CN1274823C - Production of human interleukin 288 and recombined IL-288 engineering bacteria - Google Patents
Production of human interleukin 288 and recombined IL-288 engineering bacteria Download PDFInfo
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- CN1274823C CN1274823C CN 200410052292 CN200410052292A CN1274823C CN 1274823 C CN1274823 C CN 1274823C CN 200410052292 CN200410052292 CN 200410052292 CN 200410052292 A CN200410052292 A CN 200410052292A CN 1274823 C CN1274823 C CN 1274823C
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Abstract
The present invention discloses a production method of a human interleukin 28B and a recombinant IL-28B engineering bacterium. The method of the present invention comprises the steps: artificially synthesizing primers, amplifying an IL-28B mature peptide coding area by PCR, constructing an IL-28B prokaryotic expression vector pET44-IL-28B, converting the IL-28B prokaryotic expression vector pET44-IL-28B into colibacillus to construct and recombine the IL-28B engineering bacterium, cultivating the effective expression IL-28B of engineering bacteria, and purifying IL-28B. The present invention adopts a cloning method (LIC) which is independent from a coupled reaction to accurately and rapidly obtain the mature peptide code area of IL-28B genes, uses a stop codon preferred by colibacillus to increase expression efficiency, has the advantage of high expression amount and uses an S protein affinity chromatography column to carry out one-step purification to obtain a pure product. The preparation of IL-28B has important prospects for the clinical treatment and prevention of infectious diseases and non-infectious 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 8B (IL-28B) 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).The major function of IL-28B may comprise antiviral, inhibition of cell proliferation and immunomodulatory.IL-28B 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.IL-28B 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-28B 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-28B production method ubiquity product biological activity is low, expression amount is little, the production method of a kind of Ro 24-7472/000 (IL)-28B is provided, utilize this method can efficiently express IL-28B, products therefrom IL-28B polypeptide active height, expression amount are big.
Another object of the present invention is to provide a kind of recombinant il-2 8B engineering bacteria.
Technical scheme of the present invention is as follows:
The present invention adopts gene engineering method to produce Ro 24-7472/000 (IL)-28B, and concrete grammar comprises the steps:
(1) synthetic pcr primer thing imports the LIC sticky end in the synthetic primer,
The sequence of IL-28B upstream and downstream primer is respectively:
Upstream primer: 5-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification: with the pcDNA3.1/V5-His-TOPO-IL-28B plasmid is template, by the pcr gene amplification, obtains the IL-28B gene fragment;
(3) the IL-28B 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-28B;
(4), make up recombinant il-2 8B engineering bacteria with recombinant vectors pET-44Ek/LIC-IL-28B transform bacteria;
(5) cultivate the recombinant il-2 8B engineering bacteria that makes up, induce it to express and produce IL-28B.
Wherein, the acquisition of pcDNA3.1/V5-His-TOPO-IL-28B plasmid is seen: Li Mingcai, He Shaoheng. the clone and the sequential analysis of Ro 24-7472/000 (IL)-28 and IL-29 gene. and cell and molecular immunology magazine, 2004,20 (5): 635-637.The terminator codon TGA of the IL-28B 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-28B; Then, make up recombinant il-2 8B engineering bacteria, induce it to express with IPTG again and produce IL-28B carrier pET-44 Ek/LIC-IL-28B 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 the method for above-mentioned production Ro 24-7472/000 (IL)-28B, the present invention has made up the engineering bacteria of a kind of recombinant il-2 8B, 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-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-28B plasmid is template, by the pcr gene amplification, obtains the IL-28B gene fragment;
(3) the IL-28B 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-28B;
(4), make up recombinant il-2 8B engineering bacteria with recombinant vectors pET-44Ek/LIC-IL-28B transform bacteria;
Wherein, the terminator codon TGA of the IL-28B gene fragment that the PCR method amplification can be obtained in above-mentioned steps (3) converts the codon TAA of intestinal bacteria preference to, be connected with the pET-44Ek/LIC carrier then, make up recombinant vectors pET-44Ek/LIC-IL-28B; Then, make up recombinant il-2 8B engineering bacteria with carrier pET-44Ek/LIC-IL-28B transformed into escherichia coli.
The substratum of above-mentioned constructed recombinant il-2 8B engineering bacteria 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-28B 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-28B;
Fig. 2 is the plasmid enzyme restriction collection of illustrative plates after cloning and transforming
Fig. 3 is an IL-28B expression amount SDS-PAGE collection of illustrative plates;
Fig. 4 is the SDS-PAGE collection of illustrative plates of IL-28B behind the purifying;
Wherein, among Fig. 2,1 is the DNA standard, and 3 cut the IL-28B plasmid for Xho I enzyme; Among Fig. 3, M protein standard molecular weight, 1 is supernatant total protein behind the broken bacterium, 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; Among Fig. 4, M is the protein standard molecular weight, and 1 is the IL-28B behind the purifying, and 2 are bacterial precipitation total protein behind the broken bacterium, and 3 are supernatant total protein behind the broken bacterium.
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 among the GenBank (IL)-28BcDNA gene order and the pET-44Ek/LIC carrier (seeing the specification sheets Cat 711433 of Novagen company):
Upstream primer: 5-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification: on MJ Research PTC-200 grads PCR amplification instrument, adopt warm start (hot-start) pcr amplification.With the pcDNA3.1/V5-His-TOPO-IL-28B 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-28B plasmid is seen: Li Mingcai, He Shaoheng. the clone and the sequential analysis of Ro 24-7472/000 (IL)-28 and IL-29 gene. and cell and molecular immunology magazine, 2004,20 (5): 635-637.
(3) the PCR product is connected with the pET-44Ek/LIC carrier: the IL-28B 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-28B, 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-28B 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-28B 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-28B 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-28B albumen of content more than 98%.The SDS-PAGE collection of illustrative plates of IL-28B expression amount as shown in Figure 4 behind the purifying.
Claims (6)
1, the production method of a kind of Ro 24-7472/000 (IL)-28B is characterized in that adopting gene engineering method production, and 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-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-28B plasmid is template, by the pcr gene amplification, obtains the IL-28B gene fragment;
(3) the IL-28B 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-28B;
(4), make up recombinant il-2 8B engineering bacteria with recombinant vectors pET-44Ek/LIC-IL-28B transform bacteria;
(5) cultivate the recombinant il-2 8B engineering bacteria that makes up, induce it to express and produce IL-28B.
2, the production method of Ro 24-7472/000 IL-28B 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-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-28B plasmid is template, by the pcr gene amplification, obtains the IL-28B gene fragment;
(3) the terminator codon TGA of the IL-28B 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-28B;
(4), make up recombinant il-2 8B engineering bacteria with recombinant vectors pET-44Ek/LIC-IL-28B transformed into escherichia coli;
(5) cultivate the recombinant il-2 8B engineering bacteria that makes up, induce it to express and produce IL-28B.
3, the production method of Ro 24-7472/000 as claimed in claim 1 or 2 (IL)-28B 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 Ro 24-7472/000 as claimed in claim 1 or 2 (IL)-28B is characterized in that step (5) is to adopt IPTG to induce recombinant il-2 8B engineering bacterium expression to produce.
5, the engineering bacteria of a kind of recombinant il-2 8B 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-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-28B plasmid is template, by the pcr gene amplification, obtains the IL-28B gene fragment;
(3) the IL-28B 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-28B;
(4), make up recombinant il-2 8B engineering bacteria with recombinant vectors pET-44Ek/LIC-IL-28B transform bacteria.
6, the engineering bacteria of recombinant il-2 8B 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-GACGACGACAAGATTCCTGTCGCCAGGCTC-3
Downstream primer: 3-GAGGAGAAGCCCGGTTTAGACACACAGGTCCCCGCT-5;
(2) pcr amplification:
With the pcDNA3.1/V5-His-TOPO-IL-28B plasmid is template, by the pcr gene amplification, obtains the IL-28B gene fragment;
(3) the terminator codon TGA of the IL-28B 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-28B;
(4), make up recombinant il-2 8B engineering bacteria with recombinant vectors pET-44Ek/LIC-IL-28B transformed into escherichia coli.
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