CN103193887B - Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein - Google Patents
Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein Download PDFInfo
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Abstract
The invention provides a recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein, as well as an encoding gene and expressing, purifying and inclusion body renaturing methods of the fusion protein. The IL2 realizes a very important immunomodulatory effect in a disease generation process, but has the defects of high elimination speed in blood plasma and high industrialization cost. The invention provides the recombinant porcine IL2-Fc fusion protein by utilizing a prokaryotic expression system of escherichia coli in order to obtain a large amount of porcine IL2 with a longer half-life period, wherein a porcine IL2 part is all sequences of a porcine IL2 extracellular region, an Fc segment part comprises a hinge region, a CH2 region and a CH3 region of an antibody, and the porcine IL2 part and the Fc segment part are directly fused. The recombinant porcine IL2-Fc fusion protein provided by the invention saves most of biological activity of IL-2, greatly prolongs the half-life period and provides the guarantee for low-cost large-scale expression and the industrialization.
Description
Technical field
The invention belongs to biotechnology gene field, relate to a kind of Recombinant Swine interleukin II-Fc fusion rotein and encoding gene thereof, and its expression, purifying and renaturing inclusion bodies method.
Background technology
Interleukin II (Interleukin-2, be called for short IL-2) be a kind of important cytokine produced by the T lymphocyte activated, can break up and secretory antibody with promotion B cell by activated T cell, induction produces Interferon, rabbit, strengthen monocyte and natural killer cell (natural killer cell, be called for short NK) killing activity, in the immune response of body, have important regulating effect, be the natural immunostimulant of a class.In therapeutic treatment field, commercial recombinated interleukin-2 is for the assisting therapy of the diseases such as tumour, acquired immune deficiency syndrome (AIDS), hepatitis B.In veterinary drug, because interleukin II can improve the immunne response of vaccine, reduce the generation of disease, also show wide application prospect.At present, more to the research of chicken, cattle interleukins-2 2 gene, both express all in protokaryon and eukaryotic expression system.Research display, the recombinant interleukin 2 of chicken and multiple cause of disease vaccine conbined usage, can significantly improve immune level, minimizing stress, the side effect of reduction vaccine, thus reaches stronger therapeutic action.China is big country of raising pigs in the world, the kinds of Diseases of pig are various, particularly the M & M that causes of viral infectious is the highest, causes huge financial loss every year to pig industry, therefore the treatment of these virus diseases and prevention is become to the top priority of pig industry.Interleukin II strengthens immune, to resist virus character and makes it in the virus disease of pig, demonstrate wide application prospect, but actually rare for the research of pig interleukin 2 and 6.
On the other hand, as small molecular weight protein, pig interleukin 2 and 6 is also the same with other interleukin IIs, there is the defect of plasma clearance speed, thus causes in clinical treatment, need repeatedly medication just can reach result for the treatment of.Equally, the frequent of transformation period short and medication also will become the bottleneck using pig interleukin 2 and 6 treatment porcine viral diseases.IgG immunoglobulin (Ig) is the main antibody in humans and animals body, and its transformation period is in vivo about 20 days.Its stability is because the Fc fragment of IgG can combine with neonatal Fc receptor (FcRn), avoids IgG to enter in lysosome and is degraded.As can be seen here, can consider that the Fc fragment increasing IgG on pig interleukin 2 and 6 is to form fusion rotein, to improve pig interleukin 2 and 6 Half-life in vivo, reaches long-acting object.
Thus, the invention provides one and can retain the original activity of pig interleukin 2 and 6, the pig interleukin 2 and 6 of Half-life in vivo and the fusion rotein of Fc fragment can be extended again.
But, mostly be by the recombinant protein of escherichia coli expression do not dissolve, the intracellular aggregates of non-activity, i.e. inclusion body.The renaturation of inclusion body is a very complicated process, if denaturing conditions is not suitable for there will be the mispairing of intramolecular disulfide bond, intermolecular covalent attachment or hydrophobic binding form polymer, reduce the ratio motility rate of recombinant protein on the one hand, cause quality product defective, produce Precipitation again simultaneously, affect yield.Therefore, another technical problem to be solved by this invention adopts suitable method that the inclusion bodies of protein of escherichia coli expression is carried out renaturation.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, by codon optimized mode, provide a kind of can the Recombinant Swine interleukin II-Fc fusion rotein of high expression and its gene and expression in intestinal bacteria, purifying, refolding method.
The invention provides Recombinant Swine interleukin II-Fc fusion rotein, described fusion rotein comprises pig interleukin 2 and 6 part and Fc segment portion, wherein, pig interleukin 2 and 6 part is the full sequence of pig interleukin 2 and 6 extracellular region, Fc segment portion comprises hinge area, CH2 district and CH3 district, is directly to merge between pig interleukin 2 and 6 and Fc segment portion.
Fc fragment is wherein selected from the immunoglobulin Fc of human or animal, and be Fc total length or partial sequence, Fc is selected from IgG, IgM, IgD, IgA, and often kind of immunoglobulin class comprises each hypotype, as IgG1, IgG2, IgG3, IgG4.Fc segment portion in fusion rotein of the present invention is particularly preferably from the IgG1 of pig.
Preferably, Recombinant Swine interleukin II-Fc fusion rotein of the present invention has the aminoacid sequence shown in SEQ ID NO:2, wherein 1-134 amino acids residue is the extracellular domain sequence of pig interleukin 2 and 6, and 135-334 amino acids residue is pig IgG 1 sequence.
The invention provides the gene of Recombinant Swine interleukin II-Fc fusion rotein described above of encoding, its base sequence is as shown in SEQID NO:1.This sequence aims at escherichia expression system to carry out the codon optimized sequence obtained, and can significantly improve the expression of heterologous gene in Host Strains by contrast.
Present invention also offers the plasmid of the gene containing coding Recombinant Swine interleukin II-Fc fusion rotein described above, described plasmid is preferably prokaryotic expression plasmid, most preferably is pET21b carrier.
Present invention also offers the coli strain including plasmid described above, preferably, described bacterial strain is selected from e. coli bl21 (DE3) bacterial strain.
Present invention also offers Recombinant Swine interleukin II-Fc fusion rotein in escherichia coli expression method, comprise the steps:
Steps of the method are:
1. picking one or more contain the E. coli clones of Recombinant Swine interleukin II-Fc fusion rotein described above, access LB nutrient solution, in 37 DEG C of overnight incubation;
2. get appropriate overnight culture, access in the LB nutrient solution of 100 times amount of described overnight culture, be cultured to mid-log phase OD in 37 DEG C of concussions
600=1.0;
3. in culture, add IPTG to 1mmol/L, in 37 DEG C, after abduction delivering 4h, in 4 DEG C with rotating speed 5000rpm, centrifugal treating 15min, collect the coli somatic containing Recombinant Swine interleukin II-Fc fusion rotein.
All containing penbritin 50-100 μ g/ml in described LB nutrient solution.
Expression method described above of the present invention is through the repeated multiple times experiment of contriver and gropes and verify the effective means the most for expressing Recombinant Swine interleukin II-Fc fusion rotein obtained, the expression amount of the method is high, and express obtain renaturing inclusion bodies after activity higher.
Present invention also offers the inclusion body purification method of Recombinant Swine interleukin II-Fc fusion rotein, comprise the steps:
1. precipitate collecting obtain above-mentioned containing induction Recombinant Swine interleukin II-Fc fusion rotein intestinal bacteria, resuspended with the PBS of precooling, and in 4 DEG C of high speed centrifugation process; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A3-10mL, stirs, thalline is hanged with slicking glass rod.
3. it be the PMSF of 100mmol/L, 3-100 μ L concentration is the N,O-Diacetylmuramidase of 100mg/mL that every gram (weight in wet base) thalline adds 3-10 μ L concentration, in stirring on ice.
4. broken thalline, sample is placed on ice, ultrasonic, and in 4 DEG C of high speed centrifugation process.
5. precipitation lavation buffer solution Buffer B washing, and in 4 DEG C of high speed centrifugation process, precipitation inclusion body, repeats once.
6. inclusion body precipitation denaturation buffer Buffer C dissolves, stirred at ambient temperature 30-60min.
7. the fully rear room temperature high speed centrifugation process of mixing, abandons precipitation, gets supernatant, namely obtain Recombinant Swine interleukin II-Fc fusion rotein denaturing soln.
This purification process preferred steps is as follows:
1. precipitate collecting obtain above-mentioned containing induction Recombinant Swine interleukin II-Fc fusion rotein intestinal bacteria, resuspended with the PBS of precooling, in 4 DEG C, with the centrifugal 15min of the rotating speed of 12000rpm; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A5mL, stirs, thalline is hanged with slicking glass rod.
3. it is the PMSF of 100mmol/L that every gram (weight in wet base) thalline adds 5 μ L concentration, and 5 μ L concentration are the N,O-Diacetylmuramidase of 100mg/mL, stir 20min on ice.
4. with the broken thalline of probe type ultrasonication ripple instrument, sample is placed on ice, ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, wait for sample cooling.In 4 DEG C, with the centrifugal 15min of the rotating speed of 12000rpm, abandon supernatant.
5. precipitation lavation buffer solution Buffer B washing, in 4 DEG C, with the centrifugal 15min of the rotating speed of 12000rpm, precipitation inclusion body, repeats once.
6. inclusion body precipitation denaturation buffer Buffer C dissolves, stirred at ambient temperature 30min.
7. fully after mixing under room temperature with the centrifugal 15min of the rotating speed of 12000rpm, abandon precipitation, get supernatant, namely obtain Recombinant Swine interleukin II-Fc fusion rotein denaturing soln.
Present invention also offers the renaturing inclusion bodies method of the Recombinant Swine interleukin II-Fc fusion rotein after optimization, comprise the steps:
Get the Recombinant Swine interleukin II-Fc fusion rotein denaturing soln that appropriate denaturation buffer Buffer C dissolves, with renaturation buffer Buffer D, protein concentration is diluted to 0.2mg/mL, when 4 DEG C of renaturation are to 24h, by the 0.45 μm of membrane filtration of recombinant protein solution after renaturation, namely obtain the Recombinant Swine interleukin II-Fc fusion rotein solution of lower concentration.With the super filter tube desalination of molecular weight cut-off 10KDa, concentrated, low-temperature vacuum drying, namely obtains Recombinant Swine interleukin II-Fc fusion rotein powder.Composition and the content thereof of each damping fluid are as shown in the table:
Present invention also offers Recombinant Swine interleukin II-Fc fusion rotein treat in preparation and prevent the purposes in the medicine of porcine reproductive and respiratory syndrome, porcine influenza and pig blue-ear disease disease.In Animal Medicine, the immunne response of vaccine can be improved due to interleukin II and reduce the incidence of disease, Recombinant Swine interleukin II-Fc fusion rotein and multiple cause of disease vaccine conbined usage, immune level can be significantly improved, minimizing stress, reduce the side effect of vaccine, thus reach stronger therapeutic action.
Recombinant Swine interleukin II-Fc fusion rotein also can be used as immunologic adjuvant and uses, the untoward reaction of standard adjuvant can not only be avoided, can also significantly enhancing body to the immune efficacy of virus, bacterium and parasite vaccine, improve antibody reguarity and extend the antibody time length, and the side effect of some vaccine can be reduced.
Of the present invention through optimised Recombinant Swine interleukin II-Fc fusion rotein recombination sequence, be more suitable for the expression of escherichia expression system, expressed Recombinant Swine interleukin II-Fc fusion rotein is far above the expression amount of pig interleukin 2 and 6-Fc fusion rotein native gene sequence at escherichia expression system, and, compared with pig interleukin 2 and 6, Recombinant Swine interleukin II-Fc fusion rotein of the present invention on the basis that ensure that pig interleukin 2 and 6 activity largely on extend its transformation period in vivo, achieve the long-acting object with avoiding medication repeatedly.Simultaneously, provided by the present invention is a boar source type fusion rotein, be more suitable for the preparation for Swine virus disease veterinary drug, and the procaryotic cell expression of this fusion rotein provided by the present invention, purifying and refolding method also control its preparation cost greatly, for its industrialization provides guarantee.
Accompanying drawing explanation
Fig. 1 represents Recombinant Swine interleukin II-Fc fusion rotein codon optimized front and back nucleotide sequence comparison
Wherein, odd-numbered line (row that namely " original series " is corresponding) is pig interleukin 2 and 6-Fc fusion rotein natural gene nucleotide sequence, i.e. codon optimized front sequence; Even number line (i.e. " majorizing sequence " corresponding row) is the gene nucleotide series of Recombinant Swine interleukin II-Fc fusion rotein of the present invention, the sequence after namely codon optimized.
Fig. 2-a, Fig. 2-b are the restructuring codon optimized front and back of pig interleukin 2 and 6-Fc fusion rotein CAI index in escherichia coli expression host.
Wherein, Fig. 2-a represent Recombinant Swine interleukin II-Fc fusion rotein codon optimized before in escherichia coli expression host CAI index be 0.64; Fig. 2-b represent Recombinant Swine interleukin II-Fc fusion rotein codon optimized after in escherichia coli expression host CAI index be 0.87.
Fig. 3-a, Fig. 3-b are restructuring pig interleukin 2 and 6-Fc fusion rotein codon optimal codon frequency distribution areal map in escherichia coli expression host.
Wherein, Fig. 3-a represent Recombinant Swine interleukin II-Fc fusion rotein codon optimized before in escherichia coli expression host optimal codon frequency distribution areal map, as can be seen from the figure: the poor efficiency codon (<30%) of the codon optimized presequence of Recombinant Swine interleukin II-Fc fusion rotein occurs that per-cent is 9%; Fig. 3-b represent Recombinant Swine interleukin II-Fc fusion rotein codon optimized after in escherichia coli expression host optimal codon frequency distribution areal map, the poor efficiency codon (<30%) of the codon optimized presequence of Recombinant Swine interleukin II-Fc fusion rotein occurs that per-cent is 0.
Fig. 4-a, Fig. 4-b in restructuring pig interleukin 2 and 6-Fc fusion rotein codon in escherichia coli expression host average GC base contents distributed areas figure.
Wherein, Fig. 4-a represent Recombinant Swine interleukin II-Fc fusion rotein codon optimized before in escherichia coli expression host average GC base contents be: 51.19%; Fig. 4-b represent Recombinant Swine interleukin II-Fc fusion rotein codon optimized after in escherichia coli expression host average GC base contents be: 51.34%.
Fig. 5-a, Fig. 5-b are the secondary structure prediction figure of the codon optimized front and back mRNA of pig interleukin 2 and 6-Fc fusion rotein.
The secondary structure prediction figure of the codon optimized premessenger RNA of Fig. 5-a pig interleukin 2 and 6-Fc fusion rotein, Fig. 5-b is the secondary structure prediction figure of the codon optimized rear mRNA of pig interleukin 2 and 6-Fc fusion rotein.
Fig. 6 is restructuring pig interleukin 2 and 6-Fc fusion protein expression plasmid building process figure.
Fig. 7 is restructuring pig interleukin 2 and 6-Fc fusion rotein optimized gene agarose gel electrophoresis figure.
Wherein, swimming lane 1 is 500bp DNA Ladder; Swimming lane 2 is the pET21b carrier after NdeI and XhoI double digestion; Swimming lane 3 is for containing the Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene PCR primer of NdeI and XhoI restriction enzyme site in two ends.
Fig. 8 is the SDS-PAGE gel electrophoresis figure of restructuring pig interleukin 2 and 6-Fc fusion rotein and corresponding western blot figure.
Fig. 8-a is restructuring pig interleukin 2 and 6-Fc fusion protein S DS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 is the Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate not adding IPTG induction; Swimming lane 3 is for adding the Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate of IPTG induction.
Fig. 8-b is restructuring pig interleukin 2 and 6-Fc fusion protein immunization trace figure.
Wherein, swimming lane 1(10-230KDa) the albumen loading Marker of pre-dyed of wide region, swimming lane 2 is the Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate not adding IPTG induction: swimming lane 3 is for adding the Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate of IPTG induction.
Fig. 9 Recombinant Swine interleukin II-Fc fusion rotein high expression optimum induction SDS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 is 0.5mmol/L IPTG induction 1h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 3 is 0.5mmol/L IPTG induction 2h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 4 is 0.5mmol/L IPTG induction 3h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 5 is 0.5mmol/L IPTG induction 4h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 6 is 1mmol/L IPTG induction 1h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 7 is 1mmol/L IPTG induction 2h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 8 is 1mmol/L IPTG induction 3h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 9 is 1mmol/L IPTG induction 4h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 10 is 1.5mmol/L IPTG induction 1h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 11 is 1.5mmol/L IPTG induction 2h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 12 is 1.5mmol/L IPTG induction 3h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate; Swimming lane 13 is 1.5mmol/L IPTG induction 4h Recombinant Swine interleukin II-Fc fusion rotein E. coli lysate.
Figure 10 is the pig interleukin 2 and 6-Fc fusion rotein inclusion body SDS-PAGE electrophorogram after renaturation
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 contains the full bacterium lysate of Recombinant Swine interleukin II-Fc fusion rotein for ultrasonic degradation; Swimming lane 3 is for cleaning rear Recombinant Swine interleukin II-Fc fusion rotein inclusion body precipitation with Buffer B first time; Swimming lane 4 is Recombinant Swine interleukin II-Fc fusion rotein inclusion body precipitation after the cleaning of Buffer B second time.
Figure 11 is recombinant interleukin 2-Fc stability western blot figure in porcine blood serum.
Wherein, swimming lane 1 is the albumen loading Marker of the pre-dyed of (10-230kDa) wide region; Swimming lane 2 is not for add pig interleukin 2 and 6 and Recombinant Swine interleukin II-Fc fusion rotein pig anteserum sample; Swimming lane 3 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 0h; Swimming lane 4 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 0.25h; Swimming lane 5 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 0.5h; Swimming lane 6 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 1.5h; Swimming lane 7 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 3h; Swimming lane 8 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 6h; Swimming lane 9 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 24h; Swimming lane 10 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 48h; Swimming lane 11 is for adding the pig anteserum sample of 1 μm of ol/ml pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 72h; Swimming lane 12 is for adding the pig anteserum sample of 1 μm of ol/mL pig interleukin 2 and 6 and 1 μm of ol/mL Recombinant Swine interleukin II-Fc fusion rotein 120h.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further, should be understood that quoting embodiment is only not used in for illustration of the present invention and limits the scope of the invention.
embodiment 1 Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene optimization design
1. codon optimized
Genetic codon has 64 kinds, but most biological tendency is in utilizing the part in these codons.Those are called best codon (optimal codons) by what the most frequently utilize, and those are not called by what often utilize the codon (rare or low-usage codons) that rare or utilization ratio is low.In fact, the conventional often kind of biology (comprising intestinal bacteria, yeast, mammalian cell, vegetable cell and insect cell) doing protein expression or production all shows difference or the preference of codon utilization to a certain degree.To the expression efficiency of the expression efficiency of the gene containing best codon apparently higher than the gene of the codon containing poor efficiency in intestinal bacteria, yeast and fruit bat.Therefore, in heterologous expression system, the preferences of codon have impact on the expression of recombinant protein to a great extent.Utilize preference codon (preferred codons) and avoid utilizing rare codon to carry out gene chemical synthesis, the redesign of this gene is codon optimized.Optimizing process fully takes into account the Various Complex factor that protein expression different steps may run into, as: codon adaptability, mRNA structure and transcribe and different cis element in translation process.Therefore, the gene design of the present invention to pig interleukin 2 and 6-Fc fusion rotein not only comprises codon optimized, also comprises the optimization etc. of mRNA structural modifications, translation initiation site.
2. codon-bias optimization
Codon-bias has been proved to be a very important influence factor in Prokaryotic gene expression, and it causes same codon between different organism, the change of utilization ratio between the expression level of albumen and between the different sites of same operon.The major cause of this preferences difference is caused to be the difference that tRNAs available in different cell measures.Therefore the method optimizing translation system the best is exactly keep the balance between the frequency of utilization of codon and homology tRNA.Be unpredictable and have challenge at expression in escherichia coli mammalian genes, as in intestinal bacteria, just seldom, this species diversity clearly can affect the expression of gene to the tRNA molecule corresponding to AGG and AGA.
3. the codon of poor efficiency is replaced to host and commonly use codon
The utilization ratio of the codon comprised in usual gene in specific host is lower, and the expression amount of this kind of albumen is also fewer, even when this codon exists and between protein clusters or N-terminal time expression amount can be less.The codon of poor efficiency is replaced with host under the prerequisite not changing aminoacid sequence and commonly use the expression level that codon can improve functional protein.
If the utilization ratio of the codon in any source in host organisms lower than 5% to 10% time, just there will be expression inhibiting, when these poor efficiency codons close on or are connected, larger on the impact of protein expression.The codon of the poor efficiency of cluster inhibits ribosomal motion, and this is the obvious mechanism that gene can not be expressed with proper level.Rrna translation is slower than the speed of the courier translating the same length not containing poor efficiency codon by movement velocity time nine molecular couriers of password (containing several poor efficiency codon or be all poor efficiency codon).Even if poor efficiency password submanifold is positioned at 3 ' end, courier finally also can be damaged by rrna " crowded ", and 5 ' end got back to again by rrna.The retarding effect of 3 ' end poor efficiency password submanifold can be all the same large by the molecular retarding effect of poor efficiency password with whole courier.If poor efficiency password submanifold is positioned at 5 ' end, its effect is comprehensive minimizing of initial rrna number, the poor efficiency of courier in causing albumen to synthesize.Remove the codon of poor efficiency or easily can be prevented low expression by the codon misreading as termination signal or not express.
4. expression vector and transcripting promoter
Although codon-bias plays an important role in genetic expression, the selection no less important of expression vector and transcripting promoter, the protein expression of N terminal nucleotide sequence for poor efficiency codon and the codon AUG close to initiation site very responsive.Also there is reciprocal influence between the stability of translation and mRNA, mRNA can be made more easily to be decomposed by endo-RNAses owing to having lacked ribosomal protection although reduce translation efficiency, also there is no the complete explanation affected between them at present.
Other factors also can affect protein expression, comprise and make mRNA go stable sequence.Stable mRNA secondary structure and close to 5' end molecule also have important impact to genetic expression.Utilize the open reading frame of goal gene upstream during translation successfully can improve the expression efficiency of difficulty gene.
Contriver is according to hinge area, CH2 district and CH3 district in the cDNA sequence (GenBank accession number: NM_213828.1) of the cDNA sequence (GenBank accession number: NM_213861.1) of the published pig interleukin 2 and 6 of GenBank (Sus scrofa interleukin2) and pig IgG Fc fragment (Sus scrofa IgG heavy chain), these 2 genes are directly merged and carries out the codon optimized gene obtaining Recombinant Swine interleukin II-Fc fusion rotein of the present invention, as shown in SEQ ID No:1.
Here carries out codon optimized concrete steps to restructuring pig interleukin 2 and 6-Fc antigen-4 fusion protein gene:
To this gene carry out codon optimized after obtain Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene of the present invention, as shown in SEQID No:1.
Here is carried out codon optimized to restructuring pig interleukin 2 and 6-Fc fusion rotein, and before and after optimizing, each parameter comparison is as follows:
1. codon adaptation indexI (CAI)
From Fig. 2-a, before codon is not optimized, Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene codon adaptation indexI (codon adaptation index, CAI) in intestinal bacteria is 0.64.From Fig. 2-b, after codon optimized, Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene CAI index in intestinal bacteria is made to be 0.87.Be considered to this gene during usual CAI=1 is optimal high expression state in this expression system, CAI index is lower shows that this gene expression level in this host is poorer, therefore can find out have passed through codon optimized after the gene order that obtains can improve the expression level of Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene in intestinal bacteria.
2. optimal codon frequency of utilization (FOP)
From Fig. 3-a, based on coli expression carrier, before codon is not optimized, the poor efficiency codon of pig interleukin 2 and 6-Fc antigen-4 fusion protein gene sequence occurs that per-cent is 9%.The gene that this is not optimized adopts series connection rare codon,
These codons may reduce translation efficiency, even can dismiss translation assemblage.From Fig. 3-b, after codon optimized,
Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene occurs that in E. coli system the frequency of poor efficiency codon is 0.
3.GC base contents (GC curve)
GC content ideal distribution region is 30%-70%, all can affect to some extent transcribe and translation efficiency at this any peak of extra-regional appearance.Contrasted from the GC base average content distributed areas figure of the pig interleukin 2 and 6-Fc antigen-4 fusion protein gene of Fig. 4-a, Fig. 4-b, be 51.19% by showing in pig interleukin 2 and 6-Fc antigen-4 fusion protein gene GC base average content before optimization in Fig. 4-a, by demonstrating the GC of the sequence elimination after optimization content in Fig. 4-b in extra-regional 60 bases of 30%-70%, after being finally optimized, the GC base average content of Recombinant Swine interleukin II-Fc fusion rotein is 51.34%.
3. cis-acting elements
| Cis-acting elements | Before optimization | After optimization |
| E.coli_RBS(AGGAGG) | 1 | 0 |
| PolyT(TTTTTT) | 0 | 0 |
| PolyA(AAAAAAA) | 0 | 0 |
| Ch site (GCTGGTGG) | 0 | 0 |
| T7Cis(ATCTGTT) | 0 | 0 |
4. remove tumor-necrosis factor glycoproteins
The secondary structure prediction figure of 5.mRNA
After DNA is transcribed into mRNA, because mRNA is strand linear molecule, by folded back on itself, complementary base pair is met, by the hairpin structure (Hairpin) of hydrogen bonded.5 ' hairpin structure can play regulating and controlling effect in the translation initiation stage.If but hairpin structure is very long, the required energy that unwinds is very high, just likely has influence on translation.So need the sequence expressed should avoid long and that energy is high hairpin structure as far as possible.After codon optimized, from the secondary structure prediction figure of Fig. 5-a, the codon optimized front and back mRNA of Fig. 5-b pig interleukin 2 and 6-Fc fusion rotein, 5 ' hairpin structure after optimization and the required energy that unwinds are more suitable for the expression of target protein.
embodiment 2: the expression plasmid of Recombinant Swine interleukin II-Fc antigen-4 fusion protein gene builds
By the fragment that the Recombinant Swine interleukin II-Fc fusion rotein full genome (as shown in SEQ ID No:1) after optimizing synthesizes, be building up in pUC57 plasmid (Jin Sirui Science and Technology Ltd. provides by Nanjing), obtain one and preserve plasmid for a long time, be designated as pUC57-pIL2-Fc plasmid.With pUC57-pIL2-Fc plasmid for template, upstream and downstream introduce NdeI and XhoI restriction enzyme site respectively, carry out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1:GGAATTCCATATGGCTCCGACCTCGTCGTCC
Downstream primer:
P2:GCCGCTCGAGTTATTTGCCCTGGGTTTTGCTG
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, and archaeal dna polymerase Phusion High-Fidelity DNA polymerase(used is purchased from Theromo-Fisher scientific), 2U/ μ L, adds 0.5 μ L.Reaction conditions is 98 DEG C of 5s, 55 DEG C of 45s, 72 DEG C of 30s, and after 25 circulations, product is through 1.0% agarose gel electrophoresis analysis, and product size is consistent with expection size (1098bp).(as shown in Figure 7)
The gene product DNA gel obtained is reclaimed test kit (purchased from Beijing Tian Gen biochemical technology company limited) purifying.After purifying, with NdeI and XhoI(purchased from New England Biolabs company) double digestion, with T4 ligase enzyme (purchased from New England Biolabs company), the product after double digestion is connected in pET21b plasmid (purchased from Merck company), be transformed in DH5 α competent cell (purchased from Beijing Tian Gen biochemical technology company limited), 37 DEG C of overnight incubation in the LB flat board of the penbritin (purchased from Amresco company) containing 100 μ g/mL.Second day screening positive clone bacterium, order-checking, comparison result display is completely the same with expected sequence, namely obtains the expression plasmid of a kind of form of Recombinant Swine interleukin II-Fc fusion rotein, is designated as pET21b-pIL2-Fc.
the high expression of embodiment 3 Recombinant Swine interleukin II-Fc fusion rotein in intestinal bacteria and qualification
Concrete steps are as follows:
1. by pET21b-pIL2-Fc Plastid transformation correct for comparison of checking order in embodiment 2 in e. coli bl21 (DE3) competence bacterial strain (purchased from Beijing Tian Gen biochemical technology company limited), incubated overnight in 37 DEG C of ampicillin plate.
2. within second day, choose 1-4 the restructuring bacterium colony containing pET21b-pIL2-Fc plasmid, the LB nutrient solution (purchased from Amresco company) of access containing 100 μ g/mL penbritins, 37 DEG C of overnight incubation.
3. get overnight culture in 50 μ L steps 2, access 5mL contains the LB nutrient solution of 100 μ g/mL penbritins, 37 DEG C of shaking culture.
4. survey bacterium liquid OD every 1h after inoculation
600value, treats OD
600when=1.0, with the IPTG(of 1mmol/L purchased from Amresco company) carry out abduction delivering.
5. collect bacterium liquid after abduction delivering 4h, high speed centrifugation (rotating speed: 12000rpm/min) 3min, by the PBS washing and precipitating of precooling, add 5 × sds gel sample loading buffer, 100 DEG C of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm/min) 1min, gets supernatant.Do not add the recombination bacillus coli culture of IPTG by this step process yet.
6. respectively get 10 μ L do not add IPTG and add IPTG induction by step 5 process after sample, 10%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis, moves to bottom separation gel to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining and immunoblotting, observes expression product band, sees Fig. 8-a and Fig. 8-b.
embodiment 4 Recombinant Swine interleukin II-Fc fusion rotein high expression optimum induction
Much research shows that cell growth rate has a strong impact on the expression of foreign protein, therefore must to inoculation amount of bacteria, culture temperature, after cell growth time and induction, cell density control before induction, overgrowth or overrun and all can affect the expression amount of Recombinant Swine interleukin II-Fc fusion rotein inclusion body in intestinal bacteria.Use Three factors four level, set up IPTG concentration and induction time orthogonal table, by SDS-PAGE gel electrophoresis analysis induction Recombinant Swine interleukin II-Fc fusion protein expression.
Concrete steps are as follows:
1. by pET21b-pIL2-Fc Plastid transformation correct for comparison of checking order in embodiment 2 to BL21(DE3) in competence bacterial strain (purchased from Beijing Tian Gen biochemical technology company limited), incubated overnight in 37 DEG C of ampicillin plate.
2. next day, picking 1-4 the restructuring bacterium colony containing pET21b-pIL2-Fc plasmid, the LB nutrient solution of access containing 100 μ g/mL penbritins, 37 DEG C of overnight incubation.
3. get overnight culture access 5mL in 50 μ l steps 2 and contain the LB induction broth of 100 μ g/mL penbritins, 37 DEG C of shaking culture.
4. survey bacterium liquid OD after inoculation
600value, treats OD
600when=1.0, adding concentration according to following table is that 0.5m mol/L, 1.0mmol/L, 1.5m mol/L IPTG carries out abduction delivering.
Table 1 investigates inducer concentrations and the induction time of expression of recombinant proteins
5.1,2,3, Recombinant Swine interleukin II-Fc fusion rotein bacterium liquid is collected successively after 4h, high speed centrifugation (rotating speed: 12000rpm) 3min, by the PBS washing and precipitating of precooling, adds 5 × sds gel sample loading buffer, 100 DEG C of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm) 1min.
6. get the Recombinant Swine interleukin II-Fc fusion rotein culture suspension that 10 μ L do not add IPTG induction and add different concns IPTG, different induction time, 10%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis, moves to bottom separation gel to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining, observes Recombinant Swine interleukin II-Fc fusion protein expression products band.(see figure 9)
9. the expression of Recombinant Swine interleukin II-Fc fusion rotein content qualification pig interleukin 2 and 6-Fc fusion rotein is expressed in the analysis of gel imaging system thin layer scanning.Finally determine that the applicable inductive condition that this is implemented is 1m mol/L IPTG, induction time is 4h.
embodiment 5 Recombinant Swine interleukin II-Fc fusion rotein inclusion body purification and renaturation
1. will collect the intestinal bacteria precipitation containing induction Recombinant Swine interleukin II-Fc fusion rotein obtained in embodiment 3, resuspended with the PBS of precooling, in 4 DEG C with 12000rpm, centrifugal 15min; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A5mL, stirs, thalline is hanged with slicking glass rod.
3. every gram (weight in wet base) thalline adds 5 μ L100mmol/L PMSF, and 5 μ L100mg/mL N,O-Diacetylmuramidases, stir 20min on ice.
4. with the broken thalline of probe type ultrasonication ripple instrument, sample is placed on ice, ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, wait for sample cooling.4 DEG C, 12000rpm, centrifugal 15min.
5. precipitation lavation buffer solution Buffer B washing, 4 DEG C, 12000rpm, centrifugal 15min, precipitation inclusion body, repeats once.
6. inclusion body precipitation denaturation buffer Buffer C dissolves, stirred at ambient temperature 30min.
7. room temperature 12000rpm after fully mixing, centrifugal 15min, abandons precipitation, gets supernatant, namely obtains Recombinant Swine interleukin II-Fc fusion rotein denaturing soln.
8. adopt dilution refolding method to carry out renaturation to the Recombinant Swine interleukin II-Fc fusion rotein denaturing soln in step 7.
Dilution refolding: get the Recombinant Swine interleukin II-Fc fusion rotein denaturing soln that appropriate denaturation buffer Buffer C dissolves, with bio-rad company of the Quick Start Bradford1x Dye Reagent(U.S.) survey its concentration, then with renaturation buffer BufferD, protein concentration is diluted to 0.2mg/mL, when 4 DEG C of renaturation are to 24h, the 0.45 μm of filter membrane (Merck Millipore company) of recombinant protein solution after renaturation is filtered, namely obtains the Recombinant Swine interleukin II-Fc fusion rotein solution of lower concentration.With super filter tube (the Merck Millipore company) desalination of molecular weight cut-off 10KDa, concentrated, in vacuum freeze drier (Beijing Sihuan Scientific Instrument Factory Co., Ltd) low-temperature vacuum drying, namely obtain Recombinant Swine interleukin II-Fc fusion rotein powder.
Each damping fluid according to the form below preparation:
The each buffer components of table 2
9. carry out SDS-PAGE gel electrophoresis (Figure 10) with lavation buffer solution Buffer B twice washed product in step 5 respectively, at the visible obviously band of object scope.
embodiment 6 Recombinant Swine interleukin II-Fc fusion rotein Determination of biological activity
The biological activity assay of interleukin II is mainly by detecting interleukin II to the ability of the proliferation of target cell (or reacting cells).NK92 clone is the stable strain of people's natural killer cell (Natural Killer cell, NK), under its normal proliferative needs the condition of 100IU/mL human interleukin-12, otherwise, just can mortality after 72h.This experiment is main using NK92 cell (buying from ATCC) as reacting cells, in order to detect the biological activity after Recombinant Swine interleukin II-Fc fusion rotein renaturation.
The homology of human interleukin-12 and pig interleukin 2 and 6, higher than 80%, has larger consistence in biologic activity.First this experiment adopts the positive control human interleukin-12 of 8 kinds of different concns (to buy from Jiangsu spun gold profit pharmaceutcal corporation, Ltd, the accurate word S10970058 of traditional Chinese medicines) detect its proliferation function to people's natural killer cell NK92 clone, prove the feasibility of experimental program.Then after detecting renaturation, Recombinant Swine interleukin II-Fc fusion rotein and negative control do not contain the substratum of interleukin II to the impact of NK92 cel l proliferation.Finally by mensuration positive control pig interleukin 2 and 6 (Recombinant Procine Interteukin-2, article No. 907RPIL201.Buy from ProSpec company, PO Box6591, East Brunswick, 08816 NJ, USA) to the proliferation function drawing standard curve of people's natural killer cell NK92 clone, the vigor by four parametric regression Equation for Calculating Recombinant Swine interleukin II of the present invention-Fc fusion rotein:
Recombinant Swine interleukin II-Fc fusion rotein biologic activity
P
rfor positive control pig interleukin 2 and 6 standard substance biologic activity, 1 × 10
7iU/mL
D
sfor pig interleukin 2 and 6-Fc fusion rotein extension rate of the present invention;
D
rfor positive control pig interleukin 2 and 6 standard substance extension rate;
E
sfor pig interleukin 2 and 6-Fc fusion rotein of the present invention is equivalent to the extension rate of standard substance median effective dose;
E
rfor the extension rate of positive control pig interleukin 2 and 6 median effective dose.
The data presentation of table 3 and table 4, pig interleukin 2 and 6 is the same with human interleukin-12, to NK92 cel l proliferation, namely can detect its biologic activity by detecting Recombinant Swine interleukin II-Fc fusion rotein to NK92 cel l proliferation.In addition, the Recombinant Swine interleukin II-Fc fusion rotein prepared of the present invention and positive control pig interleukin 2 and 6, the growth of human interleukin-12 to NK92 cell all have promoter action.Compared with the cell without interleukin II process, under concentration is 100ng/mL Recombinant Swine interleukin II-Fc fusion protein sample treatment condition, NK92 cell proliferation about 4 times (table 5).By formulae discovery, the Recombinant Swine interleukin II-Fc fusion rotein vigor of gained of the present invention is 0.8x107IU/mL, and 80% of its active positive control pig interleukin 2 and 6 for not connecting Fc fragment, maintains its original biologic activity substantially.
The positive control human interleukin-12 of table 3 different concns is to NK92 cel l proliferation
The positive control pig interleukin 2 and 6 of table 4 different concns is to NK92 cel l proliferation
Table 5 Recombinant Swine interleukin II-Fc fusion rotein is to NK92 cel l proliferation
the preparation of embodiment 7 Recombinant Swine interleukin II(in detail can see the earlier application of applicant: 201210584912.9)
Concrete steps are as follows:
1. build the recombination bacillus coli can expressing Recombinant Swine interleukin II.
According to the cDNA sequence (GenBank accession number: NM_213861.1) of the published pig interleukin 2 and 6 of GenBank (Sus scrofa interleukin2), according to escherichia expression system to this gene carry out codon optimized after obtain Recombinant Swine Interleukin-2 Gene, as shown in SEQ ID No:3.By the fragment that the Recombinant Swine interleukin II full genome after optimizing synthesizes, be building up in pUC57 plasmid, obtain pUC57-prIL2 plasmid.
With pUC57-prIL2 plasmid for template, upstream and downstream primer introduces NdeI and XhoI restriction enzyme site respectively, carries out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1:GGAATTCCATATGGCTCCGACCTCGTCGTCC
Downstream primer:
P2:GCCGCTCGAGTTACGTCAGGGTAGAGTAAATGC
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μm of ol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, and archaeal dna polymerase Phusion High-Fidelity DNA polymerase used, 2U/ μ L, adds 0.5 μ L.Reaction conditions is 98 DEG C of 5s, 55 DEG C of 20s, 72 DEG C of 30s, 25 circulations.PCR primer with NdeI and XhoI double digestion, is connected in pET21b plasmid with T4 ligase enzyme, and is increased in DH5 α competent cell by Plastid transformation after reclaiming kits with DNA gel.The expression plasmid pET21b-prIL2 of amplification is transformed in escherichia coli BL21(DE3) expression bacterial strain.
2. the expression of Recombinant Swine interleukin II inclusion body
By e. coli bl21 (DE3) incubated overnight in 37 DEG C of ampicillin plate containing pET21b-prIL2.Choose 1-4 the restructuring bacterium colony containing pET21b-prIL2 plasmid next day, the LB nutrient solution of access containing 100 μ g/mL penbritins, 37 DEG C of overnight incubation.Get the LB induction broth of 50 μ L overnight culture access 5mL containing 100 μ g/mL penbritins, 37 DEG C of shaking culture.Treat OD
600when=1.0, induce with the IPTG of 1mmol/L.Bacterium liquid is collected, high speed centrifugation, by the PBS washing and precipitating of precooling after 4h.
3. the renaturation of Recombinant Swine interleukin II inclusion body and purifying
By resuspended for the PBS of the PBS washing and precipitating precooling through precooling in step 2, in 4 DEG C with 12000rpm, centrifugal 15min; Repeat once.Abandon supernatant, add lysis buffer Buffer A5mL by every gram (thalline weight in wet base), thalline is hanged.Every gram of (thalline weight in wet base) thalline adds 5 μ L100mmol/L PMSF, and 5 μ L100mg/mL N,O-Diacetylmuramidases, stir 20min on ice.With the broken thalline of probe type ultrasonication ripple instrument, sample is placed on ice, ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, wait for sample cooling.4 DEG C, 12000rpm, centrifugal 15min.Precipitation lavation buffer solution Buffer B washing, 4 DEG C, 12000rpm, centrifugal 15min, precipitation inclusion body, repeats once.Inclusion body precipitation denaturation buffer Buffer C dissolves, stirred at ambient temperature 30min.Room temperature 12000rpm after abundant mixing, centrifugal 15min, abandons precipitation, gets supernatant, namely obtains Recombinant Swine interleukin II denaturing soln.Adopt dialysis renaturation method renaturation inclusion body: use denaturation buffer Buffer C by Recombinant Swine interleukin II denaturing soln concentration dilution to 0.2mg/mL, inject the dialysis card of molecular weight cut-off 10KDa, 4 DEG C of dialysis renaturations, change a renaturation buffer Buffer D every 6h.When renaturation is to 24h, recombinant protein solution after renaturation is crossed 0.45 μm of filter membrane, namely obtain the Recombinant Swine interleukin II renaturation solution of lower concentration.With the super filter tube desalination of molecular weight cut-off 10KDa, concentrated, in vacuum freeze drier low-temperature vacuum drying, namely obtain Recombinant Swine interleukin II powder.Each damping fluid according to the form below preparation is as shown in table 2.Recombinant Swine interleukin II powder 4 DEG C of refrigerators are deposited stand-by.
the stability test of embodiment 8 Recombinant Swine interleukin II-Fc fusion rotein
In order to detect the stability of pig interleukin 2 and 6-Fc fusion rotein in serum, contriver is specially designed a kind of in-vitro simulated porcine blood serum environment, will prepare Recombinant Swine interleukin II and 1 μm of ol/ml Recombinant Swine of the present invention interleukin II-Fc fusion rotein is placed in fresh porcine blood serum 50uL jointly in the embodiment 7 of 1 μm of ol/ml, 37 DEG C, 120rpm, reacts 0 respectively, 0.25,0.5,1.5,3,6,24,48,-20 DEG C are preserved after 72,120h.With mouse-anti porcine interleukin 2 monoclonal antibody (Porcine IL-2Antibody, Monoclonal Mouse IgG2B Clone#100312, Catalog Number:MAB6521, R & DSystems) protein immunoblot test is carried out to the Recombinant Swine interleukin II in porcine blood serum and pig interleukin 2 and 6-Fc fusion rotein for primary antibodie, as shown in Figure 11 Recombinant Swine interleukin II after reaction 3h by the enzymic digestion in serum totally, and Recombinant Swine interleukin II-Fc fusion rotein 120h in serum still keeps stable.As can be seen here, pig interleukin 2 and 6-Fc fusion rotein prepared by the present invention has higher stability compared with Recombinant Swine interleukin II, extends its Half-life in vivo, reaches the effect of long-acting administration.
Claims (8)
1. an encoding gene for Recombinant Swine interleukin II-Fc fusion rotein, its base sequence is as shown in SEQ ID NO:1.
2. a carrier, described carrier contains the gene of claim 1.
3. carrier as claimed in claim 2, described carrier is pET21b.
4. intestinal bacteria, described intestinal bacteria have the carrier of Claims 2 or 3.
5. intestinal bacteria as claimed in claim 4, described intestinal bacteria are BL21 (DE3) bacterial strain.
6. a procaryotic cell expression method for Recombinant Swine interleukin II-Fc fusion rotein, comprises the steps:
(1) in substratum, the intestinal bacteria described in claim 4 or 5 are cultivated under suitable conditions;
(2) from intestinal bacteria and/or substratum, be separated restructuring pig interleukin 2 and 6-Fc fusion rotein.
7. expression method as claimed in claim 6, comprises the steps:
(1) picking is one or more containing the E. coli clones described in claim 4 or 5, and access contains antibiotic LB nutrient solution, overnight incubation;
(2) get overnight culture to transfer in containing in antibiotic fresh LB nutrient solution, be cultured to mid-log phase OD in 37 DEG C of concussions
600=1.0;
(3) in culture, add the IPTG that concentration is 1m mol/L, 37 DEG C, after abduction delivering 4h, the coli somatic that centrifugal treating is collected containing Recombinant Swine interleukin II-Fc fusion rotein precipitates.
8. the purifying of Recombinant Swine interleukin II-Fc fusion rotein and a refolding method, is characterized in that, comprise following steps:
(1) precipitate collecting obtain as claimed in claim 7 containing induction Recombinant Swine interleukin II-Fc fusion rotein intestinal bacteria, resuspended with the PBS of precooling, and in 4 DEG C of high speed centrifugation process, repeat once;
(2) suck supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A 3-10mL, stirs, thalline is hanged with slicking glass rod;
(3) it be the PMSF of 100mmol/L, 3-100 μ L concentration is the N,O-Diacetylmuramidase of 100mg/mL that every gram (weight in wet base) thalline adds 3-10 μ L concentration, in stirring on ice;
(4) broken thalline, sample is placed on ice, ultrasonic, and in 4 DEG C of high speed centrifugation process;
(5) precipitation lavation buffer solution Buffer B washing, and in 4 DEG C of high speed centrifugation process, precipitation inclusion body, repeats once;
(6) inclusion body precipitation denaturation buffer Buffer C dissolves, stirred at ambient temperature 30-60min;
(7) the fully rear room temperature high speed centrifugation process of mixing, abandons precipitation, gets supernatant, namely obtain Recombinant Swine interleukin II-Fc fusion rotein denaturing soln;
(8) the Recombinant Swine interleukin II-Fc fusion rotein denaturing soln that appropriate denaturation buffer Buffer C dissolves is got, with denaturation buffer Buffer D by the concentration dilution of Recombinant Swine interleukin II-Fc fusion rotein denaturing soln to 0.2mg/mL, 4 DEG C of dialysis renaturation 24h, by the 0.45 μm of membrane filtration of recombinant protein solution after renaturation, namely obtain Recombinant Swine interleukin II-Fc fusion rotein renaturation solution;
Described Recombinant Swine interleukin II-Fc fusion rotein renaturation solution can further with the ultrafiltration and concentration of molecular weight cut-off 10KDa, desalination to minimum volume, low-temperature vacuum drying, namely obtains Recombinant Swine interleukin II powder;
The Tris – HCl of described buffer B uffer A to be content be 20mmol/L, content is the NaCl of 0.15mol/L, and content is the EDTA of 1mmol/L, and content is the PMSF of 0.1mmol/L, and solution matrix is distilled water, and pH is 7.5;
The Tris – HCl of described buffer B uffer B to be content be 20mmol/L, content is the NaCl of 0.01mol/L, and content is the EDTA of 1mmol/L, content is the Triton X-100 of 0.5% (v/v), and content is the urea of 0.5mol/L, solution matrix is distilled water, and pH is 7.5;
The Tris – HCl of described buffer B uffer C to be content be 20mmol/L, content is the NaCl of 0.01mol/L, and content is the EDTA of 1mmol/L, content is the PMSF of 0.1mmol/L, and content is the urea of 8mol/L, and content is the DTT of 20mmol/L, solution matrix is distilled water, and pH is 7.8;
The Tris – HCl of described buffer B uffer D to be content be 20mmol/L, content is the EDTA of 1mmol/L, and containing GSH, GSSG, described GSH:GSSG mol ratio is 10:1, and solution matrix is distilled water, and pH is 7.5.
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