CN103232545A

CN103232545A - Recombination porcine interferon alpha1-Fc fusion protein as well as coding gene and expression method thereof

Info

Publication number: CN103232545A
Application number: CN2013101828556A
Authority: CN
Inventors: 马永; 王安良; 章成昌; 徐春林; 陈晨; 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: 2013-05-16
Filing date: 2013-05-16
Publication date: 2013-08-07
Anticipated expiration: 2033-05-16
Also published as: CN103232545B

Abstract

The invention provides a recombination porcine IFN (interferon) alpha1-Fc fusion protein as well as a coding gene and expression, purification and inclusion body renaturation methods of the recombination porcine IFN alpha1-Fc fusion protein. IFN alpha1 is the most common interferon and has remarkable anti-virus, anti-tumor, hematopoietic cell proliferation inhibition, immune adjustment functions and the like. But natural IFN alpha1 is expressed in an organism in a very small amount, lots of IFN alpha1 is difficult to directly extract in vivo for clinical research and application, and the defect of fast clearing speed of the IFN alpha 1 in blood plasma exists. Therefore, the invention provides the recombination porcine IFN alpha1-Fc fusion protein suitable for an escherichia coli prokaryotic expression system, wherein a porcine IFN alpha1 part is an entire sequence of a porcine IFN alpha1 extracellular region, an Fc fragment part comprises a hinge region, a CH2 region and a CH3 region of an antibody, and the porcine IFN alpha1 part and the Fc fragment part are directly fused. The fusion protein provided by the invention maintains the most of biological activity of the IFN alpha 1, the half-life period of the fusion protein is greatly prolonged, and conditions are provided for the industrialization of the fusion protein.

Description

A kind of reorganization pig interferon α 1-Fc fusion rotein and encoding gene and expression method

Technical field

The invention belongs to biotechnology gene field, relate to a kind of reorganization pig interferon α 1-Fc fusion rotein and encoding gene thereof, with and expression, purifying and renaturing inclusion bodies method.

Background technology

(Interferon is one group of active protein (mainly being glycoprotein) with multiple function IFN) to Interferon, rabbit, is a kind of cytokine that is produced by monocyte and lymphocyte.They allogenic cell have wide spectrum antiviral, influence cell growth, and differentiation, regulate multiple biological activity such as immunologic function.According to the source of IFN be the character of animal species, cell type, inducer with to induce condition different, can be divided into three kinds of α, β, γ.Wherein IFN-α is one group of similar, the approaching low molecular saccharides albumen of function that immunocyte produces by the antiviral response reaction.In numerous hypotypes, interferon alpha 1 is a modal class, it have significantly antiviral, antitumor, suppress functions such as hematopoietic cell proliferation and immunomodulatory, be applicable to treatment of diseases such as disease of viral infection (as hepatitis), myeloproliferative disease, lymphocytic tumors and other tumour.

China is the big country of raising pigs, and pig virus transmissible disease kind is many, harm is big, though China generally inoculates the swine disease vaccine at present, but still can not the better controlled eqpidemic disease.Genetically engineered reorganization pig interferon has antiviral effect widely, and have have no side effect, advantage such as drug residue free, be with a wide range of applications in field of veterinary.Bibliographical information pig interferon α 1 is suppressing to have tangible effect aspect the foot and mouth disease virus vigor, and is very little but natural pig interferon α 1 expresses in body, is difficult to directly extract for clinical study and application in a large number in body.Therefore the present invention provides a kind of with low cost and can great expression reorganization pig interferon α 1 expression system and expression method by genetic engineering means.

On the other hand, as small molecular weight protein, pig interferon α 1 is also the same with other Interferon, rabbit, has plasma clearance speed defective faster, just can reach result for the treatment of thereby cause needing to repeat repeatedly medication in clinical treatment.Equally, the frequent bottleneck that uses pig interferon α 1 treatment pig virus disease that also will become of transformation period weak point and medication.The IgG immunoglobulin (Ig) is the main antibody in the humans and animals body, and its transformation period in vivo is about 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 interferon α 1 increases IgG constitutes fusion rotein, to improve the transformation period in pig interferon α 1 body, reaches long-acting purpose.Thus, the object of the invention provides and a kind ofly can keep pig interferon α 1 original activity, again can extension body in the fusion rotein of pig interferon α 1 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 recombination fusion protein that can in intestinal bacteria, efficiently express with and gene and expression, purifying, refolding method.

The invention provides reorganization pig interferon α 1-Fc fusion rotein, described fusion rotein comprises pig interferon α 1 part and Fc fragment part, wherein, pig interferon α 1 part is the full sequence of pig interferon α 1 extracellular region, the Fc fragment partly comprises hinge area, CH2 district and CH3 district, is directly to merge between pig interferon α 1 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 interferon α 1-Fc fusion rotein of the present invention has the aminoacid sequence shown in the SEQ ID NO:2, and wherein 2-167 amino acids residue is the extracellular region sequence of pig interferon α 1, and 168-397 amino acids residue is pig IgG 1 sequence.

The invention provides the gene of coding reorganization pig interferon α 1-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 interferon α 1-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 interferon α 1-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 interferon α 1-Fc fusion rotein described above, insert the LB nutrient solution, in 37 ℃ of overnight incubation;

2. get an amount of overnight culture, insert in the LB nutrient solution of 100 times of amounts of described overnight culture, 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 interferon α 1-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 interferon α 1-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 interferon α 1-Fc fusion rotein, comprises the steps:

1. above-mentioned the containing that collection is obtained induced reorganization pig interferon α 1-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 A 3-10mL, 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 interferon α 1-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 interferon α 1-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 with the slicking glass rod, and 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 interferon α 1-Fc fusion rotein denaturing soln namely obtains recombinating.

The present invention also provides the renaturing inclusion bodies method of the reorganization pig interferon α 1-Fc fusion rotein after optimizing, and comprises the steps:

Get an amount of reorganization pig interferon α 1-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 interferon α 1-Fc fusion rotein solution of lower concentration.And can be further with the ultrafiltration desalination of molecular weight cut-off 10KDa, concentrate, low-temperature vacuum drying namely obtains reorganization pig interferon α 1-Fc fusion rotein powder.Composition and the content thereof of each damping fluid are as shown in the table:

The present invention also provides the purposes of reorganization pig interferon α 1 – Fc fusion rotein in the medicine of preparation treatment and prevention porcine reproductive and respiratory syndrome, porcine influenza and pig blue-ear disease disease.In porkling disease treatment process, reorganization pig interferon α 1 – Fc fusion rotein can nonspecific performance antiviral effect widely, improve immune response and strengthen defence capability to virus.Simultaneously, reorganization pig interferon α 1 – Fc fusion rotein also can be united use with other vaccines, alleviates the untoward reaction of vaccine, strengthens whole antiviral, bacterium, parasitic effectiveness.Reorganization pig interferon α 1 – Fc fusion rotein also can be united use with multiple cause of disease vaccine, can significantly improve immune level, minimizing stress, reduce the side effect of vaccine, thereby reach stronger therapeutic action.

Reorganization pig interferon α 1-Fc fusion rotein recombination sequence through optimizing of the present invention, be more suitable for the expression of escherichia expression system, expressed reorganization pig interferon α 1-Fc fusion rotein is far above the expression amount of pig interferon α 1-Fc fusion rotein natural gene sequence at escherichia expression system, and, compare with reorganization pig interferon α 1, reorganization pig interferon α 1-Fc fusion rotein of the present invention has prolonged its transformation period in vivo largely on the basis that has guaranteed reorganization pig interferon α 1 activity, realized long-acting and avoid the purpose of medication repeatedly.Simultaneously, provided by the present invention is a boar source type fusion rotein, be more suitable for using the preparation at Swine virus disease veterinary drug, and the procaryotic cell expression of this fusion rotein provided by the present invention, purifying and refolding method have also been controlled its preparation cost greatly, for its industrialization provides guarantee.

Description of drawings

Fig. 1 represents to recombinate the codon optimized front and back of pig interferon α 1-Fc fusion rotein nucleotide sequence relatively

Wherein, odd-numbered line (i.e. " original series " corresponding row) is pig interferon α 1-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 interferon α 1-Fc fusion rotein of the present invention, the sequence after namely codon optimized.

Fig. 2-a, Fig. 2-b are the reorganization pig interferon codon optimized front and back of α 1-Fc fusion rotein CAI index in the escherichia coli expression host.

Wherein, Fig. 2-a represent to recombinate pig interferon α 1-Fc fusion rotein codon optimized before in the escherichia coli expression host CAI index be 0.61; Fig. 2-b codon optimized the back of pig interferon α 1-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 interferon α 1-Fc fusion rotein codon optimum codon frequency distributed areas figure in the escherichia coli expression host.

Wherein, Fig. 3-a represent to recombinate pig interferon α 1-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 10% that per-cent appears in the poor efficiency codon (＜30%) of reorganization pig interferon α 1-Fc fusion rotein codon optimized presequence; Fig. 3-b codon optimized the back of pig interferon α 1-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 interferon α 1-Fc fusion rotein.

Fig. 4-a, Fig. 4-b in the reorganization pig interferon α 1-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 interferon α 1-Fc fusion rotein codon optimized before in the escherichia coli expression host average GC base contents be: 59.32%; Fig. 4-b codon optimized the back of pig interferon α 1-Fc fusion rotein average GC base contents in the escherichia coli expression host of representing to recombinate is: 54.10%.

Fig. 5-a, Fig. 5-b are the secondary structure prediction figure of the codon optimized front and back mRNA of reorganization pig interferon α 1-Fc fusion rotein.

The secondary structure prediction figure of Fig. 5-codon optimized premessenger RNA of a reorganization pig interferon α 1-Fc fusion rotein, Fig. 5-b is the secondary structure prediction figure of reorganization pig interferon α 1-Fc fusion rotein codon optimized back mRNA.

Fig. 6 is reorganization pig interferon α 1-Fc fusion protein expression plasmid building process figure.

Fig. 7 is reorganization pig interferon α 1-Fc fusion rotein optimized gene agarose gel electrophoresis figure.

Wherein, swimming lane 1 contains the reorganization pig interferon α 1-Fc antigen-4 fusion protein gene PCR product of NdeI and XhoI restriction enzyme site for two ends; Swimming lane 2 is 500bp DNA Ladder; Swimming lane 3 is the pET21b carrier behind NdeI and the XhoI double digestion.

Fig. 8 is SDS-PAGE gel electrophoresis figure and the corresponding western blot figure of reorganization pig interferon α 1-Fc fusion rotein.

Fig. 8-a is reorganization pig interferon α 1-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 interferon α 1-Fc fusion rotein intestinal bacteria lysate that IPTG induces; Swimming lane 3 is for adding the reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate that IPTG induces.

Fig. 8-b is reorganization pig interferon α 1-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 interferon α 1-Fc fusion rotein intestinal bacteria lysate that IPTG induces: swimming lane 3 is for adding the reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate that IPTG induces.

Fig. 9 pig interferon α 1-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 interferon α 1-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 3 is induced 2h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 4 is induced 3h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 5 is induced 4h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 0.5mmol/L IPTG; Swimming lane 6 is induced 1h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 7 is induced 2h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 8 is induced 3h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 9 is induced 4h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1mmol/L IPTG; Swimming lane 10 is induced 1h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG; Swimming lane 11 is induced 2h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG; Swimming lane 12 is induced 3h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG; Swimming lane 13 is induced 4h reorganization pig interferon α 1-Fc fusion rotein intestinal bacteria lysate for 1.5mmol/L IPTG.

Figure 10 is the pig interferon α 1-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 contains the full bacterium lysate of reorganization pig interferon α 1-Fc fusion rotein for ultrasonic degradation; Swimming lane 3 is for cleaning back reorganization pig interferon α 1-Fc fusion rotein inclusion body precipitation for the first time with Buffer B; Swimming lane 4 cleans back reorganization pig interferon α 1-Fc fusion rotein inclusion body precipitation for the second time for Buffer B; Swimming lane 5 is reorganization pig interferon α 1-Fc fusion rotein supernatant solution after the B renaturation.

Figure 11 is recombinantinterferon 1-Fc 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 interferon α 1 and recombinantinterferon 1-Fc fusion rotein pig anteserum sample; Swimming lane 3 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 0h; Swimming lane 4 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 1h; Swimming lane 5 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 2h; Swimming lane 6 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 8h; Swimming lane 7 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 24h; Swimming lane 8 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 72h; Swimming lane 9 is for adding the pig anteserum sample of 1 μ mol/mL pig interferon α 1 and 1 μ mol/mL reorganization pig interferon α 1-Fc fusion rotein 120h.

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.

Embodiment 1 reorganization pig interferon α 1-Fc antigen-4 fusion protein gene optimization design

The contriver is according to the disclosed pig interferon α of GenBank 1(Sus scrofa interferon, alpha1) cDNA sequence (GenBank accession number: NM_214393.1) 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 interferon α 1-Fc fusion rotein of the present invention, shown in SEQ ID No:1.

Be reorganization pig interferon α 1-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 interferon α 1-Fc antigen-4 fusion protein gene in intestinal bacteria codon adaptation indexI (codon adaptation index is 0.61 CAI).By Fig. 2-b as can be known, after codon optimized, make that reorganization pig interferon α 1-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 interferon α 1-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 10% that per-cent appears in the poor efficiency codon of pig interferon α 1-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 interferon α 1-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 interferon α 1-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 interferon α 1-Fc antigen-4 fusion protein gene that GC base average content is 59.32% before optimization, eliminated GC content in extra-regional 60 bases 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 interferon α 1-Fc fusion rotein that finally is optimized is 54.10%.

3. cis-acting elements

Cis-acting elements	Before the optimization	After the optimization
			E.coli_RBS(AGGAGG)	4	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 interferon α 1-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 interferon α 1-Fc antigen-4 fusion protein gene makes up

With the synthetic fragment of the full gene of reorganization pig interferon α 1-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 pUC57-pIFN α 1-Fc plasmid.Be template with pUC57-pIFN α 1-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:GGAATTCCATATGTGTGACCTGCCGCAAACG

Downstream primer:

P2:CCGCTCGAGTCATTTGCCCTGGGTTTTGGAG

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 (1194bp).(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 interferon α 1-Fc fusion rotein that namely obtains recombinating is designated as pET21b-pIFN α 1-Fc.

Embodiment 3 reorganization pig interferon α 1-Fc fusion rotein efficiently expressing and identifying in intestinal bacteria

Concrete steps are as follows:

1. the correct pET21b-pIFN α 1-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) incubated overnight in 37 ℃ of penbritin flat boards.

2. choose 1-4 reorganization bacterium colony that contains pET21b-pIFN α 1-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, 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.

Embodiment 4 reorganization pig interferon α 1-Fc fusion roteins efficiently express inductive condition optimization

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 interferon α 1-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 interferon α 1-Fc fusion protein expression by the SDS-PAGE gel electrophoresis analysis.

Concrete steps are as follows:

1. the correct pET21b-pIFN α 1-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 pET21b-pIFN α 1-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 0.5mmol/L, 1.0mmol/L, 1.5mmol/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 interferon α 1-Fc fusion rotein bacterium liquid behind the 4h successively, 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, room temperature high speed centrifugation (rotating speed: 12000rpm) 1min.

6. getting 10 μ L does not add IPTG and induces and the reorganization pig interferon α 1-Fc fusion rotein culture suspension that adds different concns IPTG, different induction times, 10%SDS-PAGE gel electrophoresis analysis.

8. coomassie brilliant blue staining is observed reorganization pig interferon α 1-Fc fusion protein expression products band.(see figure 9)

9. the gel imaging system thin layer scanning is analyzed express recombinant pig interferon α 1-Fc fusion rotein content and is identified pig interferon α 1-Fc Expression of Fusion Protein.Final definite inductive condition that is fit to this enforcement is 1mmol/L IPTG, and induction time is 4h.

Embodiment 5 reorganization pig interferon α 1-Fc fusion rotein inclusion body purification and renaturation

1. will be among the embodiment 3 collect the intestinal bacteria precipitation of inducing reorganization pig interferon α 1-Fc fusion rotein that contains that obtains, resuspended with the PBS of precooling, in 4 ℃ with 12000rpm, centrifugal 15min; Repeat once.

3. every gram (weight in wet base) thalline adds 5 μ L 100mmol/L PMSF, and 5 μ L 100mg/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.

7. abundant room temperature 12000rpm behind the mixing, centrifugal 15min abandons precipitation, gets supernatant, and pig interferon α 1-Fc fusion rotein denaturing soln namely obtains recombinating.

8. adopt the dilution refolding method that the reorganization pig interferon α 1-Fc fusion rotein denaturing soln in the step 7 is carried out renaturation.

Dilution refolding: get an amount of reorganization pig interferon α 1-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 interferon α 1-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 interferon α 1-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 twice washed product of lavation buffer solution Buffer B in the step 5 respectively, at the visible obviously band of purpose scope.

The preparation of embodiment 6 reorganization pig interferon α 1(in detail can referring to the applicant in first to file: 201310025176.8)

Concrete steps are as follows:

1. but make up the recombination bacillus coli of express recombinant pig interferon α 1.

According to the disclosed pig interferon α of GenBank 1(Sus scrofa interferon, alpha1) cDNA sequence (GenBank accession number: NM_214393.1), according to escherichia expression system this gene is carried out obtaining recombinating after codon optimized pig interferon α 1 gene, shown in SEQ ID No:3.The synthetic fragment of reorganization pig interferon α 1 full gene with after optimizing is building up in the pUC57 plasmid, obtains pUC57-prIFN α 1 plasmid.

Be template with pUC57-prIFN α 1 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：GGAATTCCATATGTGTGACCTGCCGCAAACGC

Downstream primer:

P2：CCGCTCGAGTCATTCCTTTTTGCGCAGACGATC

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-prIFN α 1 of amplification is transformed in the escherichia coli BL21(DE3) expression bacterial strain.

2. the expression of reorganization pig interferon α 1 inclusion body

E. coli bl21 (DE3) incubated overnight in 37 ℃ of penbritin flat boards that will contain pET21b-prIFN α 1.Choose 1-4 reorganization bacterium colony that contains pET21b-prIFN α 1 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 interferon α 1 inclusion body

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 L 100mmol/L PMSF, and 5 μ L 100mg/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, is circulated between the cooling sample at every turn and waits for 2min, 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 interferon α 1 denaturing soln namely obtains recombinating.Adopt dialysis renaturation method renaturation inclusion body: ufferC will recombinate pig interferon α 1 denaturing soln concentration dilution to 0.2mg/mL with the sex change buffer B, 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 interferon α 1 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 interferon α 1 powder.Each damping fluid preparation is as shown in table 2.4 ℃ of refrigerators of reorganization pig interferon α 1 powder are deposited stand-by.

Embodiment 7 reorganization pig interferon α 1-Fc fusion rotein Determination of biological activity

(VSV, its TCID50 are 5 * 10 to the pig vesicular stomatitis virus ⁷/ 100 μ L; academy of agricultural sciences, Guangdong Province veterinary institute provides) but the nephrocyte (PK-15 of infected pigs; academy of agricultural sciences, Guangdong Province veterinary institute provides); the present invention utilizes the antiviral mechanism of pig interferon α 1 to detect at porcine kidney cell defense reaction to the pig vesicular stomatitis virus under pig interferon α 1 or reorganization pig interferon α 1-Fc fusion rotein existence condition; obtain pig interferon α 1 or reorganization pig interferon α 1-Fc fusion rotein to the protective effect curve of PK-15 cell by the pathology situation that detects the PK-15 cell, thereby measure reorganization pig interferon α 1 or reorganization pig interferon α 1-Fc fusion rotein biologic activity.

1). the positive reference substance formulations prepared from solutions: (pig genetically engineered recombinant cytokine: IFN-LLS-2 is available from the graceful general animal nutrition in Hong Kong company limited to get the pig interferon positive reference substance.), after by specification redissolves, press 10 times of one-level stepwise dilutions with the MEM cell culture fluid (Gibico product) that contains 6% foetal calf serum.

2). reorganization pig interferon α 1-Fc fusion protein sample formulations prepared from solutions: take by weighing the pig interferon α 1-Fc fusion rotein dry powder sample 500ug that recombinates after the renaturation among the embodiment 5, do 10 times of one-level stepwise dilutions with cell culture fluid, get 50 μ g/mL, 5 μ g/mL, 0.5 μ g/mL, 5 * 10 respectively ^-2μ g/mL, 5 * 10 ^-3μ g/mL, 5 * 10 ^-4μ g/mL recombinantinterferon 1 – Fc fusion rotein solution.

4). every hole adds positive controls sample or the reorganization pig interferon α 1-Fc fusion protein sample solution of different concns on 96 orifice plates, and (cell concn is about 1.8 * 10 to add the fresh PK-15 cell suspension that goes down to posterity of 50 μ L again ⁶-2.2 * 10 ⁶Individual/mL), total system 100 μ L, 37 ℃, 5%CO ₂Hatch about 24h under the condition, the PK-15 cell attachment grows to individual layer.Discard the cell culture fluid supernatant, every hole adds the MEM nutrient solution of 2% foetal calf serum that 100 μ L contain the VSV virus of 100 TCID50.Set up negative control virus group (virus that only adds PK-15 cell and same dose does not add reorganization pig interferon α 1) and blank cell control group (only add PK-15 cell and cell culture fluid, do not add reorganization pig interferon α 1 and virus) simultaneously.37 ℃, 5%CO ₂Cultivate 24h under the condition, observations when treating the cytopathy 90%-100% in virus control hole.Amount with the cytopathic Interferon, rabbit that causes the half hole is a unit (being designated as " U ").

Experimental result shows that obvious pathology all takes place the cell in the negative control virus group, and the cell growth is normal in the blank cell control group.Compare with positive control, when the concentration of reorganization pig interferon α 1-Fc fusion rotein during at 1 μ g/mL-0.1ug/mL, begin to produce and eliminate VSV virus to the phenomenon of the influence of PK-15 cell normal growth.Convert by the result and to determine that tiring of positive control Interferon, rabbit is 3.2 * 10 ⁴U/mL, it is 1.2 * 10 that reorganization pig interferon α 1-Fc fusion protein sample is tired ³U/mL.This shows, the present invention pig interferon α 1-Fc that recombinates has antiviral activity, show wide application prospect in porkling disease prevention and treatment application reverse side, and preparation technology is simple, makes may being achieved of the long-acting genetically engineered reorganization pig interferon α 1-Fc of suitability for industrialized production.

Embodiment 8 reorganization pig interferon α 1-Fc fusion rotein stability tests

In order to detect the stability of pig interferon α 1-Fc fusion rotein in serum, contriver's particular design a kind of in-vitro simulated porcine blood serum environment, the embodiment 5 reorganization pig interferon α 1-Fc fusion roteins of the present invention of preparation reorganization pig interferon α 1 and 1 μ mol/ml among the embodiment 6 of 1 μ mol/ml are placed fresh porcine blood serum 50 μ L, 37 ℃ jointly, 120rpm, react 0,1,2 respectively, 8, preserve-20 ℃ behind 24,72, the 120h.With mouse-anti reorganization pig interferon α 1 monoclonal antibody (IFN alpha Antibody (K9), Catalog Number:271001, Thermo Pierce) for primary antibodie the reorganization pig interferon α 1 in the porcine blood serum and reorganization pig interferon α 1-Fc fusion rotein are carried out the protein immunoblot test, by the enzymic digestion in the serum totally, and reorganization pig interferon α 1-Fc fusion rotein 120h in serum still keeps stable to pig interferon α 1 behind reaction 24h as shown in Figure 11.This shows that the pig interferon α 1-Fc fusion rotein of the present invention's preparation is compared with reorganization pig interferon α 1 has advantages of higher stability, has prolonged the transformation period in its body, has reached the effect of long-acting administration.

Claims

1. reorganization pig interferon α 1-Fc fusion rotein, it is characterized in that, described fusion rotein comprises pig interferon α 1 part and Fc fragment part, described pig interferon α 1 part is the full sequence of pig interferon α 1 extracellular region, described Fc fragment partly comprises hinge area, CH2 district and CH3 district, is directly to merge between pig interferon α 1 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 interferon α 1-Fc fusion rotein of recombinating comprises the steps:

(1) under appropriate condition, in substratum, cultivates the intestinal bacteria described in the claim 6 or 7;

(2) from intestinal bacteria and/or substratum, separate reorganization pig interferon α 1-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 claim 6 or 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 interferon α 1-Fc fusion rotein.

10. purifying and the refolding method of pig interferon α 1-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 interferon α 1-Fc fusion rotein intestinal bacteria precipitation, and PBS is resuspended, and handles 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 A 3-10mL, 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 interferon α 1-Fc fusion rotein denaturing soln namely obtains recombinating;

(8) get an amount of reorganization pig interferon α 1-Fc fusion rotein denaturing soln with sex change buffer B uffer C dissolving, to recombinate the concentration dilution of pig interferon α 1-Fc fusion rotein denaturing soln to 0.2mg/mL with renaturation buffer Buffer D, 4 ℃ of dialysis renaturation 24h, with 0.45 μ m membrane filtration, pig interferon α 1-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 interferon α 1-Fc fusion rotein renaturation solution, and low-temperature vacuum drying namely obtains reorganization pig interferon α 1-Fc powder.