CN103265637A - Recombinant porcine interleukin 4-Fc fusion protein as well as coding gene and expression method thereof - Google Patents
Recombinant porcine interleukin 4-Fc fusion protein as well as coding gene and expression method thereof Download PDFInfo
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Abstract
The invention provides recombinant porcine interleukin (IL) 4-Fc fusion protein, a coding gene and an expression, purification and inclusion body renaturation method thereof, which belong to the field of the biological genetic engineering. Porcine IL4 can be used for treating porcine chronic infectious diseases and parasitic diseases and can be further popularized and applied to preventing and treating diseases such as porcine anaphylactic reaction and the like which are related to immunity. However, the porcine IL4 has the defects of high clearing speed in plasma and high industrialization cost. The invention provides long-acting recombinant porcine IL4-Fc fusion protein by adopting an escherichia coli prokaryotic expression system, wherein the porcine IL4 part includes the whole sequence of a porcine IL4 extracellular region, the Fc segment part includes a hinge region of antibodies, a CH2 region and a CH3 region, and the porcine IL4 part and the Fc segment part are directly fused. According to the recombinant porcine IL4-Fc fusion protein provided by the invention, the biological activity of the IL4 is improved, the half-life period of the IL4 is greatly prolonged, and the guarantee is provided for the low-cost mass expression and the industrialization of the expression.
Description
Technical field
The invention belongs to biotechnology gene field, relate to a kind of reorganization pig interleukin 4-Fc fusion rotein and encoding gene thereof, with and expression, purifying and renaturing inclusion bodies method.
Background technology
Interleukin-4 (Interleukin4, IL-4) by activated T cell, mastocyte and basophilic granulocyte synthesis secretion, be a kind of multi-functional lymphokine, have the biologic activity of multiple complexity, comprise and regulate the lymphocytic growth of T, breeding and differentiation; Adjusting is subjected to the T cell differentiation procedure of antigenic stimulation; The T cell of regulating after breaking up can produce cytokines such as IL-4, IL-5, IL-10 and IL-13; The specificity of control immunoglobulin class conversion etc.In addition, the induced reaction of the T cell of IL-4, B cell and NK cell is being removed helminth infection, and important regulatory role is also being brought into play in aspects such as treatment tumour, autoimmune disorder.This shows that IL-4 can be used as the diagnosis index of immunological adjuvant, therapeutical agent and disease infection, has great importance at aspects such as prevention and control of diseases and diagnosis.At present, IL-4 all there is report in mouse, rabbit, sheep, ox and people's biologic activity functional study, but less relatively to the research of pig IL-4, and also rare at its special detection system and bio-evaluation systematic research.
China is the world big country of raising pigs, in the face of the great eqpidemic disease of current pig industry happens occasionally and the situation of prevailing disease more sophisticated, we must use new scientific theory and production technology to develop the industry of raising pigs, and adopt the efficient vaccine prevention and control the generation of various eqpidemic diseases and popular.Pig interleukin 4 is important component parts of pig vaccine research, yet that natural pig interleukin 4 is expressed in body is very little, is difficult to extract confession clinical studyes and practical application in a large number in the body.But genetic engineering technique mass production foreign protein satisfies research and demands of applications.Therefore the present invention provides a kind of with low cost and can great expression reorganization pig interleukin 4 expression systems and expression method by genetic engineering means.
On the other hand, as small molecular weight protein, pig interleukin 4 is also the same with other interleukins, has plasma clearance speed defective faster, causes needing to repeat repeatedly medication in clinical treatment and just can reach result for the treatment of.The IgG immunoglobulin (Ig) is the main antibody in the humans and animals body, and its transformation period in vivo can reach 20 days.Its stability be since the Fc fragment of IgG can with newborn Fc acceptor (FcRn) combination, avoid IgG to enter in the lysosome and be degraded.In view of this, the present invention attempts considering that the Fc fragment in that pig interleukin 4 increases IgG constitutes fusion rotein, to improve the transformation period in pig interleukin 4 bodies, reaches long-acting purpose.Thus, the purpose of this invention is to provide and a kind ofly can keep pig interleukin 4 original activity, again can extension body in the fusion rotein of pig interleukin 4 and Fc fragment of transformation period.
Yet, the recombinant protein by escherichia coli expression mostly be do not dissolve, aggregation, i.e. inclusion body in the cell of non-activity.The renaturation of inclusion body is a very complicated process, if the renaturation condition is not suitable for occurring the mispairing of intramolecular disulfide bond, intermolecular covalent attachment or hydrophobic in conjunction with forming polymer, reduce the ratio motility rate of recombinant protein on the one hand, cause quality product defective, produce precipitation simultaneously again and separate out, influence yield.Therefore, another technical problem to be solved by this invention is to adopt suitable method that the albumen inclusion body of escherichia coli expression is carried out renaturation.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, by codon optimized mode, provide a kind of reorganization pig interleukin 4-Fc fusion rotein that can in intestinal bacteria, efficiently express with and gene and expression, purifying, refolding method.
The invention provides reorganization pig interleukin 4-Fc fusion rotein, described fusion rotein comprises pig interleukin 4 parts and Fc fragment part, wherein, pig interleukin 4 parts are the full sequence of pig interleukin 4 extracellular regions, the Fc fragment partly comprises hinge area, CH2 district and CH3 district, is directly to merge between pig interleukin 4 and the Fc fragment part.
Fc fragment wherein is selected from human or animal's immunoglobulin Fc, is Fc total length or partial sequence, and Fc is selected from IgG, IgM, IgD, IgA, and every kind of immunoglobulin class comprises each hypotype, as IgG1, IgG2, IgG3, IgG4.The preferred especially IgG1 from pig of Fc fragment part in the fusion rotein of the present invention.
Preferably, reorganization pig interleukin 4-Fc fusion rotein of the present invention has the aminoacid sequence shown in the SEQ ID NO:2, wherein 2-110 amino acids residue is the extracellular region sequence of pig interleukin 4, and 111-340 amino acids residue is pig IgG 1 sequence.
The invention provides the gene of coding reorganization pig interleukin 4-Fc fusion rotein described above, its base sequence is shown in SEQ ID NO:1.This sequence is to aim at escherichia expression system to carry out the codon optimized sequence that obtains, and can significantly improve the expression of heterologous gene in the host bacterium by contrast.
The present invention also provides the plasmid of the gene that has comprised coding reorganization pig interleukin 4-Fc fusion rotein described above, and described plasmid is preferably prokaryotic expression plasmid, most preferably is the pET21b carrier.
The present invention also provides the coli strain that includes plasmid described above, and preferably, described bacterial strain is selected from e. coli bl21 (DE3) bacterial strain.
The present invention also provides reorganization pig interleukin 4-Fc fusion rotein in the escherichia coli expression method, comprises the steps:
Steps of the method are:
Picking one or more contain the intestinal bacteria bacterium colony of reorganization pig interleukin 4-Fc fusion rotein described above, insert the LB nutrient solution, in 37 ℃ of overnight incubation;
2. get an amount of overnight culture, in the ratio access LB nutrient solution in 1:100, be cultured to mid-log phase OD in 37 ℃ of concussions
600=1.0;
3. adding concentration in culture is the IPTG of 1m mol/L, in 37 ℃, behind the abduction delivering 4h, in 4 ℃ with rotating speed 5000rpm, centrifugal treating 15min collects the coli somatic that contains reorganization pig interleukin 4-Fc fusion rotein.
All contain penbritin 50-100 μ g/ml in the described LB nutrient solution.
Expression method described above of the present invention is that the effective means the most that is used for express recombinant pig interleukin 4-Fc fusion rotein that obtains is groped and verified in experiment through contriver's repeated multiple times, the expression amount height of this method, and express obtain renaturing inclusion bodies after activity higher.
The present invention also provides the inclusion body purification method of reorganization pig interleukin 4-Fc fusion rotein, comprises the steps:
1. above-mentioned the containing that collection is obtained induced reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria precipitation, and be resuspended with the PBS of precooling, and handle in 4 ℃ of high speed centrifugations; Repeat once.
2. inhale and remove supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A3-10mL, stirs damping fluid, and thalline is hanged.
3. every gram (weight in wet base) thalline adding 3-10 μ L concentration is the PMSF of 100mmol/L, and 3-100 μ L concentration is the N,O-Diacetylmuramidase of 100mg/mL, in stirring on ice.
4. broken thalline, sample places on ice, and is ultrasonic, and handles in 4 ℃ of high speed centrifugations.
5. precipitation is washed with lavation buffer solution Buffer B, and handles in 4 ℃ of high speed centrifugations, and the precipitation inclusion body repeats once.
6. the inclusion body precipitation stirs 30-60min with sex change buffer B uffer C dissolving under the room temperature.
7. the room temperature high speed centrifugation is handled behind the abundant mixing, abandons precipitation, gets supernatant, and pig interleukin 4-Fc fusion rotein denaturing soln namely obtains recombinating.
This purification process preferred steps is as follows:
1. above-mentioned the containing that collection is obtained induced reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria precipitation, and be resuspended with the PBS of precooling, in 4 ℃, with the centrifugal 15min of the rotating speed of 12000rpm; Repeat once.
2. inhale and remove supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A5mL, stirs solution thalline is hanged.
3. to add 5 μ L concentration be the PMSF of 100mmol/L to every gram (weight in wet base) thalline, and 5 μ L concentration are the N,O-Diacetylmuramidase of 100mg/mL, stir 20min on ice.
4. with the broken thalline of sonde-type ultrasonoscope, sample places on ice, and ultrasonic 120 times, each 5s is 5s at interval, circulates three times, is circulated between the cooling sample at every turn and waits for 2min, waits for the sample cooling.In 4 ℃, with the centrifugal 15min of the rotating speed of 12000rpm, abandon supernatant.
5. precipitation is with lavation buffer solution Buffer B washing, and in 4 ℃, with the centrifugal 15min of the rotating speed of 12000rpm, the precipitation inclusion body repeats once.
6. the inclusion body precipitation stirs 30min with sex change buffer B uffer C dissolving under the room temperature.
Fully behind the mixing under the room temperature with the centrifugal 15min of the rotating speed of 12000rpm, abandon precipitation, get supernatant, pig interleukin 4-Fc fusion rotein denaturing soln namely obtains recombinating.
The present invention also provides the renaturing inclusion bodies method of the reorganization pig interleukin 4-Fc fusion rotein after optimizing, and comprises the steps:
Get an amount of reorganization pig interleukin 4-Fc fusion rotein denaturing soln with sex change buffer B uffer C dissolving, with renaturation buffer Buffer D protein concentration is diluted to 0.2mg/mL, 4 ℃ of renaturation are during to 24h, recombinant protein solution after the renaturation with 0.45 μ m membrane filtration, is namely obtained the reorganization pig interleukin 4-Fc fusion rotein solution of lower concentration.And further ultrafiltration desalination, concentrated, low-temperature vacuum drying namely obtains reorganization pig interleukin 4-Fc fusion rotein powder.Composition and the content thereof of each damping fluid are as shown in the table:
According to prepared vaccine form difference, the embodiment of pig interleukin 4 of the present invention can have multiple: as vaccines such as aluminium glue adjuvant, oily adjuvant, liposome adjuvants; Injection system can adopt multitude of different ways such as subcutaneous injection, intradermal injection and intramuscular injection; If live vector vaccine, except above injection system, also can adopt different modes such as oral, suction, collunarium, eye drip.
Pig interleukin 4-Fc fusion rotein recombination sequence through optimizing of the present invention, be more suitable for the expression of escherichia expression system, compare with pig interleukin 4-Fc fusion rotein natural gene sequence, the expression efficiency of pig interleukin 4-Fc fusion rotein in intestinal bacteria after the optimization obviously improves.Expressed pig interleukin 4-Fc fusion rotein is far above the expression amount of pig interleukin 4-Fc fusion rotein natural gene sequence at escherichia expression system.And, compare with pig interleukin 4, reorganization pig interleukin 4-Fc fusion rotein of the present invention has prolonged its transformation period in vivo largely on the basis that has guaranteed pig interleukin 4 activity, realized long-acting and avoid the purpose of medication repeatedly.
Description of drawings
Fig. 1 represents to recombinate the codon optimized front and back of pig interleukin 4-Fc fusion rotein nucleotide sequence relatively
Wherein, odd-numbered line (i.e. " original series " corresponding row) is pig interleukin 4-Fc fusion rotein natural gene nucleotide sequence, i.e. codon optimized preceding sequence; Even number line (i.e. " majorizing sequence " corresponding row) is the gene nucleotide series of reorganization pig interleukin 4-Fc fusion rotein of the present invention, the sequence after namely codon optimized.
Fig. 2-a, Fig. 2-b are the reorganization codon optimized front and back of pig interleukin 4-Fc fusion rotein CAI index in the escherichia coli expression host.
Wherein, Fig. 2-a represent to recombinate pig interleukin 4-Fc fusion rotein codon optimized before in the escherichia coli expression host CAI index be 0.62; Fig. 2-b codon optimized the back of pig interleukin 4-Fc fusion rotein CAI index in the escherichia coli expression host of representing to recombinate is 0.86.
Fig. 3-a, Fig. 3-b are reorganization pig interleukin 4-Fc fusion rotein codon optimum codon frequency distributed areas figure in the escherichia coli expression host.
Wherein, Fig. 3-a represent to recombinate pig interleukin 4-Fc fusion rotein codon optimized before in the escherichia coli expression host optimum codon frequency distributed areas figure, as can be seen from the figure: it is 9% that per-cent appears in the poor efficiency codon (＜30%) of reorganization pig interleukin 4-Fc fusion rotein codon optimized presequence; Fig. 3-b codon optimized the back of pig interleukin 4-Fc fusion rotein optimum codon frequency distributed areas figure in the escherichia coli expression host that represents to recombinate, it is 0 that per-cent appears in the poor efficiency codon (＜30%) of the codon optimized presequence of reorganization pig interleukin 4-Fc fusion rotein.
Fig. 4-a, Fig. 4-b in the reorganization pig interleukin 4-Fc fusion rotein codon in the escherichia coli expression host average GC base contents distributed areas figure.
Wherein, Fig. 4-a represent to recombinate pig interleukin 4-Fc fusion rotein codon optimized before in the escherichia coli expression host average GC base contents be: 57.22%; Fig. 4-b codon optimized the back of pig interleukin 4-Fc fusion rotein average GC base contents in the escherichia coli expression host of representing to recombinate is: 52.19%.
Fig. 5-a, Fig. 5-b are the secondary structure prediction figure of the codon optimized front and back mRNA of reorganization pig interleukin 4-Fc fusion rotein.
The secondary structure prediction figure of Fig. 5-codon optimized premessenger RNA of a reorganization pig interleukin 4-Fc fusion rotein, Fig. 5-b is the secondary structure prediction figure of reorganization pig interleukin 4-Fc fusion rotein codon optimized back mRNA.
Fig. 6 is reorganization pig interleukin 4-Fc fusion protein expression plasmid building process figure.
Fig. 7 is reorganization pig interleukin 4-Fc fusion rotein optimized gene agarose gel electrophoresis figure.
Wherein, swimming lane 1 is 500bp DNA Ladder; Swimming lane 2 is the pET21b carrier behind NdeI and the XhoI double digestion; Swimming lane 3 contains the reorganization pig interleukin 4-Fc antigen-4 fusion protein gene PCR product of NdeI and XhoI restriction enzyme site for two ends.
Fig. 8 is SDS-PAGE gel electrophoresis figure and the corresponding western blot figure of reorganization pig interleukin 4-Fc fusion rotein.
Fig. 8-a is reorganization pig interleukin 4-Fc fusion rotein SDS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is sample Marker on the albumen that dyes in advance of (10-230kDa) wide region; Swimming lane 2 is not for adding the reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate that IPTG induces; Swimming lane 3 is for adding the reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate that IPTG induces.
Fig. 8-b is reorganization pig interleukin 4-Fc fusion protein immunization trace figure.
Wherein, swimming lane 1(10-230KDa) sample Marker on the albumen that dyes in advance of wide region, swimming lane 2 is not for adding the reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate that IPTG induces: swimming lane 3 is for adding the reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate that IPTG induces.
Fig. 9 pig interleukin 4-Fc fusion rotein of recombinating efficiently expresses inductive condition and optimizes the SDS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is sample Marker on the albumen that dyes in advance of (10-230kDa) wide region; Swimming lane 2 is induced 1h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 3 is induced 2h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 4 is induced 3h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 5 is induced 4h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 6 is induced 1h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 7 is induced 2h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 8 is induced 3h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 9 is induced 4h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 10 is induced 1h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG; Swimming lane 11 is induced 2h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG; Swimming lane 12 is induced 3h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG; Swimming lane 13 is induced 4h reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG.
Figure 10 is the reorganization pig interleukin 4-Fc fusion rotein inclusion body SDS-PAGE electrophorogram after the renaturation.
Wherein, swimming lane 1 is sample Marker on the albumen that dyes in advance of (10-230kDa) wide region; Swimming lane 2 is for containing the full bacterium lysate of reorganization pig interleukin 4-Fc fusion rotein after the ultrasonication; Swimming lane 3 is for cleaning back reorganization pig interleukin 4-Fc fusion rotein inclusion body precipitation for the first time with Buffer B; Swimming lane 4 cleans back reorganization pig interleukin 4-Fc fusion rotein inclusion body precipitation for the second time for Buffer B, and swimming lane 5 is reorganization pig interleukin 4-Fc fusion rotein after the Buffer D renaturation.
Figure 11 pig interleukin 4 vitality test curves of recombinating.
Figure 11-a represents the influence of 4 pairs of TF-1 cell proliferation rates of positive control pig interleukin, and Figure 11-b represents to compare with reorganization pig interleukin 4 with negative control, and reorganization pig interleukin 4-Fc fusion rotein of the present invention is to the influence of TF-1 cell proliferation rate.
Wherein, by Figure 11-a, 11-b as can be seen, reorganization pig interleukin 4-Fc fusion rotein of the present invention is the same with the positive control medicine, and the propagation of TF-1 cell is had promoter action; By 11-b as can be seen, compare with the TF-1 cell of handling without interleukin-4, under the effect of 1ug/mL reorganization pig interleukin 4-Fc fusion protein sample, TF-1 cell proliferation about 2 times.
Figure 12 is reorganization pig interleukin 4-Fc fusion rotein stable western blot figure in porcine blood serum.
Wherein, swimming lane 1 is sample Marker on the albumen that dyes in advance of (10-230kDa) wide region; Swimming lane 2 is not for adding pig interleukin 4 and reorganization pig interleukin 4-Fc fusion rotein pig anteserum sample; Swimming lane 3 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 0h; Swimming lane 4 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 1h; Swimming lane 5 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 2h; Swimming lane 6 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 4h; Swimming lane 7 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 8h; Swimming lane 8 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 24h; Swimming lane 9 is for adding the pig anteserum sample of 2 μ mol/mL pig interleukins 4 and 2 μ mol/mL reorganization pig interleukin 4-Fc fusion rotein 48h.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, should be understood that quoting embodiment only is used for explanation the present invention and is not used in and limits the scope of the invention.
The contriver is according to the disclosed pig interleukin 4(Sus of GenBank scrofa interleukin4) cDNA sequence (GenBank accession number: NM_214123.1) and the cDNA sequence of pig IgG Fc fragment (Sus scrofa IgG heavy chain) (GenBank accession number: hinge area, CH2 district and CH3 district NM_213828.1), these 2 genes are directly merged and carry out the codon optimized gene that obtains reorganization pig interleukin 4-Fc fusion rotein of the present invention, shown in SEQ ID No:1.
Be reorganization pig interleukin 4-Fc fusion rotein to be carried out codon optimized below, each parameter comparison is as follows before and after optimizing:
1. codon adaptation indexI (CAI)
By Fig. 2-a as can be known, before codon is not optimized, reorganization pig interleukin 4-Fc antigen-4 fusion protein gene in intestinal bacteria codon adaptation indexI (codon adaptation index is 0.62 CAI).By Fig. 2-b as can be known, after codon optimized, make that reorganization pig interleukin 4-Fc antigen-4 fusion protein gene CAI index in intestinal bacteria is 0.86.Usually being considered to this gene during CAI=1 is the optimal state that efficiently expresses in this expression system, therefore the CAI index is more low to show that this gene expression level in this host is more poor, has passed through the gene order that obtains after codon optimized as can be seen and can improve the expression level of reorganization pig interleukin 4-Fc antigen-4 fusion protein gene in intestinal bacteria.
2. optimum codon usage frequency (FOP)
By Fig. 3-a as can be known, based on coli expression carrier, before codon was not optimized, it was 9% that per-cent appears in the poor efficiency codon of pig interleukin 4-Fc antigen-4 fusion protein gene sequence.The gene that this is not optimized adopts the series connection rare codon, and these codons may reduce translation efficiency, even can dismiss the translation assemblage.By Fig. 3-b as can be known, after codon optimized, the poor efficiency codon appears in reorganization pig interleukin 4-Fc antigen-4 fusion protein gene in the intestinal bacteria system frequency is 0.
3.GC base contents (GC curve)
GC content ideal distribution zone is 30%-70%, all can influence to some extent at this any peak of extra-regional appearance and transcribe and translation efficiency.By the GC base average content distributed areas figure contrast of the pig interleukin 4-Fc antigen-4 fusion protein gene of Fig. 4-a, Fig. 4-b as can be known, by showing among Fig. 4-a in the pig interleukin 4-Fc antigen-4 fusion protein gene that GC base average content is 57.22% before optimization, eliminated GC content in the extra-regional base of 30%-70% by the sequence that demonstrates among Fig. 4-b after the optimization, the GC base average content of the back reorganization pig interleukin 4-Fc fusion rotein that finally is optimized is 52.19%.
3. cis-acting elements
| Cis-acting elements | Before the optimization | After the 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. removal tumor-necrosis factor glycoproteins
5.mRNA secondary structure prediction figure
After DNA is transcribed into mRNA, because mRNA is the strand linear molecule, make complementary base pair meet by self inflection, the hairpin structure (Hairpin) that forms by 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 might have influence on translation.So the sequence that needs to express should be avoided long and the high hairpin structure of energy as far as possible.After codon optimized, by the secondary structure prediction figure of Fig. 5-a, Fig. 5-codon optimized front and back mRNA of b pig interleukin 4-Fc fusion rotein as can be known, 5 ' hairpin structure after the optimization and the required energy that unwinds are more suitable for the expression of target protein.
Embodiment 2: the expression plasmid of reorganization pig interleukin 4-Fc antigen-4 fusion protein gene makes up
With the synthetic fragment of the full gene of reorganization pig interleukin 4-Fc fusion rotein (shown in SEQ ID No:1) after optimizing, be building up in the pUC57 plasmid (being provided by Nanjing Jin Sirui Science and Technology Ltd.), obtain a kind of prolonged preservation plasmid, be designated as the pUC57-prIL4-Fc plasmid.Be template with the pUC57-prIL4-Fc plasmid, upstream and downstream are introduced NdeI and XhoI restriction enzyme site respectively, carry out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1:GGGAATTCCATATGCATAAGTGTGATATTACGC
Downstream primer:
P2:CCGCTCGAGTCATTTGCCCTGGGTTTTGC
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μ mol/L primers respectively add 2.5 μ L, and concentration is that the dNTP of 10mmol/L adds 1 μ L, and used archaeal dna polymerase Phusion High-Fidelity DNA polymerase(is available from Theromo-Fisher scientific), 2U/ μ L adds 0.5 μ L.Reaction conditions is 98 ℃ of 5s, 55 ℃ of 45s, 72 ℃ of 30s, and after 25 circulations, product is through 1.0% agarose gel electrophoresis analysis, and the product size is consistent with expection size (1023bp).(as shown in Figure 7)
The gene product that obtains is reclaimed test kit (available from sky, Beijing root biochemical technology company limited) purifying with dna gel.Behind the purifying, with NdeI and XhoI(available from New England Biolabs company) double digestion, (available from New England Biolabs company) is connected to the product behind the double digestion in the pET21b plasmid (available from Merck company) with the T4 ligase enzyme, be transformed in the DH5 α competent cell (available from sky, Beijing root biochemical technology company limited) 37 ℃ of overnight incubation in the LB flat board of the penbritin that contains 100 μ g/mL (available from Amresco company).Second day screening positive clone bacterium, order-checking, comparison result shows and expected sequence are in full accord, and the expression plasmid of a kind of form of pig interleukin 4-Fc fusion rotein that namely obtains recombinating is designated as pET21b-prIL4-Fc.
Concrete steps are as follows:
1. the correct pET21b-pIL4-Fc plasmid of order-checking comparison among the embodiment 2 is transformed in e. coli bl21 (DE3) the competence bacterial strain (available from sky, Beijing root biochemical technology company limited), in 37 ℃, incubated overnight in containing the penbritin flat board.
2. choose 1-4 reorganization bacterium colony that contains the pET21b-prIL4-Fc plasmid in second day, insert the LB nutrient solution (available from Amresco company) that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation.
3. get overnight culture in the 50 μ L steps 2, insert the LB nutrient solution that 5mL contains 100 μ g/mL penbritins, 37 ℃ of shaking culture.
4. bacterium liquid OD is surveyed every 1h in the inoculation back
600Value is treated OD
600=1.0 o'clock, with the IPTG(of 1mmol/L available from Amresco company) carry out abduction delivering.
5. collect bacterium liquid behind the abduction delivering 4h, and high speed centrifugation (rotating speed: 12000rpm) 3min, with the PBS washing and precipitating of precooling, add 5 * sds gel sample loading buffer, 100 ℃ of heating 10min, the room temperature high speed centrifugation (rotating speed: 12000rpm) 1min, get supernatant.Do not add the recombination bacillus coli culture of IPTG by this step process yet.
6. 5 the samples after handling set by step of getting respectively that 10 μ L do not add IPTG and add that IPTG induces, the 10%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis is moved to separation gel bottom to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining and immunoblotting are observed the expression product band, see Fig. 8-a and Fig. 8-b.
Many cell growth rates that studies show that have a strong impact on the expression of foreign protein, therefore must be to inoculation amount of bacteria, culture temperature, induce before cell growth time and induce the back cell density to control, overgrowth or overrun and all can influence the expression amount of reorganization pig interleukin 4-Fc fusion rotein inclusion body in the intestinal bacteria.Use three factors, four levels, set up IPTG concentration and induction time orthogonal table, induce reorganization pig interleukin 4-Fc fusion protein expression by the SDS-PAGE gel electrophoresis analysis.
Concrete steps are as follows:
1. the correct pET21b-prIL4-Fc plasmid of order-checking comparison among the embodiment 2 is transformed into BL21(DE3) in the competence bacterial strain (available from sky, Beijing root biochemical technology company limited), incubated overnight in 37 ℃ of penbritin flat boards.
2. next day, picking 1-4 reorganization bacterium colony that contains the pET21b-prIL4-Fc plasmid inserts the LB nutrient solution that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation.
3. get overnight culture in the 50 μ l steps 2 and insert the LB inducing culture liquid that 5mL contains 100 μ g/mL penbritins, 37 ℃ of shaking culture.
4. bacterium liquid OD is surveyed in the inoculation back
600Value is treated OD
600=1.0 o'clock, adding concentration according to following table was that 0.5m mol/L, 1.0m mol/L, 1.5m mol/L IPTG carry out abduction delivering.
Table 1 is investigated inductor concentration and the induction time of expression of recombinant proteins
5.1,2,3, collect reorganization pig interleukin 4-Fc fusion rotein bacterium liquid behind the 4h successively, (rotating speed: 12000rpm) 3min with the PBS washing and precipitating of precooling, adds 5 * sds gel sample loading buffer to high speed centrifugation, 100 ℃ of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm) 1min.
6. getting 10 μ L does not add IPTG and induces and the reorganization pig interleukin 4-Fc fusion rotein culture suspension that adds different concns IPTG, different induction times, 10%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis is moved to separation gel bottom to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining is observed reorganization pig interleukin 4-Fc fusion protein expression products band under each condition.(see figure 9)
9. the gel imaging system thin layer scanning is analyzed express recombinant pig interleukin 4-Fc fusion rotein content and is identified pig interleukin 4-Fc Expression of Fusion Protein.Final definite inductive condition that is fit to this enforcement is 1m mol/L IPTG, and induction time is 4h.
1. will be among the embodiment 3 collect the intestinal bacteria precipitation of inducing reorganization pig interleukin 4-Fc fusion rotein that contains that obtains, resuspended with the PBS of precooling, in 4 ℃ with 12000rpm, centrifugal 15min; Repeat once.
2. inhale and remove supernatant, claim bacterial sediment weight, every gram (weight in wet base) adds lysis buffer Buffer A5mL, stirs with the slicking glass rod, and thalline is hanged.
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 sonde-type ultrasonoscope, sample places on ice, and ultrasonic 120 times, each 5s is 5s at interval, circulates three times, is circulated between the cooling sample at every turn and waits for 2min, waits for the sample cooling.4 ℃, 12000rpm, centrifugal 15min.
5. precipitation is washed with lavation buffer solution Buffer B, and 4 ℃, 12000rpm, centrifugal 15min, the precipitation inclusion body repeats once.
6. the inclusion body precipitation stirs 30min with sex change buffer B uffer C dissolving under the room temperature.
7. abundant room temperature 12000rpm behind the mixing, centrifugal 15min abandons precipitation, gets supernatant, and pig interleukin 4-Fc fusion rotein denaturing soln namely obtains recombinating.
8. adopt the dilution refolding method that the reorganization pig interleukin 4-Fc fusion rotein denaturing soln in the step 7 is carried out renaturation.
Dilution refolding: get an amount of reorganization pig interleukin 4-Fc fusion rotein denaturing soln with sex change buffer B uffer C dissolving, with Quick Start Bradford1x Dye Reagent(U.S. Bio-Rad company) survey its concentration, with renaturation buffer BufferD protein concentration is diluted to 0.2mg/mL then, 4 ℃ of renaturation are during to 24h, recombinant protein solution after the renaturation is filtered with 0.45 μ m filter membrane (Merck Millipore company), namely obtain the reorganization pig interleukin 4-Fc fusion rotein solution of lower concentration.With ultrafiltration pipe (the Merck Millipore company) desalination of molecular weight cut-off 10KDa, concentrate, in vacuum freeze drier (Beijing Sihuan Scientific Instrument Factory Co., Ltd) low-temperature vacuum drying, namely obtain reorganization pig interleukin 4-Fc fusion rotein powder.
Each damping fluid according to the form below preparation:
Each damping fluid composition of table 2
9. carry out SDS-PAGE gel electrophoresis (Figure 10) with the product behind the dilution refolding in twice washed product of lavation buffer solution Buffer B and the step 8 in the step 5 respectively, at the visible obviously band of purpose scope.
The preparation of embodiment 6 reorganization pig interleukins 4 (in detail can referring to the applicant in first to file: 201210585655.0 examine
In)
Concrete steps are as follows:
1. but make up the recombination bacillus coli of express recombinant pig interleukin 4.
According to the disclosed pig interleukin 4(Sus of GenBank scrofa interleukin4) cDNA sequence (GenBank accession number: NM_214123.1), according to escherichia expression system this gene is carried out obtaining recombinating after codon optimized pig interleukin 4 genes, shown in SEQ ID No:3.The synthetic fragment of reorganization pig interleukin 4 full genes with after optimizing is building up in the pUC57 plasmid, obtains the pUC57-prIL4 plasmid.
Be template with the pUC57-prIL4 plasmid, the upstream and downstream primer is introduced NdeI and XhoI restriction enzyme site respectively, carries out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1：GGGAATTCCATATGCATAAGTGTGATATTACGC
Downstream primer:
P2：CCGCTCGAGTCAGCATTTGCTGTACTTTTC
Reaction cumulative volume 50 μ L, wherein concentration is that 10 μ mol/L primers respectively add 2.5 μ L, concentration is that the dNTP of 10mmol/L adds 1 μ L, used archaeal dna polymerase Phusion High-Fidelity DNA polymerase, 2U/ μ L adds 0.5 μ L.Reaction conditions is 98 ℃ of 5s, 55 ℃ of 20s, 72 ℃ of 30s, 25 circulations.The PCR product with NdeI and XhoI double digestion, is connected in the pET21b plasmid with the T4 ligase enzyme after reclaiming the test kit purifying with dna gel, and plasmid is transformed in the DH5 α competent cell increases.The expression plasmid pET21b-prIL4 of amplification is transformed in the escherichia coli BL21(DE3) expression bacterial strain.
2. the expression of reorganization pig interleukin 4 inclusion bodys
E. coli bl21 (DE3) incubated overnight in 37 ℃ of penbritin flat boards that will contain pET21b-prIL4.Choose 1-4 reorganization bacterium colony that contains the pET21b-prIL4 plasmid next day, insert the LB nutrient solution that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation.Get 50 μ L overnight culture and insert the LB inducing culture liquid that 5mL contains 100 μ g/mL penbritins, 37 ℃ of shaking culture.Treat OD
600=1.0 o'clock, induce with the IPTG of 1mmol/L.Collect bacterium liquid behind the 4h, high speed centrifugation is with the PBS washing and precipitating of precooling.
3. renaturation and the purifying of reorganization pig interleukin 4 inclusion bodys
Will be in the step 2 resuspended with the PBS of precooling through the PBS of precooling washing and precipitating, in 4 ℃ 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 (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 sonde-type ultrasonoscope, sample places on ice, and ultrasonic 120 times, each 5s is 5s at interval, circulates three times, waits for 2min between each circulation, waits for the sample cooling.4 ℃, 12000rpm, centrifugal 15min.Precipitation is washed with lavation buffer solution Buffer B, and 4 ℃, 12000rpm, centrifugal 15min, the precipitation inclusion body repeats once.The inclusion body precipitation stirs 30min with sex change buffer B uffer C dissolving under the room temperature.Room temperature 12000rpm behind the abundant mixing, centrifugal 15min abandons precipitation, gets supernatant, and pig interleukin 4 denaturing solns namely obtain recombinating.Adopt dialysis renaturation method renaturation inclusion body: will recombinate pig interleukin 4 denaturing soln concentration dilutions to 0.2mg/mL with sex change buffer B uffer C, inject the dialysis card of molecular weight cut-off 10KDa, 4 ℃ of dialysis renaturation are changed renaturation buffer Buffer D one time every 6h.Renaturation is crossed 0.45 μ m filter membrane with recombinant protein solution after the renaturation during to 24h, namely obtains the reorganization pig interleukin 4 renaturation solution of lower concentration.With the ultrafiltration pipe desalination of molecular weight cut-off 10KDa, concentrate, in the vacuum freeze drier low-temperature vacuum drying, namely obtain reorganization pig interleukin 4 powder.Each damping fluid preparation is as shown in table 2.4 ℃ of refrigerators of reorganization pig interleukin 4 powder are deposited stand-by.
The HRBC leukemia cell's of IL-4 (being called for short the TF-1 cell, available from ATCC CRL-2003) propagation has promoter action.In view of human interleukin 4 by using and pig interleukin 4 homologys greater than 80%, so present embodiment is with pig interleukin 4(Recombinant Procine Interteukin-4, article No. 907RPIL401, purchase is from ProSpec company, PO Box6591, East Brunswick, 08816NJ, USA) as positive control, relatively under the different concns condition gained reorganization pig interleukin 4 or reorganization pig interleukin 4-Fc fusion rotein to the influence of people TF-1 cell proliferation rate, thereby measure its biologic activity.
The biological activity of the reorganization pig interleukin 4 of dialysis renaturation method gained among dilution refolding gained reorganization pig interleukin 4-Fc fusion rotein and the embodiment 6 is measured positive control pig interleukin 4 and negative control simultaneously and is not contained the substratum of interleukin-4 to the influence of the proliferation function of people TF-1 cell among the embodiment 5 of mensuration different concns.By the recombinate vigor of pig interleukin 4-Fc fusion rotein of four parametric regression Equation for Calculating the present invention:
Reorganization pig interleukin 4-Fc fusion rotein biologic activity
The positive contrast pig interleukin 4 standard substance biologic activity of Pr, 2.5x10
5～1x10
6IU/ml
Ds is pig interleukin 4-Fc fusion rotein extension rate of the present invention;
The positive contrast pig interleukin 4 standard substance extension rates of Dr;
Es is equivalent to the extension rate of standard substance median effective dose for pig interleukin 4-Fc fusion rotein of the present invention;
The extension rate of positive contrast pig interleukin 4 median effective doses of Er.
Experimental result shows: compare with positive control, reorganization pig interleukin 4, reorganization pig interleukin 4-Fc fusion rotein all have the effect that promotes propagation to the TF-1 cell, and have dosage effect.Comparing with the cell of handling without interleukin-4, is under the 1ug/ml reorganization pig interleukin 4-Fc fusion protein sample treatment condition in concentration, TF-1 cell proliferation about 2 times.In addition, the reorganization pig interleukin 4-Fc fusion rotein vigor of gained of the present invention is 3.75x10
5～1.5x10
6IU/ml is nearly 1.5 times of pig interleukin 4 standard substance vigor, and active in reorganization pig interleukin 4(such as Figure 11-a, 11-b and table 3,4).As seen product vigor of the present invention has the value that extension is produced no less than the famous cytokine recombinant protein in whole world company development.
4 pairs of TF-1 cel l proliferations of the positive control pig interleukin of table 3 different concns
Table 4 reorganization pig interleukin 4-Fc fusion rotein is to the TF-1 cel l proliferation
In order to detect the stability of reorganization pig interleukin 4-Fc fusion rotein in serum, contriver's particular design a kind of in-vitro simulated porcine blood serum environment, reorganization pig interleukin 4-Fc fusion rotein of the present invention among the embodiment 5 of preparation reorganization pig interleukin 4 and 2 μ mol/ml among the embodiment 6 of 2 μ mol/ml is placed fresh porcine blood serum 50 μ L, 37 ℃ jointly, 120rpm, react 0,1,2 respectively, 4,8,24,48 backs are preserved-20 ℃.With mouse-anti pig interleukin 4 monoclonal antibodies (Porcine IL-4Antibody, Monoclonal Mouse IgG1Clone#99605, Catalog Number:MAB6543, MAB6521, R﹠amp; D Systems) for primary antibodie the pig interleukin 4 in the porcine blood serum and reorganization pig interleukin 4-Fc fusion rotein are carried out the protein immunoblot test, pig interleukin 4 just can't detect in serum in that reaction is very fast after 2 hours as shown in Figure 12, and reorganization pig interleukin 4-Fc fusion rotein still kept stablizing in serum in 48 hours.This shows that the reorganization pig interleukin 4-Fc fusion rotein of the present invention's preparation is compared with pig interleukin 4 has advantages of higher stability, has prolonged the transformation period in its body, has reached the effect of long-acting administration.
Claims (10)
1. reorganization pig interleukin 4-Fc fusion rotein, it is characterized in that, described fusion rotein comprises pig interleukin 4 parts and Fc fragment part, described pig interleukin 4 parts are the full sequence of pig interleukin 4 extracellular regions, described Fc fragment partly comprises hinge area, CH2 district and CH3 district, is directly to merge between pig interleukin 4 and the Fc fragment part.
2. fusion rotein as claimed in claim 1 is characterized in that, the aminoacid sequence of described fusion rotein is shown in SEQ ID NO:2.
3. gene of the fusion rotein described in the claim 2 of encoding, its base sequence is shown in SEQ ID NO:1.
4. carrier, described carrier contains the gene of claim 3.
5. carrier as claimed in claim 4, described carrier is pET21b.
6. intestinal bacteria, described intestinal bacteria have the carrier of claim 4 or 5.
7. intestinal bacteria as claimed in claim 6, described intestinal bacteria are BL21(DE3) bacterial strain.
8. the procaryotic cell expression method of pig interleukin 4-Fc fusion rotein of recombinating comprises the steps:
(1) under appropriate condition, in substratum, cultivates the intestinal bacteria described in the claim 7;
(2) from intestinal bacteria and/or substratum, separate reorganization pig interleukin 4-Fc fusion rotein.
9. expression method as claimed in claim 8 comprises the steps:
(1) picking is one or more contains the intestinal bacteria bacterium colony described in the claim 7, inserts to contain antibiotic LB nutrient solution, overnight incubation;
(2) get overnight culture and transfer in containing antibiotic fresh LB nutrient solution, be cultured to mid-log phase OD in 37 ℃ of concussions
600=1.0;
(3) adding concentration in culture is the IPTG of 1m mol/L, and 37 ℃, behind the abduction delivering 4h, centrifugal treating is collected the coli somatic precipitation that contains reorganization pig interleukin 4-Fc fusion rotein.
10. purifying and the refolding method of pig interleukin 4-Fc fusion rotein of recombinating is characterized in that, comprises following steps:
(1) as claimed in claim 9 the containing that collection is obtained induced reorganization pig interleukin 4-Fc fusion rotein intestinal bacteria precipitation, and be resuspended with the PBS of precooling, and handle in 4 ℃ of high speed centrifugations, repeats once;
(2) supernatant is removed in suction, claims bacterial sediment weight, and every gram (weight in wet base) adds lysis buffer Buffer A3-10mL, stirs damping fluid, and thalline is hanged;
(3) every gram (weight in wet base) thalline adding 3-10 μ L concentration is the PMSF of 100mmol/L, and 3-100 μ L concentration is the N,O-Diacetylmuramidase of 100mg/mL, in stirring on ice;
(4) broken thalline, sample places on ice, and is ultrasonic, and handles in 4 ℃ of high speed centrifugations;
(5) precipitation is washed with lavation buffer solution Buffer B, and handles in 4 ℃ of high speed centrifugations, and the precipitation inclusion body repeats once;
(6) the inclusion body precipitation stirs 30-60min with sex change buffer B uffer C dissolving under the room temperature;
(7) the room temperature high speed centrifugation is handled behind the abundant mixing, abandons precipitation, gets supernatant, and pig interleukin 4-Fc fusion rotein denaturing soln namely obtains recombinating;
(8) get an amount of reorganization pig interleukin 4-Fc fusion rotein denaturing soln with sex change buffer B uffer C dissolving, to recombinate the concentration dilution of pig interleukin 4-Fc fusion rotein denaturing soln to 0.2mg/mL with sex change buffer B uffer D, 4 ℃ of dialysis renaturation 24h, with 0.45 μ m membrane filtration, pig interleukin 4-Fc fusion rotein renaturation solution namely obtains recombinating with recombinant protein solution after the renaturation;
Further ultrafiltration and concentration, desalination are to minimum volume for described reorganization pig interleukin 4-Fc fusion rotein renaturation solution, and low-temperature vacuum drying namely obtains reorganization pig interleukin 4-Fc powder.
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| CN106046177A (en) * | 2016-08-19 | 2016-10-26 | 北华大学 | P-5m-Fc fusion protein and expression gene thereof, and preparation method and applications of P-5m-Fc fusion protein |
| CN110055272A (en) * | 2019-04-18 | 2019-07-26 | 西北农林科技大学 | A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication |
| CN111565747A (en) * | 2018-09-19 | 2020-08-21 | 巴伊沃爱普有限公司 | Antigen fused with porcine FC fragment, and vaccine composition comprising same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104558100A (en) * | 2015-02-04 | 2015-04-29 | 吉林农业大学 | Inclusion body pretreatment method |
| CN106046177A (en) * | 2016-08-19 | 2016-10-26 | 北华大学 | P-5m-Fc fusion protein and expression gene thereof, and preparation method and applications of P-5m-Fc fusion protein |
| CN106046177B (en) * | 2016-08-19 | 2020-02-21 | 北华大学 | P-5m-Fc fusion protein and expression gene, preparation method and application thereof |
| CN111565747A (en) * | 2018-09-19 | 2020-08-21 | 巴伊沃爱普有限公司 | Antigen fused with porcine FC fragment, and vaccine composition comprising same |
| CN111565747B (en) * | 2018-09-19 | 2023-08-15 | 巴伊沃爱普有限公司 | Antigen fused to porcine FC fragment and vaccine composition comprising the same |
| CN110055272A (en) * | 2019-04-18 | 2019-07-26 | 西北农林科技大学 | A kind of targeting antibodies therapeutic agent inhibiting PRRS virus multiplication |
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