CN104561021A - Method for modifying chicken interleukin 2 expressing gene and gene modified by virtue of method - Google Patents
Method for modifying chicken interleukin 2 expressing gene and gene modified by virtue of method Download PDFInfo
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- CN104561021A CN104561021A CN201410811347.4A CN201410811347A CN104561021A CN 104561021 A CN104561021 A CN 104561021A CN 201410811347 A CN201410811347 A CN 201410811347A CN 104561021 A CN104561021 A CN 104561021A
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Abstract
The invention provides a method for modifying a chicken interleukin 2 expressing gene and the gene modified by virtue of the method, aiming at an expression system taking escherichia coli as a host. According to the method for modifying the chicken interleukin 2 expressing gene, rare codons in escherichia coli are replaced by non-rare codons based on a natural chicken interleukin 2 expressing gene, so that the expression efficiencies of escherichia coli on chicken interleukin 2 and derived protein of the chicken interleukin 2 are improved. According to the gene provided by the invention, the expression efficiency of the gene in escherichia coli can be improved, chicken interleukin 2 protein expressed by the gene efficiently expresses in a soluble protein manner in escherichia coli, after the expression, recombinant protein accounts for over 50% of the total bacterial protein, and furthermore, the steps of processing inclusion bodies and renaturation are omitted; furthermore, the production cycle is short, the cost is low, and a good foundation is laid for the scale production of the chicken interleukin 2.
Description
Technical field
The present invention relates to gene engineering technology field, the gene be specifically related to a kind of chicken interleukin-2 2 expressing gene remodeling method, utilizing the method transformation to obtain.
Background technology
Interleukin II (interleukin 2, IL-2) be the glycoprotein that T cell and natural killer sexual cell (NK cell) produce, it is the cytokine that in animal body, a class is important, have and activate Lymphatic circulation and natural killer cell, stimulate T accessory cell and natural killer cell to produce cytokine and promote the breeding of cytokine, playing an important role in the immunne response of body.
The researchdevelopment of people and many Mammals IL-2 is very fast, treatment and the vaccine immunity of various disease are widely used in, and chicken IL-2 gene researchdevelopment is relatively slow, 1997, Sundick and Gill-Dixon is by the method in construction cDNA library, the IL-2cDNA of chicken is obtained first, for the research of chicken IL-2 gene is opened up the game from the T lymphocyte that Con A activates.Afterwards, domestic numerous investigators have then cloned the IL-2 gene of more than ten Local Chicken Breeds such as gu-shi chicken, Xiaoshan chicken, white plumage Gallus Domesticus in succession.At present, achieved chicken IL-2 gene gene to express by intestinal bacteria, yeast or eukaryotic cell.In above phraseology, utilize that intestinal bacteria are higher as expression vector stability, technique is easy to control, be most widely used, but there is more intestinal bacteria rare codon due to chicken interleukin-2 2 expressing gene, therefore have impact on productive rate.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, and the gene provide a kind of chicken interleukin-2 2 expressing gene remodeling method, utilizing the method transformation to obtain, to solve in prior art the technical problem utilizing escherichia coli expression chicken interleukin-2 2 productive rate lower.
Another technical problem that the present invention solves is by promoting the effect of the chook Interleukin-2 protein expressed by it to exocytosis to the improvement of chicken interleukin-2 2 expressing gene.
The another technical problem that the present invention solves is by the improvement of chicken interleukin-2 2 expressing gene thus the chook Interleukin-2 protein later-period purification be convenient to expressed by it.
The technical problem again that the present invention solves is by providing the method utilizing above-mentioned modifying gene to express chicken interleukin-2 2, to promote expression efficiency.
For realizing above technical purpose, the present invention by the following technical solutions:
A kind of chicken interleukin-2 2 expressing gene remodeling method, with the non-rare codon replacement of intestinal bacteria intestinal bacteria rare codon wherein on the basis of the natural expressing gene of chicken interleukin-2 2.
Preferably, the Nucleotide that the protein N terminal expressed by described chicken interleukin-2 2 expressing gene is corresponding is connected with signal peptide or utilizes the signal peptide on expression vector; Can do preferably following on this basis further: signal peptide is pelB, by expression vector pET-22b (+) is with, be positioned at multiple clone site N end.
Preferably, described chicken interleukin-2 2 expressing gene end has label protein; Can do preferably following on this basis further: described label protein is the wherein a kind of of His label, Arg label, FLAG label or GST label.
Meanwhile, present invention also offers chicken interleukin-2 2 expressing gene utilizing the transformation of one of aforesaid method to obtain, its nucleotide sequence is as shown in SEQ ID NO2.
Meanwhile, present invention also offers chicken interleukin-2 2 expressing gene utilizing the transformation of one of aforesaid method to obtain, its nucleotide sequence is as shown in SEQ ID NO3.
Simultaneously, present invention also offers a kind of nucleotide sequence that utilizes as the method for the genetic expression chicken interleukin-2 2 of SEQ ID NO2, the method by nucleotide sequence as the gene of SEQ ID NO2 inserts in expression vector, then this recombinant expression vector is imported intestinal bacteria, with isopropyl-beta D-thio galactopyranoside for inductor carries out abduction delivering, induced concentration is 0.1 ~ 0.3mmol/L, inducing temperature is 14 ~ 20 DEG C, the abduction delivering time is 14 ~ 20 hours, and described expression vector is any one of pBV220, pET serial carrier or pQE serial carrier.
On the basis of this expression method:
Preferably, described pET serial carrier is pET22b (+) carrier.
Preferably, described intestinal bacteria are bacillus coli DH 5 alpha, BL21 (DE3) or JM109.
Method provided by the invention and gene are specially for prokaryotic expression system, especially be the expression system of host with intestinal bacteria, therefore genetic modification method of the present invention wherein will replace with non-rare codon by colibacillary rare codon on the basis of the natural expressing gene of chicken interleukin-2 2, thus improve the expression efficiency of intestinal bacteria to chicken interleukin-2 2 and derived protein thereof.On this basis, the albumen after the present invention achieves transformation by the replacement to chicken interleukin-2 2 expressing gene codon, interpolation or disappearance expressed by gene has the biological activity of chicken interleukin-2 2.In addition, the present invention by increasing signal peptide gene thus make expressed albumen n end be connected with signal peptide on chicken interleukin-2 2 expressing gene, and then promotes that protein excretion is in periplasmic or substratum.In addition, the present invention by setting up label protein gene thus make expressed albumen n end or C end be connected with label protein on chicken interleukin-2 2 expressing gene, and then is convenient to later stage separation and purification.
The Nucleotide quantity of the gene of sequence provided by the invention as shown in SEQ ID NO2 and the natural expressing gene of chicken interleukin-2 2 is 366, and its aminoacid sequence of expressed albumen is identical.Except can promoting the expression efficiency of this gene in intestinal bacteria; utilize chook Interleukin-2 protein expressed by this gene in intestinal bacteria with the form high expression of soluble protein; after expressing, recombinant protein accounts for more than 50% of bacterial protein; and eliminate step such as process inclusion body, renaturation etc.; and it is with short production cycle; cost is low, for the large-scale production of chicken interleukin-2 2 is had laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the natural expressing gene of chicken interleukin-2 2 (ChIL-2-N) sequence and SEQ ID NO2 sequence (ChIL-2-O) comparison diagram of the present invention;
Fig. 2 is the pcr amplification result that agarose gel electrophoresis detects ChIL-2-O gene, and wherein M is Marker, L1 is PCR primer electrophoresis result, and L2 is negative control.
Fig. 3 is after SDS-PAGE detects induction and without the expression of results of ChIL-2 albumen in intestinal bacteria of inducing, wherein M is non-pre-dyed albumen Marker.
Fig. 4 be SDS-PAGE detect ChIL-2 albumen Ni+ affinitive layer purification result wherein M be non-pre-dyed albumen Marker.
Fig. 5 is the Activity determination result that ChIL-2 albumen urgees chicken periphery blood T lymphocyte propagation.
Fig. 6 is that the gene order of adding His label protein on the present invention's improved chicken interleukin-2 2 expressing gene (ChIL-2-O) basis again (namely uses primers F 1R1, gene after PCR), wherein be scribed ss restriction enzyme site, italic is the encoding sequence of 6 × His label, and gray background part is ChIL-2-O gene.
Embodiment
Below will be described in detail the specific embodiment of the present invention.In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.
The approximating language used in following examples can be used for quantitative expression, shows to allow quantity to have certain variation when not changing basic function.Therefore, this exact value itself is not limited to the numerical value that the language such as " approximately ", " left and right " is revised.In certain embodiments, " approximately " represents and allows its numerical value revised to change in the positive and negative scope of 10 (10%), such as, and any numerical value that what " about 100 " represented can be between 90 to 110.In addition, in the statement of " about first numerical value is to second value ", revise the first and second numerical value two numerical value approximately simultaneously.In some cases, approximating language may be relevant with the precision of surveying instrument.
Apart from outside definition, technology used in following examples and scientific terminology have the identical meanings generally understood with those skilled in the art of the invention.
Test reagent consumptive material used in following examples, if no special instructions, is routine biochemistry reagent; Described experimental technique, if no special instructions, is ordinary method; Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged; % in following examples, if no special instructions, is mass percentage.
Embodiment 1
Present embodiment describes the preparation method of chicken interleukin-2 2 (ChIL-2-O) gene of natural chicken interleukin-2 2 (ChIL-2-N) gene provided by the invention and optimization.
According to the mRNA sequence of AF483600.1 in GeneBank, after removal signal peptide sequence, be the nucleotide sequence (shown in SEQ ID NO1) of natural chicken interleukin-2 2 (ChIL-2-N) gene provided by the invention.
By the analysis of intestinal bacteria (E.coli) codon analysis software, and according to the preferences of intestinal bacteria to codon, natural chicken interleukin-2 2 (ChIL-2-N) gene has been carried out to replacement rare codon, regulated the optimizations such as GC content, namely chicken interleukin-2 2 (ChIL-2-O) gene (shown in SEQ ID NO2) of optimization is obtained, can at E. coli.Then carry out synthetic according to this sequence.
Embodiment 2
Present embodiment describes the structure of recombinant plasmid containing ChIL-2-O gene and engineering bacteria.
One, the amplification of ChIL-2-O gene
According to above-mentioned ChIL-2-O gene order, utilize Primer Premire software design primer, introduce Nco I restriction enzyme site and 6 × His sequence label at 5 ' end of primer respectively, 3 ' end introduces Hind III digestion site simultaneously:
Upstream primer (F1): 5 '-
cCATGGgTCATCATCATCATCATCACGGTGGCGGTGCAAGCCTGAGCAGCG-3 ' (dashed part is Nco I restriction enzyme site);
Downstream primer (R1): 5 '-
aAGCTTtTATTTCTGCAGATAGCG-3 ' (dashed part is Hind III digestion site).
With the ChIL-2-O gene order of synthesis for template, with F1 and R1 for primer carries out pcr amplification, reaction system contains template 1 μ g, each 1 μM of upstream and downstream primer, and total reaction system is 50 μ L; Reaction conditions is: 94 DEG C, 5min; 94 DEG C, 30sec, 54 DEG C, 30sec, 72 DEG C, 45sec, 30 circulations; 72 DEG C, 10min.PCR primer detects through 1.5% agarose gel electrophoresis, and as shown in Figure 2, wherein, the electrophoresis result of M to be Marker, L1 be PCR primer, No. L2 is negative control to detected result, and result shows there is clear band at about 407bp.
Two, the structure of recombinant plasmid and engineering bacteria
1. use DNA to reclaim test kit and reclaim PCR primer.
2., with the pcr amplification product that restriction enzyme Nco I and Hind III double digestion have reclaimed, obtain digestion products;
3., with restriction enzyme Nco I and Hind III double digestion expression vector pET22b (+), utilize DNA to reclaim test kit and reclaim carrier framework;
4. by the digestion products of step 2 and the carrier framework of step 3, connect with T4DNA ligase enzyme 16 DEG C and spend the night, namely obtain connecting product;
5. will connect product conversion bacillus coli DH 5 alpha competent cell, picking list bacterium colony carries out bacterium colony PCR and enzyme cuts qualification: 1) bacterium colony PCR uses primer to be F1 and R1, and result has the bacterium colony of clear single band to be positive bacterium colony at about 407bp; 2) enzyme is cut and is identified that enzyme used is restriction enzyme Nco I and Hind III, has the bacterium colony of clear single band to be positive bacterium colony at about 407bp and 5400bp simultaneously; 3) be all that positive recombinant plasmid carries out DNA sequencing by above verification result.Check order correct namely for the purpose of recombinant plasmid, called after ChIL-2-pET22b (+).
6. by recombinant plasmid ChIL-2-pET22b transformation of E. coli BL21 (DE3), the single bacterium colony of rear screening, and carry out digestion verification.Result is positive bacterium colony, and recombinant bacterial strain namely, the Host Strains of called after is BL21 (DE3)-ChIL-2-pET22b (+).
Embodiment 3
Present embodiment describes the abduction delivering of recombinant bacterial strain BL21 (DE3)-ChIL-2-pET22b (+), and the purifying of the recombination chicken interleukin-22 (ChIL-2) of expressing.
One, the abduction delivering of recombinant bacterial strain BL21 (DE3)-ChIL-2-pET22b (+)
1. recombinant bacterial strain BL21 (DE3)-ChIL-2-pET22b (+) is inoculated in 100mL containing ammonia benzyl resistance (Amp
+) LB liquid medium in, 37 DEG C, on the shaking table of 200rpm concussion cultivate, to OD
600when value is 0.8, move to 16 DEG C of shaking tables after adding inductor IPTG (isopropyl-beta D-thio galactopyranoside, final concentration 0.2mM), 150rpm continues inducing culture, and induction time is 16 hours; Establish control group, except not adding inductor IPTG, other operations are identical simultaneously.
2. get induction respectively and respectively get 5mL with the fermentation culture of not inducing, the centrifugal 20min of 8000-10000rpm collects thalline.Then add 500 μ L Tris-HCl (PH 7.9) resuspended thalline, after ultrasonication, the centrifugal 10min of 14000g, collects supernatant and precipitation (precipitate still use 500 μ L Tris-HCl resuspended) respectively.
3. get each 60 μ L of above 4 kinds of samples, add 20 μ L 4 × albumen sample-loading buffer (containing DTT) respectively, after mixing, boil 5-10 minute.Then each loading 10 μ L carries out SDS-PAGE electrophoresis detection, and electrophoresis detection result as shown in Figure 3, locates appearance protein band at about 15kD in the thalline supernatant after induction, conforms to expection size.
Two, the purifying of recombination chicken interleukin-22 (ChIL-2)
1., after collecting thalline, it is resuspended that every 100mg thalline (weight in wet base) adds 1-5mL Tris-HCl (PH 7.9), ultrasonic degradation thalline.
2.14000g, 4 DEG C centrifugal 10 minutes, collect centrifuged supernatant.
3., after the Binding Buffer of enough volumes to be used balances chromatography column, loading can be started.With Binding Buffer by cellular lysate liquid equimultiple dilution back loading upper prop, flow velocity be 10 times of column volumes/hour.
4. use the Binding Buffer of 15 times of column volumes to rinse pillar, wash away foreign protein.
5. use the Elution Buffer (50mM imidazoles) of 5 times of column volumes to wash foreign protein.
6. use Elution Buffer (200mM imidazoles) wash-out of 5 times of column volumes, collect elution peak.
Attention: elution peak can be in charge of collection, every 1ml collects 1 pipe, and adopts albumen monitor to monitor, and collects elution peak.
7. use Elution Buffer (500mM imidazoles) thoroughly to clean albumen on post.
8. in purge process, often step need get the sample of 60 μ L, adds 20 μ L 4 × albumen sample-loading buffer (containing DTT) respectively, boils 5-10 minute after mixing.Then each loading 10 μ L carries out SDS-PAGE electrophoresis detection, and electrophoresis detection result as shown in Figure 4.Result shows: after two kinds of different buffer solution foreign proteins, use 200mM imidazoles Elution Buffer can completely by by target protein (recombination chicken interleukin-22, ChIL-2) wash-out, and the purity of ChIL-2 can reach more than 95%.
9. adopt Bradford method to measure the protein concentration of ChIL-2 protein solution before and after purifying, calculating the rate of recovery rate of recovery can reach more than 90%, also reaches good concentrated effect simultaneously.
Table 1 protein purification buffer formulation
| Composition | Tris-HCl(PH 7.9) | Imidazoles | NaCl |
| Binding Buffer | 20mM | 10mM | 0.5M |
| Elution Buffer | 20mM | 50/200/500mM | 0.5M |
Embodiment 4
Present embodiment describes the bioactive mensuration of recombination chicken interleukin-22 (ChIL-2) chicken peripheral T lymphocyte propagation.IL-2 produces primarily of activating T cell, and again by T cell propagation is required, therefore IL-2 can urge chicken peripheral blood lymphocyte and breeds in a large number, carries out the level of biological activity of IL-2 in mensuration measuring samples with this.
1) new freshly-slaughtered poultry anticoagulation 5mL is got, after 1:1 mixes by volume with Hank ' s liquid, be added on the face of cellular segregation liquid of 10mL gently, with 1500-2000rpm centrifugal (radius 15cm horizontal rotor) 15 minutes, get oyster white ring-type buffy coat and put into centrifuge tube containing 10mL Hank ' s liquid, after abundant mixing, with the centrifugal 10-30 minute of 1500-2000rpm, sedimentation cell is washed repeatedly and namely obtains required cell 2 times;
2) cell of precipitation has again been hanged with RPIM1640 cell culture medium, adjustment cell concn to 5 × 10
6individual/mL, and add con A (ConA, final concentration 5 μ g/mL), in 5%CO
2, 42 DEG C cultivate 42 hours;
3) collected by centrifugation periphery blood T lymphocyte, resuspended with RPIM1640 cell culture medium, its concentration of counting adjustment is 5 × 10
6individual/mL, as responsive cell; Get aseptic 96 orifice plate kind plates, the every hole of experimental port adds responsive cell 50 μ L, and zeroing hole does not add cell;
4) ChIL-2 protein sample is suitably diluted, after making adjustment, protein concentration reaches 100ng/mL, then dilutes successively with 2 times of dilution methods with RPIM1640 cell culture medium, and every hole adds 50 μ L samples, each dilution gradient does 6 repeating holes, 5%CO2,42 DEG C of cultivation 36h; Zeroing hole (do not add cell, mend it with cell culture medium) and negative control hole (do not add protein sample, mend it with cell culture medium) are set simultaneously;
5) every hole adds MTS solution 30 μ L, and after continuing to cultivate 3h, the shaking table being placed in low speed concussion shakes 10min, and microplate reader detector measures A
490value, result as shown in Figure 5.
Result as can be seen from figure, the ChIL-2 albumen dilution 2 of purifying
7times time, the ability of its induction chicken periphery blood T lymphocyte propagation, still significantly improve (P < 0.01 than blank group, with * mark in figure), and there is significant concn dependency, namely along with the increase of ChIL-2 protein concentration, it promotes that the activity of chicken periphery blood T lymphocyte propagation strengthens gradually.Criterion is (experimental port OD
490value-zeroing hole OD
490value)/(the empty OD of negative control
490value-zeroing hole OD
490value)>=1.4, for having significantly short T lymphocyte proliferation activity.
Above embodiments of the invention have been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a chicken interleukin-2 2 expressing gene remodeling method, is characterized in that: with the non-rare codon replacement of intestinal bacteria intestinal bacteria rare codon wherein on the basis of the natural expressing gene of chicken interleukin-2 2.
2. a kind of chicken interleukin-2 2 expressing gene remodeling method according to claim 1, is characterized in that: the Nucleotide that the protein N terminal expressed by described chicken interleukin-2 2 expressing gene is corresponding is connected with signal peptide or utilizes the signal peptide on expression vector.
3. a kind of chicken interleukin-2 2 expressing gene remodeling method according to claim 2, is characterized in that described signal peptide is: pelB, by expression vector pET-22b (+) is with, is positioned at multiple clone site N end.
4. a kind of chicken interleukin-2 2 expressing gene remodeling method according to claim 1, is characterized in that: described chicken interleukin-2 2 expressing gene end has label protein.
5. a kind of chicken interleukin-2 2 expressing gene remodeling method according to claim 4, is characterized in that described label protein is the wherein a kind of of His label, Arg label, FLAG label or GST label.
6. utilize chicken interleukin-2 2 expressing gene that method described in claim 1 transformation obtains, its nucleotide sequence is as shown in SEQ ID NO2.
7. utilize chicken interleukin-2 2 expressing gene that method described in claim 2 or 3 transformation obtains, its nucleotide sequence is as shown in SEQ ID NO3.
8. one kind utilizes genetic expression chicken interleukin-2 2 method described in claim 6 or 7, it is characterized in that comprising the following steps: this gene is inserted in expression vector, then this recombinant expression vector is imported intestinal bacteria, with isopropyl-beta D-thio galactopyranoside for inductor carries out abduction delivering, induced concentration is 0.1 ~ 0.3mmol/L, inducing temperature is 14 ~ 20 DEG C, and the abduction delivering time is 14 ~ 20 hours.
9. method according to claim 8, is characterized in that described expression vector is any one of pBV220, pET serial carrier or pQE serial carrier.
10. method according to claim 8, is characterized in that described intestinal bacteria are bacillus coli DH 5 alpha, e. coli bl21 (DE3) or e. coli jm109.
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| CN105154497A (en) * | 2015-07-14 | 2015-12-16 | 天津瑞普生物技术股份有限公司 | Method for preparing chicken interleukin 2 by aid of recombinant Escherichia coli |
| CN106434685A (en) * | 2016-11-28 | 2017-02-22 | 大连海洋大学 | Gene and method for preparing recombination fugu rubripe interleukin-2 protein |
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Application publication date: 20150429 |