CN1305902C - Method for preparing human interleukin 29 and recombinant IL-29 engineering strain - Google Patents
Method for preparing human interleukin 29 and recombinant IL-29 engineering strain Download PDFInfo
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- CN1305902C CN1305902C CN 200410052377 CN200410052377A CN1305902C CN 1305902 C CN1305902 C CN 1305902C CN 200410052377 CN200410052377 CN 200410052377 CN 200410052377 A CN200410052377 A CN 200410052377A CN 1305902 C CN1305902 C CN 1305902C
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Abstract
The present invention discloses a method for producing human interleukin 29 and recombinant IL-29 engineering bacteria. The present invention comprises the following steps that a primer is artificially synthesized; an encoding region of IL-29 mature peptide is amplified by PCR; the pET44-IL-29 of an IL-29 prokaryotic expression carrier is constructed; recombinant IL-29 engineering bacteria are constructed after colibacillus is converted; the high-efficiency expression IL-29 of engineering bacteria is cultivated; the IL-29 is purified. The present invention accurately and rapidly obtains an encoding region of mature peptide of an IL-29 gene because of the adoption of a clone method (LIC) independent of connection reaction, expression efficiency is improved by a termination codon preferred by colibacillus, expression quantity is high, and a pure product can be obtained by one step purification of an S protein affinity column. The preparation of the IL-29 has an important prospect for clinically treating and preventing infection diseases and non-infection diseases.
Description
Technical field
The present invention relates to the genetically engineered field, relate in particular to a kind of production method and recombinant bacterial strain thereof of Ro 24-7472/000.
Background technology
Human interleukin-12 9 (IL-29) is by some viruses or double-stranded RNA (dsRNA) activation people's various kinds of cell such as the cytokine (Sheppard that peripheral blood lymphocytes (PMBC), dendritic cell (DC) and HeLa cell etc. produce, P et al, 2003, NatureImmunology, 4 (1): 63-68).IL-29 and Interferon, rabbit (IFN) and IL-10 have low-level homology, and IL-29 and IL-28A have 81% homology; Chromosomal localization 19q13.13.IL-29 with interferons seemingly produces albumen in the various kinds of cell by inducing cell, its biologic activity of these protein mediations, and can optionally act on dissimilar target cells.Therefore, IL-29 can be used as the substitute of IFN, is used for the treatment of tumour, virus disease etc., and the potential application prospect is arranged in clinical application.Problems such as but the production ubiquity product biological activity for IL-29 is low at present, expression amount is little.
Summary of the invention
The objective of the invention is to overcome the existing problem that IL-29 production method ubiquity product biological activity is low, expression amount is little, a kind of human IL-2's 9 production method is provided, utilize this method can efficiently express IL-29, products therefrom IL-29 polypeptide active height, expression amount are big.
Another object of the present invention is to provide a kind of recombinant il-2 9 engineering bacterias.
Technical scheme of the present invention is as follows:
The present invention adopts gene engineering method to produce human IL-2 9, and concrete grammar comprises the steps:
(1) synthetic pcr primer thing imports the LIC sticky end in the synthetic primer,
The sequence of IL-29 upstream and downstream primer is respectively:
5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5；
(2) pcr amplification: with the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template, by the pcr gene amplification, obtains the IL-29 gene fragment;
(3) the IL-29 gene fragment that above-mentioned pcr amplification is obtained is connected with the pET-44Ek/LIC carrier, makes up recombinant vectors pET-44Ek/LIC-IL-29;
(4), make up recombinant il-2 9 engineering bacterias with recombinant vectors pET-44Ek/LIC-IL-29 transform bacteria;
(5) cultivate recombinant il-2 9 engineering bacterias that make up, induce it to express and produce IL-29.
Wherein, the acquisition of pcDNA3.1/V5-His-TOPO-IL-29 plasmid is seen: Li Mingcai, He Shaoheng.The clone of human IL-2 8 and IL-29 gene and sequential analysis.Cell and molecular immunology magazine, 2004,20 (5): 635-637.The terminator codon TGA of the IL-29 gene fragment that the PCR method amplification can be obtained in above-mentioned steps (3) converts the codon TAA of intestinal bacteria preference to, is connected with the pET-44Ek/LIC carrier then, makes up recombinant vectors pET-44Ek/LIC-IL-29; Then, make up recombinant il-2 9 engineering bacterias, induce it to express with IPTG again and produce IL-29 carrier pET-44Ek/LIC-IL-29 transformed into escherichia coli.
The reaction conditions of above-mentioned pcr amplification is: 1. 95 ℃ of sex change 15min; 2. 94 ℃ of 45s, 72 ℃ of 30s, the every circulation of annealing temperature reduces by 1.0 ℃, 72 ℃ of 45s, 5 circulations; 3. 94 ℃ of 45s, be provided with annealing temperature gradient from 68 ℃ to 70 ℃ 30s, 72 ℃ of 45s, 30 circulations; 4. 72 ℃ are extended 10min.
In above-mentioned production human IL-2's 9 method, the present invention has made up a kind of engineering bacteria of recombinant il-2 9, it is characterized in that being formed by the following steps structure:
(1) synthetic pcr primer thing, and in the synthetic primer, import the LIC sticky end,
Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template, by the pcr gene amplification, obtains the IL-29 gene fragment;
(3) the IL-29 gene fragment that above-mentioned pcr amplification is obtained is connected with the pET-44Ek/LIC carrier, makes up recombinant vectors pET-44Ek/LIC-IL-29;
(4), make up recombinant il-2 9 engineering bacterias with recombinant vectors pET-44Ek/LIC-IL-29 transform bacteria;
Wherein, the terminator codon TGA of the IL-29 gene fragment that the PCR method amplification can be obtained in above-mentioned steps (3) converts the codon TAA of intestinal bacteria preference to, is connected with the pET-44Ek/LIC carrier then, makes up recombinant vectors pET-44Ek/LIC-IL-29; Then, make up recombinant il-2 9 engineering bacterias with carrier pET-44Ek/LIC-IL-29 transformed into escherichia coli.
The substratum of above-mentioned constructed recombinant il-2 9 engineering bacterias is: (1) LB liquid nutrient medium: 1% peptone, 0.5% yeast extract, 1% sodium-chlor, PH7.0; (2) LB solid medium: the agar powder that adds 1.5% (W/V) at above-mentioned LB liquid nutrient medium.
Beneficial effect of the present invention: (1) is adopted and is contained the primer that does not rely on ligation, accurately obtains the mature peptide cDNA fragment of IL-29 gene, and is cloned on the IPTG inductive pET carrier; (2) terminator codon of using intestinal bacteria to have a preference for improves expression efficiency; (3) the thalline output height after the enlarged culturing; (4) because expression amount soluble components height makes single step purification can obtain very pure product with S albumen affinity column.
Description of drawings
Fig. 1 makes up collection of illustrative plates for recombinant plasmid pET-44-IL-29;
Fig. 2 is the plasmid enzyme restriction collection of illustrative plates after cloning and transforming
Fig. 3 is an IL-29 expression amount SDS-PAGE collection of illustrative plates;
Fig. 4 is the SDS-PAGE collection of illustrative plates of IL-29 behind the purifying;
Wherein, among Fig. 2,1 is the DNA standard, and 2 cut the IL-29 plasmid for Xho I enzyme.Among Fig. 3,1 is the protein standard molecular weight, and 2 for not inducing total bacterial protein, and 3 for inducing the back total bacterial protein, and 4 are IL-29 supernatant total protein behind the broken bacterium, and 5 are bacterium IL-29 precipitation total protein behind the broken bacterium; Among Fig. 4,1 is the protein standard molecular weight, and 2 for not inducing total bacterial protein, and 3 for inducing the back total bacterial protein, and 4 are bacterial precipitation total protein behind the broken bacterium, and 5 are supernatant total protein behind the broken bacterium, and 6 is the IL-29 behind the purifying.
Embodiment
Embodiment:
(1) synthetic pcr primer thing:, adopt the following primer of Omiga 2.0 software designs according to the LIC site sequence on Ro 24-7472/000 IL-29cDNA gene order among the GenBank and the pET-44Ek/LIC carrier (seeing the specification sheets Cat 711433 of Novagen company):
Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;
(2) pcr amplification: on MJ Research PTC-200 grads PCR amplification instrument, adopt warm start (hotstart) pcr amplification.With the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template.Annealing temperature gradient is set, and cycling condition is: 1. 95 ℃ of sex change 15min; 2. 94 ℃ of 45s, 72 ℃ of 30s, the every circulation of annealing temperature reduces by 1.0 ℃, 72 ℃ of 45s, 5 circulations; 3. 94 ℃ of 45s, be provided with annealing temperature gradient from 68 ℃ to 70 ℃ 30s, 72 ℃ of 45s, 30 circulations; 4. 72 ℃ are extended 10min.1.0% agarose gel electrophoresis is identified the PCR product.The acquisition of above-mentioned pcDNA3.1/V5-His-TOPO-IL-29 plasmid is seen: Li Mingcai, He Shaoheng.The clone and the sequential analysis of Ro 24-7472/000 (IL)-28 and IL-29 gene.Cell and molecular immunology magazine, 2004,20 (5): 635-637.
(3) the PCR product is connected with the pET-44Ek/LIC carrier: the IL-29 gene fragment that above-mentioned PCR method amplification is obtained is connected with the pET-44Ek/LIC carrier, constitute recombinant vectors pET-44Ek/LIC-IL-29, be connected in the 0.5ml plastic centrifuge tube and carry out, concrete grammar is seen the specification sheets (Novagen Cat 711433 can buy from the commercial channel) of pET-44Ek/LIC carrier.The structure collection of illustrative plates of recombinant vectors pET-44Ek/LIC-IL-29 as shown in Figure 1.
(4) connect product and transform the NovaBlue competence bacteria: behind the above-mentioned connection product mixing, be added among the competent intestinal bacteria NovaBlue, be placed in the ice bath 5 minutes, again in 42 ℃ of water-baths 30 seconds, placed on ice 2 minutes, add 250 μ l SOC nutrient solutions (not containing antibiotic), 37 ℃ are incubated 1 hour, coat and contain LB solid medium (including penbritin), 37 ℃ of overnight incubation, get the intestinal bacteria bacterium colony of positive colony, carry out bacterium colony PCR, enzyme and cut and identify and gene sequencing that the result is consistent with bibliographical information.Plasmid enzyme restriction collection of illustrative plates after clone and the conversion as shown in Figure 2.
(5) conversion BL21 (DE3) competence bacteria: extract and identify correct positive colony plasmid, be transformed in the competent e. coli bl21 (DE3) with quadrat method more than adopting, through the screening of resistance substratum, the intestinal bacteria that get positive colony are engineering bacteria.
(6) chemically inducible expression: the intestinal bacteria after screening are inoculated in the LB substratum that contains penbritin 100 μ g/ml, 37 ℃ of concussion overnight incubation.Next day, 37 ℃ of concussions were cultivated 2～3 hours, to OD in 1.5% ratio enlarged culturing
600Reach at 0.4～0.6 o'clock, add IPTG to final concentration 1mmol/L, 22 ℃ violent (210 rev/mins) concussion was cultivated 6 hours, collected thalline.
(7) SDS electrophoresis: ultrasonic broken bacterial cell, the centrifugal cell conditioned medium that obtains carries out SDS-PAGE mensuration and shows that IL-29 albumen accounts for more than 30% of thalline soluble protein, and it is proteic more than 50% to account for inclusion body.The SDS-PAGE collection of illustrative plates of IL-29 expression amount as shown in Figure 3.
(8) separation and purification of expression product: concrete grammar is seen the specification sheets (Novagen Cat 692323 can buy from the commercial channel) of S-Tag ThrombinPurification Kit.Press the S albumen column purification of Novagen company, can get the IL-29 albumen of content more than 98%.The SDS-PAGE collection of illustrative plates of IL-29 expression amount as shown in Figure 4 behind the purifying.
Claims (6)
1, a kind of production method of human interleukin-12 9 is characterized in that comprising the steps:
(1) synthetic pcr primer thing, and in the synthetic primer, import the LIC sticky end,
Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template, by the pcr gene amplification, obtains the IL-29 gene fragment;
(3) the IL-29 gene fragment that above-mentioned pcr amplification is obtained is connected with the pET-44Ek/LIC carrier, makes up recombinant vectors pET-44Ek/LIC-IL-29;
(4), make up recombinant il-2 9 engineering bacterias with recombinant vectors pET-44Ek/LIC-IL-29 transform bacteria;
(5) cultivate recombinant il-2 9 engineering bacterias that make up, induce it to express and produce IL-29.
2, the production method of human interleukin-12 9 as claimed in claim 1 is characterized in that described gene engineering method comprises the steps:
(1) synthetic pcr primer thing, and in the synthetic primer, import the LIC sticky end,
Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template, by the pcr gene amplification, obtains the IL-29 gene fragment:
(3) the terminator codon TGA of the IL-29 gene fragment that above-mentioned PCR method amplification is obtained converts TAA to, is connected with the pET-44Ek/LIC carrier then, makes up recombinant vectors pET-44Ek/LIC-IL-29;
(4), make up recombinant il-2 9 engineering bacterias with recombinant vectors pET-44Ek/LIC-IL-29 transformed into escherichia coli;
(5) cultivate recombinant il-2 9 engineering bacterias that make up, induce it to express and produce IL-29.
3, the production method of human interleukin-12 9 as claimed in claim 1 or 2 is characterized in that the reaction conditions of the described pcr amplification of step (2) is: 1. 95 ℃ of sex change 15min; 2. 94 ℃ of 45s, 72 ℃ of 30s, the every circulation of annealing temperature reduces by 1.0 ℃, 72 ℃ of 45s, 5 circulations; 3. 94 ℃ of 45s, be provided with annealing temperature gradient from 68 ℃ to 70 ℃ 30s, 72 ℃ of 45s, 30 circulations; 4. 72 ℃ are extended 10min.
4, the production method of human interleukin-12 9 as claimed in claim 1 or 2 is characterized in that step (5) is to adopt IPTG to induce recombinant il-2 9 engineering bacterium expression to produce.
5, a kind of engineering bacteria of recombinant il-2 9 is characterized in that being formed by the following steps structure:
(1) synthetic pcr primer thing, and in the synthetic primer, import the LIC sticky end,
Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template, by the pcr gene amplification, obtains the IL-29 gene fragment;
(3) the IL-29 gene fragment that above-mentioned pcr amplification is obtained is connected with the pET-44Ek/LIC carrier, makes up recombinant vectors pET-44Ek/LIC-IL-29;
(4), make up recombinant il-2 9 engineering bacterias with recombinant vectors pET-44Ek/LIC-IL-29 transform bacteria.
6, the engineering bacteria of recombinant il-2 9 as claimed in claim 5 is characterized in that being formed by the following steps structure:
(1) synthetic pcr primer thing, and in the synthetic primer, import the LIC sticky end,
Upstream primer: 5-GACGACGACAAGATCCCCACTTCCAAGCCCAC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGGTGGACTCAGGGTGGGTTG-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-29 plasmid is template, by the pcr gene amplification, obtains the IL-29 gene fragment;
(3) the terminator codon TGA of the IL-29 gene fragment that above-mentioned PCR method amplification is obtained converts TAA to, is connected with the pET-44Ek/LIC carrier then, makes up recombinant vectors pET-44Ek/LIC-IL-29;
(4), make up recombinant il-2 9 engineering bacterias with recombinant vectors pET-44Ek/LIC-IL-29 transformed into escherichia coli.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2303132T3 (en) | 2003-08-07 | 2008-08-01 | Zymogenetics, Inc. | HOMOGENEAS PREPARATIONS OF IL-29. |
| US20070020227A1 (en) | 2005-07-20 | 2007-01-25 | Sheppard Paul O | Use of truncated cysteine IL28 and IL29 mutants to treat cancers and autoimmune disorders |
| EP1931704B1 (en) * | 2005-10-04 | 2010-12-15 | ZymoGenetics, L.L.C. | Production and purification of il-29 |
| CN100408686C (en) * | 2005-11-04 | 2008-08-06 | 中国人民解放军第三军医大学 | Process for preparing human leucocyte interleukin 24 by genetic engineering and it expressing carrier and engineering bacterium |
| CN102533840A (en) * | 2011-12-13 | 2012-07-04 | 江南大学 | Method for preparing human interleukin 29 (hIL-29) mature peptide by using Pichia pastoris |
| CN102559737A (en) * | 2011-12-13 | 2012-07-11 | 江南大学 | Human interleukin 29 variant and preparation method thereof |
| CN103224951A (en) * | 2013-01-25 | 2013-07-31 | 江南大学 | Human interleukin29 mature peptide mutant (hIL-29G) with lysine produced by site-directed mutagenesis of 35th arginine and preparation method thereof |
| CN103397047A (en) * | 2013-07-29 | 2013-11-20 | 宁波大学 | Production method of human interleukin-37b |
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