000xg, abandon supernatant liquor.
In pipe, add 1ml high-salt buffer (10mM Tris-HCl, 1mM EDTA, 0.5MNaCI pH7.4) and wash,, in centrifugal 10 seconds, abandon supernatant liquor Oligo (dT)-cellulose suspension.Repeat above-mentioned steps 4 times.
Add 1ml low salt buffer (0.1MNaCl, pH 7.4 for 10mM Tris-HCl, 1mM EDTA) at Guan Zhongzai and wash repeated washing 3 times.Oligo after the washing (dT)-Mierocrystalline cellulose adds the 0.3ml low salt buffer and suspends, and puts on the micro-column, and the oligo in post (dT)-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 Glycogne solution in the 400 μ lmRNA solution that elute, 40 μ l potassium acetate solutions add 1ml-20 ℃ of cold ethanol (95%) again, and-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:
Carry out from the described method of the synthetic cDNA trial-production box of Poly (A) RNA by Boenringer company.20 μ l mRNA are put in the plastic centrifuge tube, add the synthetic damping fluid of 4 μ l, first chain in ice bath, 1 μ l RNase inhibitor, 2 μ l dATP.dCTP.dGTP.dTTP mixed solutions, 2 μ loligo (dT)
15Primer, 2 μ l AMV reversed transcriptive enzymes and an amount of redistilled water always add 20 μ l, and behind the mixing, 42 ℃ of pre-temperature 60 minutes, mixed solution was put into ice bath again.
In mixed solution, add the synthetic component of second chain again, comprise: 40 μ l, second chain synthesizes damping fluid, 1 μ l RNaseH solution, 5 μ l E.coli archaeal dna polymerase and suitable redistilled waters, cumulative volume 100 μ l mix the centrifugal 10 seconds kinds in back, and are pre-warm 10 minutes in 60 minutes 22 ℃ of pre-temperature of 12 ℃ of pre-temperature 60 minutes and 65 ℃ again, add 4 μ l T4 archaeal dna polymerases again, pre-warm 10 minutes in 37 ℃.At last, add 10 μ l EDTA solution (0.2mol/L) and 2 μ l sarkosyl 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 Tris-HCl, 15m mol/L MgCl
2, 0.1% gelatin, 1%Tritonx-100.pH8.3) 5 μ l dNTP mixed solutions, 5 μ l primers, 1 μ l template DNA solution behind the mixing centrifugal 10 minutes, is put into the pre-sex change of 95 ℃ of water-baths 10 minutes again, centrifugal 10 seconds, add 1 μ l (3 unit) Taq then, archaeal dna polymerase adds 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 pTrc carrier of cutting through EcoRI+Bam HI enzyme is connected with IL-6cDNA, should carry out in the 0.5ml plastic centrifuge tube after the connection, adds 4.5 μ l redistilled waters, 1.5 μ l ligation damping fluid (50mMol/L Tris-Hcl 10mMol/L MgCl in the pipe
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 (containing penbritin) 37 ℃ of overnight incubation on the LB solid medium of proper volume, obtain 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.
The promotor of expression vector pTrc is-35 (trpB) and-10 (lacUV in the experiment
5) hybrid promoter, this promotor has very strong initial functional transcription, the Laco site is a Lac repressor binding site, induce and can efficiently express by inductor, Anti-Term is one section E.coli rrnB sequence, can prevent premature transcription termination, 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, and the promoter of this expression vector pTrc is-35 (trpB) and-10 (lacUV5) hybrid promoter, and other characteristics make cDNA Efficiently expressed, 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 polypeptide active is the total length maturation 2.8 times of albumen.
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 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, 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) cDNA is synthetic: contain human interleukin-6 mRNA synthetic primer in the presence of, it is synthetic that the method that provides from the synthetic cDNA trial-production of Poly (A) RNA box by Boehringer company is made cDNA, 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
10ATC?TCA?GCC?CTG?AGA?AAG?GAG?ACA?TGT?AAC?AAG?AGT?AAC?ATG
20TGT?GAA?AGC?AGC?AAA?GAG?GCA?CTG?GCA?GAA?AAC?AAC?CTG?AAC
30 40CTT?CCA?AAG?ATG?GCT?GAA?AAA?GAT?GGA?TGC?TTC?CAA?TCT?GGA
50TTC?AAT?GAG?GAG?ACT?TGC?CTG?GTG?AAA?ATC?ATC?ACT?GGT?CTT
60 70TTG?GAG?TTT?GAG?GTA?TAC?CTA?GAG?TAC?CTC?CAG?AAC?AGA?TTT
80GAG?AGT?AGT?GAG?GAA?CAA?GCC?AGA?GCT?GTG?CAG?ATG?AGT?ACA
90AAA?GTC?CTG?ATC?CAG?TTC?CTG?CAG?AAA?AAG?GCA?AAG?AAT?CTA
100 110GAT?GCA?ATA?ACC?ACC?CCT?GAC?CCA?ACC?ACA?AAT?GCC?AGC?CTG
120CTG?ACG?AAG?CTG?CAG?GCA?CAG?AAC?CAG?TGG?CTG?CAG?GAC?ATG
130 140ACA?ACT?CAT?CTC?ATT?CTG?CGC?AGC?TTT?AAG?GAG?TTC?CTG?CAG
150TCC?AGC?CTG?AGG?GCT?CTT?CGG?CAA?ATG?TAG
160 164
(5) IL-6 cDNA clone and intestinal bacteria transform:
The pTrc carrier of cutting through EcoRI+BamHI enzyme is connected with IL-6cDNA, and the promotor of this expression vector pTrc is-35 (trpB) and-10 (lacUV
5) hybrid promoter; The plasmid that is loaded with the IL-6 gene obtains the intestinal bacteria of positive colony through routine conversion and resistance culture medium culturing.
(6) IL-6 purifying is measured.
2, obtain polypeptide by the described preparation method of claim 1, it is characterized in that its aminoacid sequence is: Met Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly
10Ile?Ser?Ala?Leu?Arg?Lys?Glu?Thr?Cys?Asn?Lys?Ser?Asn?Met
20Cys?Glu?Ser?Ser?Lys?Glu?Ala?Leu?Ala?Glu?Asn?Asn?Leu?Asn
30 40Leu?Pro?Lys?Met?Ala?Glu?Lys?Asp?Gly?Cys?Phe?Gln?Ser?Gly
50Phe?Asn?Glu?Glu?Thr?Cys?Leu?Val?Lys?Ile?Ile?Thr?Gly?Leu
60 70Leu?Glu?Phe?Glu?Val?Tyr?Leu?Glu?Tyr?Leu?Gln?Asn?Arg?Phe
80Glu?Ser?Ser?Glu?Glu?Gln?Ala?Arg?Ala?Val?Gln?Met?Ser?Thr
90Lys?Val?Leu?Ile?Gln?Phe?Leu?Gln?Lys?Lys?Ala?Lys?Asn?Leu
100 110Asp?Ala?Ile?Thr?Thr?Pro?Asp?Pro?Thr?Thr?Asn?Ala?Ser?Leu
120Leu?Thr?Lys?Leu?Gln?Ala?Gln?Asn?Gln?Trp?Leu?Gln?Asp?Met
130 140Thr?Thr?His?Leu?Ile?Leu?Arg?Ser?Phe?Lys?Glu?Phe?Leu?Gln
150Ser?Ser?Leu?Arg?Ala?Leu?Arg?Gln?Met?End
160 164
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| CN94104006A CN1055312C (en) | 1994-04-13 | 1994-04-13 | Method for preparation of human interleukin-6 and its products |
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| CN94104006A CN1055312C (en) | 1994-04-13 | 1994-04-13 | Method for preparation of human interleukin-6 and its products |
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| CN1055312C true CN1055312C (en) | 2000-08-09 |
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| 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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| Title |
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| 中国免疫学杂志8卷3期 1992.1.1 PBV-IL-6 重组质粒的构建及其在E.CLI细胞中的超高表达，任启生等 * |
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