CN108218979A - A kind of production method of mouse IL-38 - Google Patents
A kind of production method of mouse IL-38 Download PDFInfo
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- CN108218979A CN108218979A CN201810000545.0A CN201810000545A CN108218979A CN 108218979 A CN108218979 A CN 108218979A CN 201810000545 A CN201810000545 A CN 201810000545A CN 108218979 A CN108218979 A CN 108218979A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The present invention relates to a kind of production methods of mouse IL 38.It is produced using gene engineering method, this method includes the following steps：1) optimization of 38 gene codons of mouse IL and synthesis；2) PCR primer synthesizes；3) PCR amplification obtains 38 segments of mouse IL；4) 38 recombinant vectors of structure pET28a mIL；5) pET 28a mIL 38 are converted into e. coli bl21,38 genetic engineering bacteriums of structure mouse IL；6) 38 genetic engineering bacteriums of culture mouse IL, 38 protein expressions of inducing mouse IL, the recombination IL 38 of the engineering bacterium expression is mainly soluble protein；7) purify 38 albumen of mouse IL that can obtain higher degree to 38 albumen of mouse IL of expression with Ni NAT.The successful preparation of 38 albumen of mouse IL is laid a good foundation to probe into effects and its molecular mechanism of the IL 38 in various mouse inflammation disease models.
Description
Technical field
The invention belongs to genetic engineering fields.More particularly to a kind of production method of mouse IL-38.
Background technology
Interleukin 38 (interleukin-38, IL-38) is newly discovered IL-1 families cell factor, people IL-
38 genes schedule No. 2 chromosome 2q13-14.1, the gene position with coding IL-1Ra (receptor antagonist of IL-1) and IL-36Ra
Point is adjacent.IL-38 genes include 5 extrons, encode the protein being made of 152 amino acid residues, molecule matter
Amount is about 16.9kDa.IL-38 has expression in Various Tissues, such as heart, placenta, fetal livers, skin, spleen, thymus gland, tonsillotome
Deng.Sequence analysis shows that IL-38 albumen and the amino acid composition of IL-1Ra, IL-36Ra albumen have compared with high homology, difference
For 41% and 43%.Binding ability comparative studies with IL-36R finds that the IL-38 of high concentration is apparently higher than the IL- of same concentration
36Ra prompts may have stronger receptor antagonist compared with IL-36Ra in the inflammatory pathways that the IL-38 of high concentration mediates in IL-36R
Agent acts on.IL-38 can inhibit Mycotoruloides stimulation human peripheral blood mononuclearcell (peripheral blood mononuclear
Cell, PBMC) Th17 relevant cell factors (IL-17A and IL-22) are generated, and can reduce what IL-36 γ stimulations PBMC was generated
IL-8 inhibits inflammatory reaction.In view of important function of the IL-38 in inflammatory reaction, it is necessary to it in mouse disease model
Mechanism of action is studied, can be with great expression and the method for preparing high-purity mouse IL-38 albumen so urgent need exploitation is a kind of.
Invention content
The object of the present invention is to provide a kind of production methods of mouse IL-38 albumen.
The technical solution adopted by the present invention is：A kind of production method of mouse IL-38, is produced using technique for gene engineering,
The technique for gene engineering includes the following steps：
1) pUC57-mIL-38 plasmids are built：Mouse IL-38 genes are synthesized, are cloned into pUC57 vector plasmids, are built
pUC57-
MIL-38 plasmids；
2) PCR amplification：Using P1 and P2 as specific primer, III restriction enzyme site of EcoR I and Hind is imported in the both sides of primer,
Using pUC57-mIL-38 plasmids as template, PCR amplification is carried out；The sequence of the specific primer P1 and P2 is：
P1：5’-GGGGAATTCATGTGCTCTCTGCCGATG-3’
P2：5’-CCCAAGCTTACGGCTCATTTCAAAATAAAAT-3’
PCR reaction systems are：Each 2 μ L, pUC57-mIL-38 plasmid template of 2 × PfuMasterMix25 μ L, P1, P2 primers
0.2 μ L add distilled water to be supplemented to 50 μ L.
PCR reaction conditions are：94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C extend 35s, amplification
30 cycles；72 DEG C of extension 10min.
3) structure of pET28a-mIL-38 recombinant expression carriers：Pcr amplification product and pET28a vector plasmids are used respectively
EcoR I, Hind III carry out double digestion, gel recycling mIL-38 and pET28a carriers after double digestion, the mIL-38 after recycling with
PET28a carriers are oriented through T4 ligases and are connected, structure prokaryotic expression plasmid pET28a-mIL-38；
4) it converts：Prokaryotic expression plasmid pET28a-mIL-38 is converted into e. coli bl21, obtains IL-38 recombined engineerings
Bacterium；
5) induced expression of mouse IL-38 albumen：IL-38 recombination engineerings are inoculated in the LB culture mediums containing kanamycins
In, 37 DEG C, 200r/min shake cultures stay overnight；Next day, with 1:50 ratios expand culture, 37 DEG C, 210r/min shake cultures 1.5
~2h, until when OD600 reaches 0.6, the IPTG for adding in final concentration of 0.7mmol/L carries out induced expression, and PCR inductive conditions are：
20 DEG C, 100r/min, 20h, obtain thalline；
6) purifying of mouse IL-38 albumen：Using sonioation method by the cellular lysate of acquisition, centrifuging and taking supernatant is used
Ni-NAT purifies supernatant, obtains the IL-38 albumen of purifying.
The beneficial effects of the invention are as follows：
1. the present invention establishes the production method of mouse IL-38, using technique for gene engineering, structure obtains small according to demand
Mouse IL-38 genetic engineering bacteriums can make mouse IL-38 high efficient expressions, since the mouse IL-38 of the engineering bacterium expression is predominantly solvable
Property ingredient, opposite biological activity is higher after purification.
2. the present invention since the mouse IL-38 PROTEIN Cs end of expression is with 6 × His-Tags, is purified i.e. with Ni-NAT
It can obtain the IL-38 albumen of higher degree.
3. the present invention successfully prepares mouse IL-38 albumen, to probe into effects of the IL-38 in diseases associated with inflammation and mutually shutting down
The researchs such as system provide premise, and are that exploitation controls inflammation in advance and the novel clinical drug of autoimmune disease is laid a good foundation.
Description of the drawings
Fig. 1 is pUC57-mIL-38 plasmid double digestion results；
Wherein, 1：M：DNA standard molecular weights；PUC57-mIL-38 plasmid double digestion results.
Fig. 2 is mouse IL-38 gene magnification result figures；
Wherein, M：DNA standard molecular weights；1：IL-38PCR results.
The structure collection of illustrative plates of Fig. 3 recombinant plasmids pET-28a-mIL-38.
Fig. 4 is recombinant plasmid pET28a-mIL-38 sequencing results.
Fig. 5 is expression and the SDS-PAGE of mouse IL-38 albumen analyzes collection of illustrative plates after purification；
Wherein, M：Protein standard marker；1：Non- Induction of bacterial total protein；Total bacterial protein after 2 inductions；3：Bacterium is broken
Total protein is precipitated after wall；4：Supernatant total protein after bacterium broken wall；5：Mouse IL-38 albumen after purification.
Specific embodiment
1) pUC57-mIL-38 plasmids are built：
Optimize mouse IL-38 gene codons, entrust Sangon Biotech (Shanghai) Co., Ltd.) limited company synthesis mouse IL-
38 genes are cloned into pUC57, build pUC57-mIL-38 plasmids.The DNA of mouse IL-38 genes is as shown in SEQ ID NO.1.
Verify that the results are shown in Figure 1 for double digestion, as seen from Figure 1, through digestion, pUC57-mI through Sma I and BamHI double digestions
L-38 plasmids occur carrier ribbon and target DNA fragment, plasmid construction success in correct position.
2) synthesis of PCR primer
One couple of PCR primers is designed and synthesized according to the mouse IL-38 gene orders of optimization, EcoR is imported in the both sides of primer
I and H ind, III restriction enzyme sites：
Sense primer P1：5’-GGGGAATTCATGTGCTCTCTGCCGATG-3 ' (being EcoR I at underscore),
Downstream primer P2：5’-CCCAAGCTTACGGCTCATTTCAAAATAAAAT-3 ' (being Hind III at underscore).
3) PCR amplification mouse IL-38 genes
Using pUC57-mIL-38 plasmids as template, PCR amplification is carried out with sense primer P1 and downstream primer P2, obtains mouse
IL-38 genetic fragments.
PCR reaction systems are as follows:2 × PfuMasterMix, 25 μ L, each 2 μ L, pUC57-mIL-38 matter of upstream and downstream primer
Grain 0.2 μ L of template, add distilled water to be supplemented to 50 μ L.
PCR reaction conditions are：94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 35s, amplification
30 cycles；72 DEG C of extension 10min.
Pcr amplification product identifies PCR product through 1.0% agarose gel electrophoresis, and the results are shown in Figure 2, as shown in Figure 2,
Go out mouse IL-38 gene orders by template amplification of pUC57-mIL-38 plasmids, sequence 459bp is consistent with electrophoresis result.
4) structure of pET28a-mIL-38 recombinant expression carriers
Pcr amplification product and pET28a vector plasmids are subjected to double digestion with EcoR I, Hind III respectively.It is coagulated after double digestion
Glue recycles mIL-38 and pET28a carriers, and the mIL-38 after recycling is oriented through T4 ligases with pET28a carriers and connected, and structure is former
Nuclear expression plasmid pET28a-mIL-38.
The structure collection of illustrative plates of recombinant expression carrier pET28a-mIL-38 is as shown in Figure 3.
5) it converts
By in prokaryotic expression plasmid pET28a-mIL-38 transformed competence colibacillus recipient bacterium e. coli bl21s (DE3), will convert
Product is coated on the LB Agar Platings containing kanamycins, 37 DEG C of culture about 14h, single bacterium on next day picking culture dish
It falls, and positive recombinant is identified with bacterium colony PCR.Picking positive bacterium colony is inoculated in the LB fluid nutrient mediums containing kanamycins,
37 DEG C of shaken cultivation 14h extract recombinant plasmid, carry out digestion verification.Finally, it is positive restructuring to select digestion and PCR identifications
Plasmid carries out sequencing.Sequencing identifies that correct thalline is mouse IL-38 recombination engineerings.Sequencing result is as shown in Figure 4.
It is identified correctly through sequencing, as recombined small-mouse IL-38 genetic engineering bacteriums.
6) induced expression of mouse IL-38 albumen
Mouse IL-38 recombination engineerings are inoculated in the LB culture mediums containing kanamycins, 37 DEG C, 200r/min concussion trainings
It supports overnight；Next day, with 1:50 ratios, which expand, cultivates, 37 DEG C, 210r/min 1.5~2h of shake culture, until when OD600 reaches 0.6,
The IPTG for adding in final concentration of 0.7mmol/L carries out induced expression, and PCR inductive conditions are：It 20 DEG C, 100r/min, 20h, obtains
Thalline.
7) purifying of mouse IL-38 albumen：Using sonioation method by the cellular lysate of acquisition, centrifuging and taking supernatant is used
Ni-NAT purifies supernatant, obtains the mouse IL-38 albumen that purity reaches more than 95%.
The sample of collection is made into SDS-PAGE analyses, the results are shown in Figure 5, as shown in Figure 5, is induced through IPTG, identified
Recombined small-mouse IL-38 genetic engineering bacteriums can express IL-38 albumen, and it is primarily present in supernatant, purified through Ni-NAT, can
Obtain the IL-38 albumen of more than 95% purity.
The sample mouse IL-38 protein amino acid sequences being collected into are as shown in SEQ ID NO.2, mouse produced by the invention
IL-38 is mainly soluble component, and opposite biological activity is higher after purification.
<110>Liaoning University
<120>A kind of production method of mouse IL-38
<160> 2
<210> 1
<211> 459
<212> DNA
<213>Mouse IL-38
<400> 1
atgtgctctc tgccgatggc tcgttattat attattaaag atgctcacca gaaagctctg 60
tatacccgta acggtcagct gctgctgggt gatccggata gcgataacta ttctccggaa 120
aaagtttgca tcctgccgaa ccgtggtctg gatcgttcta aagttccgat ttttctgggt 180
atgcagggtg gttcttgctg tctggcttgt gttaaaaccc gtgaaggtcc gctgctgcag 240
ctggaagatg ttaacatcga agatctgtat aaaggtggtg aacagaccac ccgtttcacc 300
ttcttccagc gtagcctggg tagcgcgttc cgtctggaag ctgcagcttg tccgggttgg 360
ttcctgtgcg gtccggctga accgcagcag ccggttcagc tgaccaaaga atctgaaccg 420
tctacccata ccgaatttta ttttgaaatg agccgttaa 459
<210> 2
<211> 201
<212>Amino acid
<213>Mouse IL-38 albumen
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val
1 5 10 15
Pro Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met
20 25 30
Gly Arg Gly Ser Glu Phe Met Cys Ser Leu Pro Met Ala Arg Tyr
35 40 45
Tyr Ile Ile Lys Asp Ala His Gln Lys Ala Leu Tyr Thr Arg Asn
50 55 60
Gly Gln Leu Leu Leu Gly Asp Pro Asp Ser Asp Asn Tyr Ser Pro
65 70 75
Glu Lys Val Cys Ile Leu Pro Asn Arg Gly Leu Asp Arg Ser Lys
80 85 90
Val Pro Ile Phe Leu Gly Met Gln Gly Gly Ser Cys Cys Leu Ala
95 100 105
Cys Val Lys Thr Arg Glu Gly Pro Leu Leu Gln Leu Glu Asp Val
110 115 120
Asn Ile Glu Asp Leu Tyr Lys Gly Gly Glu Gln Thr Thr Arg Phe
125 130 135
Thr Phe Phe Gln Arg Ser Leu Gly Ser Ala Phe Arg Leu Glu Ala
140 145 150
Ala Ala Cys Pro Gly Trp Phe Leu Cys Gly Pro Ala Glu Pro Gln
155 160 165
Gln Pro Val Gln Leu Thr Lys Glu Ser Glu Pro Ser Thr His Thr
170 175 180
Glu Phe Tyr Phe Glu Met Ser Arg Lys Leu Ala Ala Ala Leu Glu
185 190 195
His His His His His His
200
Claims (4)
1. a kind of production method of mouse IL-38, which is characterized in that produced using technique for gene engineering, the genetic engineering
Technology includes the following steps：
1) pUC57-mIL-38 plasmids are built：Mouse IL-38 genes are synthesized, are cloned into pUC57 vector plasmids, build pUC57-
MIL-38 plasmids；
2) PCR amplification：Using P1 and P2 as specific primer, III restriction enzyme site of EcoR I and Hind is imported in the both sides of primer, with
PUC57-mIL-38 plasmids are template, carry out PCR amplification；The sequence of the specific primer P1 and P2 is：
P1：5’-GGGGAATTCATGTGCTCTCTGCCGATG-3’
P2：5’-CCCAAGCTTACGGCTCATTTCAAAATAAAAT-3’
3) structure of pET28a-mIL-38 recombinant expression carriers：Pcr amplification product and pET28a vector plasmids are used into EcoR respectively
Ith, Hind III carries out double digestion, and gel recycling mIL-38 is with pET28a carriers after double digestion, mIL-38 and pET28a after recycling
Carrier is oriented through T4 ligases and is connected, structure prokaryotic expression plasmid pET28a-mIL-38；
4) it converts：Prokaryotic expression plasmid pET28a-mIL-38 is converted into e. coli bl21, obtains IL-38 recombination engineerings；
5) induced expression of mouse IL-38 albumen：IL-38 recombination engineerings are inoculated in the LB culture mediums containing kanamycins,
37 DEG C, 200r/min shake cultures stay overnight；Next day, with 1:50 ratios expand culture, 37 DEG C, 210r/min shake cultures 1.5~
2h, until when OD600 reaches 0.6, the IPTG for adding in final concentration of 0.7mmol/L carries out induced expression, obtains thalline；
6) purifying of mouse IL-38 albumen：Using sonioation method by the cellular lysate of acquisition, centrifuging and taking supernatant uses Ni-
NAT purifies supernatant, obtains the IL-38 albumen of purifying.
2. a kind of production method of mouse IL-38 according to claim 1, which is characterized in that in step 2), PCR reactions
System is：Each 0.2 μ L of 2 μ L, pUC57-mIL-38 plasmid template of 2 × PfuMasterMix25 μ L, P1, P2 primers, add distilled water
It is supplemented to 50 μ L；PCR reaction conditions are：94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 35s,
30 cycles of amplification；72 DEG C of extension 10min.
3. a kind of production method of mouse IL-38 according to claim 1, which is characterized in that in step 5), PCR inductions
Condition：20 DEG C, 100r/min, 20h.
4. the mouse IL-38 albumen according to the production of claim 1-3 any one of them method controls inflammation and itself in advance in preparation
Application in immunity disease drug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576277A (en) * | 2019-01-17 | 2019-04-05 | 辽宁大学 | A kind of mouse truncates the production method of IL-36 α albumen |
| CN112358539A (en) * | 2020-11-16 | 2021-02-12 | 桂林医学院附属医院 | Mouse truncated IL-36 gamma protein and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397038A (en) * | 2013-07-29 | 2013-11-20 | 宁波大学 | Production method of human interleukin-38 |
| CN107007821A (en) * | 2017-05-04 | 2017-08-04 | 宁波大学 | Interleukin Ⅲ 8 is preparing the application in preventing and treating obesity and metabolic syndrome product |
-
2018
- 2018-01-02 CN CN201810000545.0A patent/CN108218979A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103397038A (en) * | 2013-07-29 | 2013-11-20 | 宁波大学 | Production method of human interleukin-38 |
| CN107007821A (en) * | 2017-05-04 | 2017-08-04 | 宁波大学 | Interleukin Ⅲ 8 is preparing the application in preventing and treating obesity and metabolic syndrome product |
Non-Patent Citations (1)
| Title |
|---|
| MAN CHUA等: "In vivo anti-inflammatory activities of novel cytokine IL-38 in Murphy Roths Large (MRL)/lpr mice", 《IMMUNOBIOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109576277A (en) * | 2019-01-17 | 2019-04-05 | 辽宁大学 | A kind of mouse truncates the production method of IL-36 α albumen |
| CN109576277B (en) * | 2019-01-17 | 2022-06-14 | 辽宁大学 | Production method of mouse truncated IL-36 alpha protein |
| CN112358539A (en) * | 2020-11-16 | 2021-02-12 | 桂林医学院附属医院 | Mouse truncated IL-36 gamma protein and preparation method thereof |
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