CN103265637B

CN103265637B - A kind of Recombinant Swine IL-4-Fc fusion rotein and encoding gene thereof and expression method

Info

Publication number: CN103265637B
Application number: CN201310222553.7A
Authority: CN
Inventors: 马永; 王安良; 章成昌; 陈晨; 徐春林; 王耀方
Original assignee: CHANGZHOU GENSUN INSTITUTE OF BIOMEDICINE Co Ltd; ZONHON BIOPHARMA INSTITUTE Inc
Current assignee: Jiangsu Jingsen Biomedical New Materials Technology Co., Ltd.
Priority date: 2013-06-04
Filing date: 2013-06-04
Publication date: 2015-10-21
Anticipated expiration: 2033-06-04
Also published as: CN103265637A

Abstract

The invention provides a kind of Recombinant Swine IL4-Fc fusion rotein and encoding gene, expression, purifying and renaturing inclusion bodies method, belong to biological gene engineering field.Pig IL4 can be used for the chronic infectious disease of pig and the treatment of parasitosis, also can be applied to anaphylaxis of pig etc. with Ia diseases prevention and treatment by further genralrlization.But there is the defect that in blood plasma, removing speed is fast, industrialization cost is high in pig IL4.The present invention adopts escherichia coli prokaryotic expression system to provide a kind of long-acting Recombinant Swine IL4-Fc fusion rotein.Wherein, pig IL4 part be the full sequence of pig IL4 extracellular region, and Fc segment portion comprises the hinge area of antibody, CH2 district and CH3 district, be therebetween direct fusion.Recombinant Swine IL4-Fc fusion rotein provided by the invention, not only improves the biological activity of IL4, and extends its transformation period greatly, for low cost great expression and industrialization thereof provide guarantee.

Description

A kind of Recombinant Swine IL-4-Fc fusion rotein and encoding gene thereof and expression method

Technical field

The invention belongs to biotechnology gene field, relate to a kind of Recombinant Swine IL-4-Fc fusion rotein and encoding gene thereof, and its expression, purifying and renaturing inclusion bodies method.

Background technology

Interleukin-4 (Interleukin4, IL-4) by the T cell activated, mastocyte and basophilic granulocyte synthesis secretion, be a kind of multi-functional lymphokine, there is the biologic activity of Various Complex, comprise and regulate the lymphocytic growth of T, breeding and differentiation; Regulate the T cell atomization by antigenic stimulation; The T cell after differentiation is regulated to produce the cytokines such as IL-4, IL-5, IL-10 and IL-13; Control the specificity etc. of immunoglobulin class conversion.In addition, IL-4 is to the induced reaction of T cell, B cell and NK cell in removing helminth infection, and the treatment aspect such as tumour, autoimmune disorder also plays important regulating effect.As can be seen here, IL-4 can be used as the diagnosis index that immunological adjuvant, therapeutical agent and disease infect, and has great importance in prevention and control of diseases and diagnosis etc.At present, all have report to IL-4 in the biologic activity functional study of mouse, rabbit, sheep, ox and people, but relatively less to the research of pig IL-4, and for its special detection system and bio-evaluation systematic research also rare.

China raises pigs big country in the world, in the face of the great epidemic 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 pig industry, adopt the generation of efficient vaccine prevention and corntrol various epidemic disease and popular.Porcine IL-4 is the important component part of pig vaccine research, but natural porcine IL-4 is very micro-at machine expression in vivo, is difficult to a large amount of in body extraction for clinical study and practical application.Genetic engineering technique can produce foreign protein in a large number, meets the demand of investigation and application.Therefore the present invention provides a kind of with low cost and can great expression Recombinant Swine interleukin-4 expression system and expression method by genetic engineering means.

On the other hand, as small molecular weight protein, porcine IL-4 is also the same with other interleukins, there is the defect of plasma clearance speed, causes in clinical treatment, need repeatedly medication just can reach result for the treatment of.IgG immunoglobulin (Ig) is the main antibody in humans and animals body, and its transformation period in vivo can reach 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.In view of this, the present invention attempts considering that the Fc fragment increasing IgG on porcine IL-4 is to form fusion rotein, to improve porcine IL-4 Half-life in vivo, reaches long-acting object.Thus, the object of this invention is to provide one and can retain the original activity of porcine IL-4, the porcine IL-4 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 IL-4-Fc fusion rotein of high expression and its gene and expression in intestinal bacteria, purifying, refolding method.

The invention provides Recombinant Swine IL-4-Fc fusion rotein, described fusion rotein comprises porcine IL-4 part and Fc segment portion, wherein, porcine IL-4 part is the full sequence of porcine IL-4 extracellular region, Fc segment portion comprises hinge area, CH2 district and CH3 district, is directly to merge between porcine IL-4 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 IL-4-Fc fusion rotein of the present invention has the aminoacid sequence shown in SEQ ID NO:2, wherein 2-110 amino acids residue is the extracellular domain sequence of porcine IL-4, and 111-340 amino acids residue is pig IgG 1 sequence.

The invention provides the gene of Recombinant Swine IL-4-Fc fusion rotein described above of encoding, its base sequence is as shown in SEQ IDNO: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 IL-4-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 IL-4-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 IL-4-Fc fusion rotein described above, access LB nutrient solution, in 37 DEG C of overnight incubation;

2. get appropriate overnight culture, in the ratio access LB nutrient solution of 1:100, 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, 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 IL-4-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 IL-4-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 IL-4-Fc fusion rotein, comprise the steps:

1. precipitate collecting obtain above-mentioned containing induction Recombinant Swine IL-4-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 damping fluid, thalline is hanged.

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 IL-4-Fc fusion rotein denaturing soln.

This purification process preferred steps is as follows:

1. precipitate collecting obtain above-mentioned containing induction Recombinant Swine IL-4-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 solution and thalline is hanged.

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 IL-4-Fc fusion rotein denaturing soln.

Present invention also offers the renaturing inclusion bodies method of the Recombinant Swine IL-4-Fc fusion rotein after optimization, comprise the steps:

Get the Recombinant Swine IL-4-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 IL-4-Fc fusion rotein solution of lower concentration.And can ultrafiltration desalination further, concentrated, low-temperature vacuum drying, namely obtains Recombinant Swine IL-4-Fc fusion rotein powder.Composition and the content thereof of each damping fluid are as shown in the table:

Porcine IL-4 of the present invention not only can be used for treating the chronic infectious disease of pig and parasitosis, also can be applied in the anaphylaxis of pig etc. and Ia disease treatment and go by further genralrlization.

Different according to prepared vaccine form, the embodiment of porcine IL-4 of the present invention can have multiple: as vaccines such as aluminium glue adjuvant, oily adjuvant, Liposome Adjuvants; Injection system can adopt the multitude of different ways such as subcutaneous injection, intradermal injection and intramuscular injection; If live vector vaccine, except above injection system, also can adopt the different modes such as oral, suction, collunarium, eye drip.

Of the present invention through optimised porcine IL-4-Fc fusion rotein recombination sequence, be more suitable for the expression of escherichia expression system, compared with porcine IL-4-Fc fusion rotein native gene sequence, the expression efficiency of porcine IL-4-Fc fusion rotein in intestinal bacteria after optimization significantly improves.Expressed porcine IL-4-Fc fusion rotein is far above the expression amount of porcine IL-4-Fc fusion rotein native gene sequence at escherichia expression system.And, compared with porcine IL-4, Recombinant Swine IL-4-Fc fusion rotein of the present invention on the basis that ensure that porcine IL-4 activity largely on extend its transformation period in vivo, achieve long-acting and avoid the object of medication repeatedly.

Accompanying drawing explanation

Fig. 1 represents Recombinant Swine IL-4-Fc fusion rotein codon optimized front and back nucleotide sequence comparison

Wherein, odd-numbered line (row that namely " original series " is corresponding) is porcine IL-4-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 IL-4-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 porcine IL-4-Fc fusion rotein CAI index in escherichia coli expression host.

Wherein, Fig. 2-a represent Recombinant Swine IL-4-Fc fusion rotein codon optimized before in escherichia coli expression host CAI index be 0.62; Fig. 2-b represent Recombinant Swine IL-4-Fc fusion rotein codon optimized after in escherichia coli expression host CAI index be 0.86.

Fig. 3-a, Fig. 3-b are restructuring porcine IL-4-Fc fusion rotein codon optimal codon frequency distribution areal map in escherichia coli expression host.

Wherein, Fig. 3-a represent Recombinant Swine IL-4-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 IL-4-Fc fusion rotein occurs that per-cent is 9%; Fig. 3-b represent Recombinant Swine IL-4-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 IL-4-Fc fusion rotein occurs that per-cent is 0.

Fig. 4-a, Fig. 4-b in restructuring porcine IL-4-Fc fusion rotein codon in escherichia coli expression host average GC base contents distributed areas figure.

Wherein, Fig. 4-a represent Recombinant Swine IL-4-Fc fusion rotein codon optimized before in escherichia coli expression host average GC base contents be: 57.22%; Fig. 4-b represent Recombinant Swine IL-4-Fc fusion rotein codon optimized after in escherichia coli expression host average GC base contents be: 52.19%.

Fig. 5-a, Fig. 5-b are the secondary structure prediction figure of the codon optimized front and back mRNA of restructuring porcine IL-4-Fc fusion rotein.

The secondary structure prediction figure of the codon optimized premessenger RNA of Fig. 5-a Recombinant Swine IL-4-Fc fusion rotein, Fig. 5-b are the secondary structure prediction figure of the codon optimized rear mRNA of restructuring porcine IL-4-Fc fusion rotein.

Fig. 6 is restructuring porcine IL-4-Fc fusion protein expression plasmid building process figure.

Fig. 7 is restructuring porcine IL-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 after NdeI and XhoI double digestion; Swimming lane 3 is for containing the Recombinant Swine IL-4-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 porcine IL-4-Fc fusion rotein and corresponding western blot figure.

Fig. 8-a is restructuring porcine IL-4-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 IL-4-Fc fusion rotein E. coli lysate not adding IPTG induction; Swimming lane 3 is for adding the Recombinant Swine IL-4-Fc fusion rotein E. coli lysate of IPTG induction.

Fig. 8-b is restructuring porcine IL-4-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 IL-4-Fc fusion rotein E. coli lysate not adding IPTG induction: swimming lane 3 is for adding the Recombinant Swine IL-4-Fc fusion rotein E. coli lysate of IPTG induction.

Fig. 9 Recombinant Swine IL-4-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 IL-4-Fc fusion rotein E. coli lysate; Swimming lane 3 is 0.5mmol/L IPTG induction 2h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 4 is 0.5mmol/L IPTG induction 3h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 5 is 0.5mmol/L IPTG induction 4h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 6 is 1mmol/L IPTG induction 1h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 7 is 1mmol/L IPTG induction 2h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 8 is 1mmol/L IPTG induction 3h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 9 is 1mmol/L IPTG induction 4h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 10 is 1.5mmol/L IPTG induction 1h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 11 is 1.5mmol/L IPTG induction 2h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 12 is 1.5mmol/L IPTG induction 3h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate; Swimming lane 13 is 1.5mmol/L IPTG induction 4h Recombinant Swine IL-4-Fc fusion rotein E. coli lysate.

Figure 10 is the Recombinant Swine IL-4-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 is the full bacterium lysate containing Recombinant Swine IL-4-Fc fusion rotein after ultrasonication; Swimming lane 3 is for cleaning rear Recombinant Swine IL-4-Fc fusion rotein inclusion body precipitation with Buffer B first time; Swimming lane 4 is Recombinant Swine IL-4-Fc fusion rotein inclusion body precipitation after the cleaning of Buffer B second time, and swimming lane 5 is Recombinant Swine IL-4-Fc fusion rotein after Buffer D renaturation.

Figure 11 Recombinant Swine interleukin-4 vitality test curve.

Figure 11-a represents the impact of positive control porcine IL-4 on TF-1 cell proliferation rate, and Figure 11-b represents and to compare with Recombinant Swine interleukin-4 with negative control, and Recombinant Swine IL-4-Fc fusion rotein of the present invention is on the impact of TF-1 cell proliferation rate.

Wherein, as can be seen from Figure 11-a, 11-b, Recombinant Swine IL-4-Fc fusion rotein of the present invention is the same with positive control medicine, has promoter action to the propagation of TF-1 cell; As can be seen from 11-b, compared with the TF-1 cell without interleukin-4 process, under the effect of 1ug/mL Recombinant Swine IL-4-Fc fusion protein sample, TF-1 cell proliferation about 2 times.

Figure 12 is restructuring porcine IL-4-Fc fusion rotein 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 porcine IL-4 and Recombinant Swine IL-4-Fc fusion rotein pig anteserum sample; Swimming lane 3 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 0h; Swimming lane 4 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 1h; Swimming lane 5 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 2h; Swimming lane 6 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 4h; Swimming lane 7 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 8h; Swimming lane 8 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 24h; Swimming lane 9 is for adding the pig anteserum sample of 2 μm of ol/mL porcine IL-4 and 2 μm of ol/mL Recombinant Swine IL-4-Fc fusion rotein 48h.

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 IL-4-Fc antigen-4 fusion protein gene optimization design

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_214123.1) of the published porcine IL-4 of GenBank (Sus scrofa interleukin4) 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 IL-4-Fc fusion rotein of the present invention, as shown in SEQ ID No:1.

Here is carried out codon optimized to restructuring porcine IL-4-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 IL-4-Fc antigen-4 fusion protein gene codon adaptation indexI (codon adaptation index, CAI) in intestinal bacteria is 0.62.From Fig. 2-b, after codon optimized, Recombinant Swine IL-4-Fc antigen-4 fusion protein gene CAI index in intestinal bacteria is made to be 0.86.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 IL-4-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 porcine IL-4-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, and these codons may reduce translation efficiency, even can dismiss translation assemblage.From Fig. 3-b, after codon optimized, Recombinant Swine IL-4-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 porcine IL-4-Fc antigen-4 fusion protein gene of Fig. 4-a, Fig. 4-b, be 57.22% by showing in porcine IL-4-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 the extra-regional base of 30%-70%, after being finally optimized, the GC base average content of Recombinant Swine IL-4-Fc fusion rotein is 52.19%.

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 porcine IL-4-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 IL-4-Fc antigen-4 fusion protein gene builds

By the fragment that the Recombinant Swine IL-4-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-prIL4-Fc plasmid.With pUC57-prIL4-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:GGGAATTCCATATGCATAAGTGTGATATTACGC

Downstream primer:

P2:CCGCTCGAGTCATTTGCCCTGGGTTTTGC

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 (1023bp).(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 EnglandBiolabs 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 IL-4-Fc fusion rotein, is designated as pET21b-prIL4-Fc.

the high expression of embodiment 3 Recombinant Swine IL-4-Fc fusion rotein in intestinal bacteria and qualification

Concrete steps are as follows:

1. by pET21b-pIL4-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), in 37 DEG C, containing incubated overnight in ampicillin plate.

2. within second day, choose 1-4 the restructuring bacterium colony containing pET21b-prIL4-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) 3min, by the PBS washing and precipitating of precooling, adds 5 × sds gel sample loading buffer, and 100 DEG C of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm) 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 IL-4-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 IL-4-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 IL-4-Fc fusion protein expression.

Concrete steps are as follows:

1. by pET21b-prIL4-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-prIL4-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, add according to following table that concentration is 0.5m mol/L, 1.0m mol/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 IL-4-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 IL-4-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.

8. coomassie brilliant blue staining, observes Recombinant Swine IL-4-Fc fusion protein expression products band under each condition.(see figure 9)

9. the expression of Recombinant Swine IL-4-Fc fusion rotein content qualification porcine IL-4-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 IL-4-Fc fusion rotein inclusion body purification and renaturation

1. will collect the intestinal bacteria precipitation containing induction Recombinant Swine IL-4-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.

7. room temperature 12000rpm after fully mixing, centrifugal 15min, abandons precipitation, gets supernatant, namely obtains Recombinant Swine IL-4-Fc fusion rotein denaturing soln.

8. adopt dilution refolding method to carry out renaturation to the Recombinant Swine IL-4-Fc fusion rotein denaturing soln in step 7.

Dilution refolding: get the Recombinant Swine IL-4-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 (MerckMillipore company) of recombinant protein solution after renaturation is filtered, namely obtains the Recombinant Swine IL-4-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 IL-4-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 the product after dilution refolding in lavation buffer solution Buffer B twice washed product in step 5 and step 8 respectively, at the visible obviously band of object scope.

the preparation of embodiment 6 Recombinant Swine interleukin-4 (in detail can see the earlier application of applicant: 201210585655.0 examine in)

Concrete steps are as follows:

1. build the recombination bacillus coli can expressing Recombinant Swine interleukin-4.

According to the cDNA sequence (GenBank accession number: NM_214123.1) of the published porcine IL-4 of GenBank (Sus scrofa interleukin4), according to escherichia expression system to this gene carry out codon optimized after obtain Recombinant Swine interleukin-4 gene, as shown in SEQ ID No:3.By the fragment that the Recombinant Swine interleukin-4 full genome after optimizing synthesizes, be building up in pUC57 plasmid, obtain pUC57-prIL4 plasmid.

With pUC57-prIL4 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：GGGAATTCCATATGCATAAGTGTGATATTACGC

Downstream primer:

P2：CCGCTCGAGTCAGCATTTGCTGTACTTTTC

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-prIL4 of amplification is transformed in escherichia coli BL21(DE3) expression bacterial strain.

2. the expression of Recombinant Swine interleukin-4 inclusion body

By e. coli bl21 (DE3) incubated overnight in 37 DEG C of ampicillin plate containing pET21b-prIL4.Choose 1-4 the restructuring bacterium colony containing pET21b-prIL4 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-4 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, waits for 2min between each circulation, waits 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-4 denaturing soln.Adopt dialysis renaturation method renaturation inclusion body: use denaturation buffer Buffer C by Recombinant Swine interleukin-4 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-4 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-4 powder.Each buffer is as shown in table 2.Recombinant Swine interleukin-4 powder 4 DEG C of refrigerators are deposited stand-by.

embodiment 7 Recombinant Swine IL-4-Fc fusion rotein Determination of biological activity

The propagation of IL-4 to HRBC leukemia cell (being called for short TF-1 cell, purchased from ATCC CRL-2003) has promoter action.In view of human interleukin 4 by using and porcine IL-4 homology are greater than 80%, so the present embodiment is with porcine IL-4 (Recombinant Procine Interteukin-4, article No. 907RPIL401, buy from ProSpec company, PO Box6591, East Brunswick, 08816NJ, USA) as positive control, under comparison different concns condition, gained Recombinant Swine interleukin-4 or Recombinant Swine IL-4-Fc fusion rotein are on the impact of people TF-1 cell proliferation rate, thus measure its biologic activity.

Measure the biological activity of the Recombinant Swine interleukin-4 of dialysis renaturation method gained in dilution refolding gained Recombinant Swine IL-4-Fc fusion rotein and embodiment 6 in the embodiment 5 of different concns, Simultaneously test positive control porcine IL-4 and negative control containing the substratum of interleukin-4 on the impact of the proliferation function of people TF-1 cell.Vigor by four parametric regression Equation for Calculating Recombinant Swine IL-4-Fc of the present invention fusion rotein:

Recombinant Swine IL-4-Fc fusion rotein biologic activity

Pr is positive control porcine IL-4 standard substance biologic activity, 2.5x10 ⁵~ 1x10 ⁶iU/ml

Ds is porcine IL-4-Fc fusion rotein extension rate of the present invention;

Dr is positive control porcine IL-4 standard substance extension rates;

Es is the extension rate that porcine IL-4-Fc fusion rotein of the present invention is equivalent to standard substance median effective dose;

Er is the extension rate of positive control porcine IL-4 median effective dose.

Experimental result shows: compared with positive control, and Recombinant Swine interleukin-4, Recombinant Swine IL-4-Fc fusion rotein all have the effect promoting propagation to TF-1 cell, and have dosage effect.Compared with the cell without interleukin-4 process, under concentration is 1ug/ml Recombinant Swine IL-4-Fc fusion protein sample treatment condition, TF-1 cell proliferation about 2 times.In addition, the Recombinant Swine IL-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 porcine IL-4 standard substance vigor, and activity is apparently higher than Recombinant Swine interleukin-4 (as Figure 11-a, 11-b and table 3,4).Visible product vigor of the present invention, no less than the whole world famous cytokine recombinant protein company development, has the value that extension is produced.

The positive control porcine IL-4 of table 3 different concns is to TF-1 cel l proliferation

Table 4 Recombinant Swine IL-4-Fc fusion rotein is to TF-1 cel l proliferation

the stability test of embodiment 8 Recombinant Swine IL-4-Fc fusion rotein

In order to detect the stability of Recombinant Swine IL-4-Fc fusion rotein in serum, contriver is specially designed a kind of in-vitro simulated porcine blood serum environment, and Recombinant Swine IL-4-Fc fusion rotein of the present invention in the embodiment 5 preparing Recombinant Swine interleukin-4 and 2 μm of ol/ml in the embodiment 6 of 2 μm of ol/ml is placed in fresh porcine blood serum 50 μ L, 37 DEG C jointly, 120rpm, react 0 respectively, 1,2,4,-20 DEG C are preserved after 8,24,48.With mouse-anti porcine IL-4 monoclonal antibody (PorcineIL-4Antibody, Monoclonal Mouse IgG1Clone#99605, Catalog Number:MAB6543, MAB6521, R & D Systems) protein immunoblot test is carried out to the porcine IL-4 in porcine blood serum and Recombinant Swine IL-4-Fc fusion rotein for primary antibodie, porcine IL-4 just cannot detect reaction is very fast after 2 hours in serum as shown in Figure 12, and Recombinant Swine IL-4-Fc fusion rotein still keeps stable in 48 hours in serum.As can be seen here, Recombinant Swine IL-4-Fc fusion rotein prepared by the present invention has higher stability compared with porcine IL-4, extends its Half-life in vivo, reaches the effect of long-acting administration.

Claims

1. encode the gene of Recombinant Swine IL-4-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 IL-4-Fc fusion rotein, comprises the steps:

(1) in substratum, the intestinal bacteria described in claim 5 are cultivated under suitable conditions;

(2) from intestinal bacteria and/or substratum, be separated restructuring porcine IL-4-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 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 IL-4-Fc fusion rotein precipitates.

8. the purifying of Recombinant Swine IL-4-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 IL-4-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 damping fluid, thalline is hanged;

(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 IL-4-Fc fusion rotein denaturing soln;

(8) the Recombinant Swine IL-4-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 IL-4-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 IL-4-Fc fusion rotein renaturation solution;

Described Recombinant Swine IL-4-Fc fusion rotein renaturation solution can ultrafiltration and concentration, desalination be to minimum volume further, and low-temperature vacuum drying, namely obtains Recombinant Swine IL-4-Fc powder;

Composition and the content of described Buffer A are: 20mmol/L Tris – HCl, 0.15mol/L NaCl, 1mmol/L EDTA, and 1mmol/L PMSF, and solution matrix is distilled water, and pH is 7.5;

Composition and the content of described Buffer B are: 20mmol/L Tris – HCl, 0. 15mol/L NaCl, 1mmol/L EDTA, and 0.5% (v/v) Triton X-100(content is), and 0.5mol/L urea, solution matrix is distilled water, and pH is 7.5;

Composition and the content of described Buffer C are: 20mmol/L Tris – HCl, 0. 15mol/L NaCl, 1mmol/L EDTA, 1mmol/L PMSF, 6mol/L Guanidinium hydrochloride, and 20mmol/L DTT, and solution matrix is distilled water, and pH is 7.8;

Composition and the content of described Buffer D are: 20mmol/L Tris – HCl, 1mmol/L EDTA, GSH:GSSG(mol/mol)=15:1, solution matrix is distilled water, and pH is 7.5.