CN103059123A - Recombinant porcine interferon beta1, encoding gene and expression method thereof - Google Patents
Recombinant porcine interferon beta1, encoding gene and expression method thereof Download PDFInfo
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Abstract
The invention provides recombinant porcine interferon beta1, an encoding gene and an expression, purification and inclusion body refolding method and belongs to the field of biological genetic engineering. The recombinant porcine interferon beta1 has wide medical prospect in the field of veterinary medicine as a nonspecific broad-spectrum anti-viral biological preparation, but has the problems of shortage of throughput, high price, different drug specifications, and the like just as most genetic engineering drugs. In order to obtain a lot of recombinant porcine interferon beta1, an escherichia coli expression system is adopted to carry out heterologous expression on the recombinant porcine interferon beta1 gene optimized by a codon. In addition, an inclusion body purifying and refolding method of the recombinant porcine interferon beta1 is also provided by the invention aiming at the problem that the porcine interferon beta1 in a prokaryotic expression system is mostly expressed in a form of an inclusion body, so that the prepared porcine interferon beta1 has high activity and reaches the standard of industrial production.
Description
Technical field
The invention belongs to biotechnology gene field, relate to a kind of Recombinant Swine interferon beta 1 and encoding gene thereof, with and expression, purifying and renaturing inclusion bodies method.
Background technology
Interferon, rabbit (Interferon, IFN) is one group of active protein (mainly being glycoprotein) with several functions, is a kind of cytokine that is produced by monocyte and lymphocyte.They allogenic cell have wide spectrum antiviral, affect Growth of Cells, and differentiation, regulate the multiple biological activity such as immunologic function.Be that the character of animal species, cell type, inducer is different with Induction Condition according to the source of IFN, can be divided into three kinds of α, β, γ.Wherein, interferon beta is mainly produced by inoblast, has in vivo the cell surface of enhancing HLAI, class Ⅱ antigens expression, improves the new butterfly cry of certain animals of serum and β
2-microglobulin level, enhancing NK cell viability and ADCC effect and periphery lymphocyte 2 ', 5 '-few gland former times synthase activity.In the clinical treatment that interferon beta is applied to multiple sclerosis, hepatitis B, hepatitis C etc.
China is that live pig produces pig big country, and the multiple pig virus transmissible diseases such as Porcine circovirus desease, porcine reproductive and respiratory syndrome, porcine pseudorabies have been brought huge financial loss to pig industry.Though China extensively inoculates various swine disease vaccines at present, still can not effectively control disease popularity.Interferon beta has stronger antiviral effect and immunoregulatory activity, the same with IFN-α, it has the broad-spectrum disease resistance toxic action, can be used as the alternative preparation of interferon alpha, and due to illness the kind of poison is different and very big-difference is arranged to the inhibition degree of virus for interferon beta, and for example interferon beta has better anti-SARS-CoV effect than interferon alpha; Interferon beta also obviously is better than interferon-gamma in the tolerance of acid, alkali, heat.Simultaneously, IFN-β also can be suppressed to the propagation of fibrocyte, epithelial cell, endotheliocyte and hematopoietic cell, suppresses and killing tumor cell, and immunity system is played regulating effect, and therefore the application in field of veterinary is more and more extensive.Yet there is obvious species variation in interferon beta, only relies on the requirement that extraction in the live hog body can not satisfying the market far away.Animal with genetic engineering interferon β drug residue free, have no side effect, be subjected to very much clinical animal doctor and raiser's favor, but on the market most similar medicines still be faced with underproduce, levels of audit quality is uneven, the problem such as expensive.Therefore, but this patent at first provides a kind of expression system and expression method of great expression Recombinant Swine interferon beta 1 with low cost, stay-in-grade.
Prokaryotic expression system is used the earliest and studies, and this also is to grasp at present the most ripe expression system.The main method of this technology is carrier (the being generally plasmid) transform bacteria (what usually select is intestinal bacteria) that will be cloned into the goal gene dna fragmentation, induces and the required target protein of final purifying acquisition by IPTG.Its advantage is to obtain gene expression product within a short period of time, and required cost is relatively cheap.The heterogenous expression that this patent has namely adopted coli expression system that restructuring pig interferon β 1 is carried out, thus a large amount of target proteins obtained.But because the system of the shortage posttranslational modification of prokaryotic expression system, the eukaryotic protein of its expression often produces with SA inclusion body form.And the renaturation of inclusion body is a very complicated process, and is not only closely bound up with renaturation condition and renaturation material, depends on more to a great extent the character of protein self.If the renaturation condition is not suitable for, will cause the mispairing of intramolecular disulfide bond, intermolecular mode with covalent attachment or hydrophobic binding to form polymer, thereby make the product Precipitation, affect yield, and reduce the ratio motility rate of recombinant protein, affect quality product.Therefore, this patent another technical problem to be solved is, adopts suitable condition that the inclusion body of the Recombinant Swine interferon beta 1 of coli expression system production is carried out renaturation, obtains having the product than higher specific activity.
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 can be in intestinal bacteria high efficient expression Recombinant Swine interferon beta 1 with and gene and expression, purifying, refolding method.
The invention provides a kind of Recombinant Swine interferon beta 1, its aminoacid sequence is shown in SEQ ID NO:2.
The invention provides the gene of coding Recombinant Swine interferon beta 1 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 by contrast the expression efficiency of heterologous gene in Host Strains.
The present invention also provides the carrier of the gene that has comprised coding Recombinant Swine interferon beta 1 described above, and described carrier is preferably prokaryotic expression plasmid, most preferably is pET21b.
The present invention also provides the coli strain that includes carrier described above, and preferably, described bacterial strain is selected from e. coli bl21 (DE3) bacterial strain.
The present invention also provides Recombinant Swine interferon beta 1 in the escherichia coli expression method, comprises the steps:
Steps of the method are:
1. one of picking contains the intestinal bacteria bacterium colony of Recombinant Swine interferon beta 1 described above, access LB nutrient solution, overnight incubation;
2. get overnight culture and access in the LB nutrient solution, concussion is cultured to mid-log phase (A
600=1.0);
3. add IPTG to 0.5-1.5mmol/L in culture, in 37 ℃, behind the abduction delivering 1-4h, centrifugal treating is collected the coli somatic precipitation that contains Recombinant Swine interferon beta 1.
All contain penbritin 50-100 μ g/mL in the described LB nutrient solution.
The present invention also provides the inclusion body purification method of Recombinant Swine interferon beta 1, comprises the steps:
1. above-mentioned the containing that collection is obtained induced Recombinant Swine interferon beta 1 coli somatic precipitation, and be resuspended with the PBS of precooling, and process in 4 ℃ of high speed centrifugations; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (thalline weight in wet base) adds lysis buffer BufferA3-10ml, stirs with the slicking glass rod, and thalline is hanged.
3. every gram (thalline 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 processes in 4 ℃ of high speed centrifugations, abandons supernatant.
5. precipitation is washed with lavation buffer solution Buffer B, and processes 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 processed behind the abundant mixing, abandons precipitation, gets supernatant, namely obtains Recombinant Swine interferon beta 1 denaturing soln.
This purification process preferred steps is as follows:
1. above-mentioned the containing that collection is obtained induced Recombinant Swine interferon beta 1 coli somatic precipitation, and be resuspended with the PBS of precooling, in 4 ℃, with the centrifugal 15min of the rotating speed of 12000rpm/min; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (thalline weight in wet base) adds lysis buffer BufferA5mL, 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 (thalline 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 interval 5s 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/min, 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/min, 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/min, abandon precipitation, get supernatant, namely obtain Recombinant Swine interferon beta 1 denaturing soln.
The present invention also provides the renaturing inclusion bodies method of the Recombinant Swine interferon beta 1 after optimizing, and comprises the steps:
Get an amount of Recombinant Swine interferon beta 1 denaturing soln described above with sex change buffer B uffer C dissolving, survey its concentration, then with renaturation buffer Buffer D protein concentration is diluted to 0.2mg/mL, during 4 ℃ of renaturation to 24 hour, recombinant protein solution after the renaturation is crossed 0.45 μ m filter membrane, namely obtain the Recombinant Swine interferon beta 1 renaturation solution of lower concentration.Molecular weight cut-off 10KDa ultrafiltration desalination, concentrated in the vacuum freeze drier low-temperature vacuum drying, namely obtains Recombinant Swine interferon beta 1 powder.
Expression described above of the present invention, purifying, refolding method are to grope and verify the most effective means that escherichia expression system is expressed Recombinant Swine interferon beta 1 that is used for that obtains through the repeated multiple times experiment of contriver, the expression amount of the method is high, and express obtain renaturing inclusion bodies after activity higher.The gene order of the Recombinant Swine interferon beta 1 through optimizing especially of the present invention is more suitable for the expression of escherichia expression system, and expressed Recombinant Swine interferon beta 1 is far above the expression amount of pig interferon β 1 natural gene sequence at escherichia expression system.
The present invention also provides the purposes of Recombinant Swine interferon beta 1 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, pig interferon β 1 can nonspecific performance antiviral effect widely, improve immune response and strengthen defence capability to virus.Simultaneously, pig interferon β 1 also can unite use with other vaccines, alleviates the untoward reaction of vaccine, strengthens whole antiviral, bacterium, parasitic effectiveness.
Description of drawings
Fig. 1 represents Recombinant Swine interferon beta 1 codon optimized front and back nucleotide sequence comparison
Wherein, even number line (i.e. row corresponding to " original series ") is pig interferon β 1 natural gene nucleotide sequence, i.e. codon optimized front sequence; Odd-numbered line (i.e. " majorizing sequence " corresponding row) is the gene nucleotide series of Recombinant Swine interferon beta 1 of the present invention, the sequence after namely codon optimized.
Fig. 2-a, Fig. 2-b are restructuring pig interferon β 1 codon optimized front and back CAI index in the escherichia coli expression host.
Wherein, Fig. 2-a represents that pig interferon β 1 natural gene nucleotides sequence is listed among the escherichia coli expression host CAI index and is calculated as 0.60 through program; Recombinant Swine interferon beta 1 codon of the present invention after Fig. 2-b represents to optimize CAI index in the escherichia coli expression host is calculated as 0.90 through program.
Fig. 3-a, Fig. 3-b are pig interferon β 1 codon optimized front and back optimal codon frequency distribution areal maps in the escherichia coli expression host.
Wherein, Fig. 3-a represents that pig interferon β 1 natural gene nucleotides sequence is listed in optimal codon frequency distribution areal map among the escherichia coli expression host, and as can be seen from the figure: it is 15% that per-cent appears in the poor efficiency codon of pig interferon β 1 natural gene nucleotide sequence; Recombinant Swine interferon beta 1 codon of the present invention optimal codon frequency distribution areal map in the escherichia coli expression host after Fig. 3-b represents to optimize, it is 0 that per-cent appears in the poor efficiency codon of the Recombinant Swine interferon beta of the present invention 1 codon sequence after the optimization.
Fig. 4-a, Fig. 4-b are restructuring pig interferon β 1 codon optimized front and back average GC base contents distributed areas figure in the escherichia coli expression host.
Wherein, Fig. 4-a represents that pig interferon β 1 natural gene nucleotides sequence is listed among the escherichia coli expression host average GC base contents and is: 46.45%; Recombinant Swine interferon beta 1 codon of the present invention after Fig. 4-b represents to optimize average GC base contents in the escherichia coli expression host is: 47.27%.
Fig. 5-a, Fig. 5-b are the secondary structure prediction figure of restructuring pig interferon β 1 codon optimized front and back mRNA.
The secondary structure prediction figure of Fig. 5-a pig interferon β 1 natural gene mRNA, Fig. 5-b are the secondary structure prediction figure of the Recombinant Swine interferon beta 1mRNA of the present invention after codon optimized.
Fig. 6 is restructuring pig interferon β 1 expression plasmid building process figure.
Fig. 7 is the agarose gel electrophoresis figure of restructuring pig interferon β 1 gene PCR product.
Wherein, swimming lane 1 is cut the pET21b carrier for NdeI and XhoI enzyme; Swimming lane 2 is 500bp DNA Ladder; Swimming lane 3 contains the Recombinant Swine interferon beta 1 gene PCR product of NdeI and XhoI restriction enzyme site for two ends.
Fig. 8-a, Fig. 8-b are SDS-PAGE gel electrophoresis figure and the corresponding western blot figure of restructuring pig interferon β 1.
Fig. 8-a is restructuring pig interferon β 1SDS-PAGE gel electrophoresis figure.
Wherein, swimming lane 1 is the albumen loading Marker that dyes in advance of (10-230kDa) wide region; Swimming lane 2 is not for adding the Recombinant Swine interferon beta 1 intestinal bacteria lysate that IPTG induces; The Recombinant Swine interferon beta 1 intestinal bacteria lysate that swimming lane 3 is induced for adding IPTG.
Fig. 8-b is restructuring pig interferon β 1 western blot figure.
Wherein, swimming lane 1(10-230KDa) the albumen loading Marker that dyes in advance of wide region, swimming lane 2 is not for adding the Recombinant Swine interferon beta 1 intestinal bacteria lysate that IPTG induces: the Recombinant Swine interferon beta 1 intestinal bacteria lysate that swimming lane 3 is induced for adding IPTG.
The SDS-PAGE gel electrophoresis figure of Fig. 9 Recombinant Swine interferon beta 1 efficient induced expression condition optimizing.
Wherein, swimming lane 1 is the albumen loading Marker that dyes in advance of (10-230kDa) wide region; 1h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 2 for 0.5mmol/L IPTG induces; 2h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 3 for 0.5mmol/L IPTG induces; 3h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 4 for 0.5mmol/L IPTG induces; 4h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 5 for 0.5mmol/L IPTG induces; 1h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 6 for 1mmol/LIPTG induces; 2h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 7 for 1mmol/L IPTG induces; 3h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 8 for 1mmol/L IPTG induces; 4h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 9 for 1mmol/L IPTG induces; 1h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 10 for 1.5mmol/LIPTG induces; 2h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 11 for 1.5mmol/L IPTG induces; 3h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 12 for 1.5mmol/L IPTG induces; 4h's contain Recombinant Swine interferon beta 1 intestinal bacteria lysate to swimming lane 13 for 1.5mmol/L IPTG induces.
Figure 10 is the Recombinant Swine interferon beta 1 inclusion body SDS-PAGE electrophorogram after the renaturation
Wherein, swimming lane 1 is the albumen loading Marker that dyes in advance of (10-230kDa) wide region; Swimming lane 2 is with inducing rear full bacterium lysate; Swimming lane 3 is rear Recombinant Swine interferon beta 1 inclusion body precipitation for Buffer B cleans for the first time; Swimming lane 4 is for cleaning for the second time rear Recombinant Swine interferon beta 1 inclusion body precipitation for Buffer B; Swimming lane 5 is the Recombinant Swine interferon beta 1 behind the dilution refolding
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, should be understood that quoting embodiment only is used for explanation the present invention and is not used in and limits the scope of the invention.
The design of embodiment 1 Recombinant Swine interferon beta 1 gene optimization
1. codon optimized
Genetic codon has 64 kinds, but the part of most biological tendencies in utilizing these codons.Those are the most frequently utilized is called best codon (optimal codons), and those are not often utilized is called codon (rare orlow-usage codons) rare or that utilization ratio is low.In fact, commonly use every kind of biology (comprising intestinal bacteria, yeast, mammalian cell, vegetable cell and insect cell) of doing protein expression or production and all show difference or the preference that codon to a certain degree utilizes.In intestinal bacteria, yeast and the fruit bat to the expression efficiency of the gene that the contains best codon expression efficiency apparently higher than the gene of the codon that contains poor efficiency.Therefore, in heterologous expression system, the preferences of codon has affected the expression of recombinant protein to a great extent.Utilize preference codon (preferred codons) and avoid utilizing rare codon to carry out gene synthetic, the redesign of this gene is codon optimized.Optimizing process fully takes into account the Various Complex factor that the protein expression different steps may run into, as: codon adaptability, mRNA structure and transcribe with translation process in different cis elements.Therefore, the present invention not only comprises codon optimized to the gene design of pig interferon β 1, also comprise the optimization of mRNA structural modifications, translation initiation site etc.
2. codon-bias optimization
Codon-bias has been proved to be a very important influence factor in Prokaryotic gene expression, it has caused same codon between different organisms, the change of utilization ratio between the protein expression level and between the different sites of same operon.The major cause that causes this preferences difference is the difference that tRNAs available in the different cells measures.Therefore the method for optimizing translation system the best is exactly the balance that keeps between Codon usage frequency and the homology tRNA.Unpredictable and have challenge at the expression in escherichia coli mammalian genes, as in intestinal bacteria, the corresponding tRNA molecule of AGG and AGA just seldom, this species diversity clearly can affect the expression of gene.
3. the codon with poor efficiency replaces to host's codon commonly used
Usually the utilization ratio of codon in specific host that comprises in the gene is lower, and this kind protein expression amount is also just fewer, even when expression amount can be still less between this codon existence and protein clusters or when N-terminal.Codon with poor efficiency under the prerequisite that does not change aminoacid sequence replaces with the expression level that host's codon commonly used can improve functional protein.
If when the utilization ratio of the codon in any source in host organisms is lower than 5% to 10%, expression inhibiting will occur, when these poor efficiency codons close on or link to each other, larger on the impact of protein expression.The codon of the poor efficiency of cluster has suppressed ribosomal motion, and this is that gene can not be obviously machine-processed with that proper level is expressed.The rrna translation movement velocity during by nine molecular couriers of password (contain several poor efficiency codons or all be the poor efficiency codon) is slower than the courier's who translates the same length that does not contain the poor efficiency codon speed.Even poor efficiency password submanifold is positioned at 3 ' end, the courier also can " be crowded " by rrna and damage at last, and rrna is got back to again 5 ' end.The retarding effect of 3 ' end poor efficiency password submanifold can be all the same large by the molecular retarding effect of poor efficiency password with whole couriers.If poor efficiency password submanifold is positioned at 5 ' end, its effect is comprehensive minimizing of initial rrna number, causes the synthetic middle courier's of albumen poor efficiency.The codon of removing the codon of poor efficiency or easily being misread as termination signal can prevent from lowly expressing or not expressing.
4. expression vector and transcripting promoter
Although the codon preference plays an important role in genetic expression, the selection no less important of expression vector and transcripting promoter, the protein expression of N terminal nucleotide sequence is for poor efficiency codon and very responsive near the codon AUG of initiation site.Exist reciprocal influence between translation and the stability of mRNA yet, decomposed by endo-RNAses although reduce translation efficiency can to make mRNA easier owing to having lacked ribosomal protection, present also do not have the complete explanation that affects between them.
Other factors also can affect protein expression, comprise making mRNA go stable sequence.The molecule of stable mRNA secondary structure and near 5 ' end also has important impact to genetic expression.The open reading frame of goal gene upstream can successfully improve the expression efficiency of difficulty gene when utilizing translation.
The contriver is according to the published pig interferon β of GenBank 1(Sus scrofa interferon, beta1) cDNA sequence (GenBank accession number: NM_001003923.1), this gene is carried out obtaining Recombinant Swine interferon beta 1 gene of the present invention after codon optimized, shown in SEQ ID No:1.
The below carries out codon optimizedly to restructuring pig interferon β 1, each parameter comparison is as follows before and after optimizing:
1. codon adaptation indexI (Codon Adaptation Index, CAI)
By Fig. 2-a as can be known, before codon was not optimized, pig interferon β 1 natural gene codon adaptation indexI (CAI) in intestinal bacteria was 0.60.By Fig. 2-b as can be known, after codon optimized, so that Recombinant Swine interferon beta 1 gene of the present invention CAI index in intestinal bacteria is 0.90.Being considered to this gene during common CAI=1 is optimal efficient expression status in this expression system, the CAI index is lower to show that this gene expression level in this host is poorer, therefore can find out through the gene order that obtains after codon optimized can improve Recombinant Swine interferon beta 1 gene Expression in Escherichia coli level.
2. optimal codon frequency of utilization (Frequency of Optimal Codons, FOP)
By Fig. 3-a as can be known, based on coli expression carrier, before codon was not optimized, it was 15% that per-cent appears in the poor efficiency codon of pig interferon β 1 natural gene sequence.This gene that is not optimized contains 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 Recombinant Swine interferon beta 1 gene of the present invention in the intestinal bacteria system frequency is 0.
3.GC base contents (GC curve)
GC content ideal distribution zone is 30%-70%, all can affect 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 pig interferon β 1 gene of Fig. 4-a, Fig. 4-b as can be known, by showing among Fig. 4-a in pig interferon β 1 natural gene that GC base average content is 46.45% before optimization, by GC content all bases outside the 30%-70% zone that demonstrated sequence elimination after the optimization among Fig. 4-b, the GC base average content of the rear Recombinant Swine interferon beta 1 that finally is optimized is 47.26%.
3. the cis-acting elements situation is as follows before and after optimizing:
| Cis-acting elements | After the optimization | Before the optimization |
| E.coli_RBS(AGGAGG) | 0 | 0 |
| PolyT(TTTTTT) | 0 | 0 |
| PolyA(AAAAAAA) | 0 | 0 |
| Ch site (GCTGGTGG) | 0 | 0 |
| T7Cis(ATCTGTT) | 0 | 2 |
4. the palindrome and tumor-necrosis factor glycoproteins situation are as follows before and after optimizing:
The secondary structure prediction figure of 5mRNA
After DNA is transcribed into mRNA because mRNA is the strand linear molecule, by self inflection so that complementary base pair meets 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-b pig interferon β 1 codon optimized front and back mRNA 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 Recombinant Swine interferon beta 1 gene makes up
The synthetic fragment of Recombinant Swine interferon beta 1 full gene (shown in SEQ ID No:1) with after optimizing is building up in the pUC57 plasmid (being provided by Nanjing Jin Sirui Science and Technology Ltd.), obtains a kind of prolonged preservation plasmid, is designated as pUC57-prIFN β 1 plasmid.Take pUC57-prIFN β 1 plasmid as template, the upstream and downstream primer is introduced respectively NdeI and XhoI restriction enzyme site, carries out pcr amplification, and the primer sequence is as follows:
Upstream primer:
P1：CGGGAATTCCATATGATGTCCTATGATGTTCTGCG
Downstream primer:
P2：CCGCTCGAGTTAATTGCGCAGATAATCCGTC
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 20s, 72 ℃ of 30s, and after 25 circulations, product is through 1.0% agarose gel electrophoresis analysis, and the result shows that the product size is consistent with expection size (498bp).(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, be connected in the pET21b plasmid (available from Merck company) with T4 ligase enzyme (available from New EnglandBiolabs company), 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).The order-checking of second day screening positive clone bacterium is compared, and is in full accord with expected sequence, namely obtains the expression plasmid of 1 one kinds of forms of Recombinant Swine interferon beta, is designated as pET21b-prIFN β 1.
Concrete steps are as follows:
1. order-checking among the embodiment 2 is compared correct pET21b-prIFN β 1 Plasmid Transformation in e. coli bl21 (DE3) competence bacterial strain (available from sky, Beijing root biochemical technology company limited), incubated overnight in 37 ℃ of penbritin flat boards.
2. second day is chosen 1-4 restructuring bacterium colony that contains pET21b-prIFN β 1 plasmid, and access contains the LB nutrient solution of 100 μ g/mL penbritins, 37 ℃ of overnight incubation.
3. get the LB inducing culture liquid that 50 μ L overnight culture access 5mL contains 100 μ g/mL penbritins, 37 ℃ of shaking culture.
4. survey bacterium liquid OD600 value every 1h after the inoculation, when treating OD600=1.0, with the IPTG(of 1mmol/L available from Amresco company) carry out abduction delivering.Simultaneously do negative control with the intestinal bacteria nutrient solution that does not add IPTG.
5.4h after collect bacterium liquid, high speed centrifugation (rotating speed: 12000rpm/min) 3min, with the PBS washing and precipitating of precooling, add the 5XSDS gel loading buffer, 100 ℃ of heating 10min, the room temperature high speed centrifugation (rotating speed: 12000rpm/min) 1min, get supernatant.Do not add the intestinal bacteria nutrient solution of IPTG by this step process yet.
6. respectively get not adding IPTG and adding the culture samples that IPTG induces, 12%SDS-PAGE gel electrophoresis analysis of 10 μ L step 5 gained.
7.8-15V/cm electrophoresis moves to separation gel bottom to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining and immunoblotting are observed the expression product band, see Fig. 8-a and Fig. 8-b.
Many cell growth rates that studies show that have a strong impact on the expression of foreign protein, therefore must be to inoculation amount of bacteria, culture temperature, induce the front Growth of Cells time and induce rear cell density to control, overgrowth or overrun and all can increase the weight of intestinal bacteria and form Recombinant Swine interferon beta 1 inclusion body.Use three factors, four levels, set up IPTG concentration and induction time orthogonal table, by
The SDS-PAGE gel electrophoresis analysis is induced Recombinant Swine interferon beta 1 expression amount.
Concrete steps are as follows:
1. pET21b-prIFN β 1 Plasmid Transformation that order-checking comparison among the embodiment 2 is correct is to 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. second day is chosen contrast bacterium and 1-4 restructuring bacterium colony that contains pET21b-prIFN β 1 plasmid, and access contains the LB nutrient solution of 100 μ g/mL penbritins, 37 ℃ of overnight incubation.
3. get the LB inducing culture liquid that 50 μ L overnight culture access 5mL contains 100 μ g/mL penbritins, 37 ℃ of shaking culture.
4. survey bacterium liquid OD600 value after the inoculation, when treating OD600=1.0, add respectively 0.5,1.0,1.5m mol/LIPTG concentration and the time is carried out abduction delivering according to table 1.Simultaneously do negative control with the intestinal bacteria nutrient solution that does not add IPTG.
Table 1 is expressed IPTG concentration and time conditions
5.1,2, collect successively Recombinant Swine interferon beta 1 bacterium liquid behind 3, the 4h, high speed centrifugation (rotating speed: 12000rpm/min) 3min, PBS washing and precipitating with precooling, must contain the intestinal bacteria precipitation of inducing Recombinant Swine interferon beta 1, add the 5XSDS gel loading buffer, 100 ℃ of heating 10min, the room temperature high speed centrifugation (rotating speed: 12000rpm/min) 1min, get supernatant.Do not add the intestinal bacteria nutrient solution of IPTG by this step process yet.
6. respectively get not adding IPTG and adding different concns IPTG that 10 μ L steps 5 are processed, Recombinant Swine interferon beta 1 sample of expressing under the different induction time conditions, 12%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis moves to separation gel bottom to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining is observed Recombinant Swine interferon beta 1 expression product band.See Fig. 9.
The expression that Recombinant Swine interferon beta 1 content is identified Recombinant Swine interferon beta 1 is expressed in the analysis of gel imaging system thin layer scanning.Final definite suitable inductive condition of the present invention is 1m mol/L IPTG, and induction time is 4h.
1. with the intestinal bacteria precipitation of inducing Recombinant Swine interferon beta 1 through the PBS of precooling washing and precipitating containing of obtaining in embodiment 4 steps 5, resuspended with the PBS of precooling, in 4 ℃ with 12000rpm/min, centrifugal 15min; Repeat once.
2. suck supernatant, claim bacterial sediment weight, every gram (thalline weight in wet base) adds lysis buffer BufferA5mL, stirs with the slicking glass rod, and thalline is hanged.
3. every gram (thalline weight in wet base) thalline adds 5 μ L100mmol/L PMSF, and 5 μ L100mg/mL N,O-Diacetylmuramidases stir 20min on ice.
4. with the broken thalline of sonde-type ultrasonoscope, sample places on ice, and ultrasonic 120 times, each 5s interval 5s circulates three times, is circulated between the cooling sample at every turn and waits for 2min, waits for the sample cooling.4 ℃, 12000rpm/min, centrifugal 15min.
5. precipitation is washed with lavation buffer solution Buffer B, and 4 ℃, 12000rpm/min, centrifugal 15min, the precipitation inclusion body repeats once.
6. the inclusion body precipitation stirs 30min with sex change buffer B uffer C dissolving under the room temperature.
7. abundant room temperature 12000rpm/min behind the mixing, centrifugal 15min abandons precipitation, gets supernatant, namely obtains Recombinant Swine interferon beta 1 denaturing soln.
8. adopt the dilution refolding method that Recombinant Swine interferon beta 1 denaturing soln in the step 7 is carried out renaturation.
Get an amount of Recombinant Swine interferon beta 1 denaturing soln with sex change buffer B uffer C dissolving, with Quick Start Bradford1x Dye Reagent(U.S. bio-rad company) survey its concentration, then with renaturation buffer BufferD protein concentration is diluted to 0.2mg/mL, during 4 ℃ of renaturation to 24 hour, recombinant protein solution after the renaturation is crossed 0.45 μ m filter membrane (MerckMillipore company), namely obtain the Recombinant Swine interferon beta 1 renaturation solution of lower concentration.Super filter tube (Merck with molecular weight cut-off 10KDa
Millipore company) desalination, concentrated in vacuum freeze drier (Beijing Sihuan Scientific Instrument Factory Co., Ltd) low-temperature vacuum drying, namely obtains Recombinant Swine interferon beta 1 powder.
Each damping fluid according to the form below preparation:
Each damping fluid preparation of table 2
9. carry out SDS-PAGE electrophoretic analysis (as shown in figure 10) with the product of twice washed product of lavation buffer solution Buffer B and renaturation gained in the step 5 respectively, at the visible obviously band of purpose range.
Pig vesicular stomatitis virus (VSV, its TCID
50Be 5 * 107/100 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 β 1 to detect at porcine kidney cell defense reaction to the pig vesicular stomatitis virus under pig interferon β 1 existence condition; obtain the protective effect curve of 1 pair of PK-15 cell of pig interferon β by the pathology situation that detects the PK-15 cell, thereby measure Recombinant Swine interferon beta 1 biologic activity.
1. biological activity primary dcreening operation experiment
Positive reference substance solution preparation: (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.Measuring that it tires is 5 * 10
4U/mL), 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.
Recombinant Swine interferon beta 1 sample solution preparation: take by weighing Recombinant Swine interferon beta 1 sample 1mg after the renaturation, after the dissolving of 500 μ L cell culture fluids, then do 10 times of one-level stepwise dilutions with cell culture fluid, respectively must 2 * 10
3μ g/mL, 2 * 10
2μ g/mL, 20 μ g/mL, 2 μ g/mL, 2 * 10
-1μ g/mL, 2 * 10
-2μ g/mL recombinant interferon β 1 solution.
Every hole adds the Recombinant Swine interferon beta 1 of above-mentioned 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
2Hatched under the condition approximately 24 hours, 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 simultaneously negative control virus group (virus that only adds PK-15 cell and same dose does not add restructuring pig interferon β 1) and blank cell control group (only add PK-15 cell and cell culture fluid, do not add restructuring pig interferon β 1 and virus).37 ℃, 5%CO
2Cultivate 24h under the condition, observations when treating the cytopathy 90%～100% in virus control hole.Take the amount of the cytopathic Interferon, rabbit that causes the half hole as a unit (being designated as " U ").
The experimental result demonstration, obvious pathology all occurs in the cell in the negative control virus group, and Growth of Cells is normal in the blank cell control group.Compare with positive control, when the concentration of Recombinant Swine interferon beta 1 in 20 μ g/mL～2 * 10
-1During μ g/mL, begin to produce and eliminate VSV virus to the phenomenon of the impact of PK-15 cell normal growth.
2. biological activity is sieved experiment again
According to the primary dcreening operation result, adjust positive control and Recombinant Swine interferon beta 1 experimental concentration:
The preparation of positive reference substance solution: get the pig interferon positive reference substance, by specification is diluted to every 1ml with cell culture fluid and approximately contains 1000IU after redissolving, by 4 times of one-level stepwise dilutions.
Recombinant Swine interferon beta 1 sample solution preparation: according to the active Preliminary detection result of embodiment 5 renaturation samples, the Recombinant Swine interferon beta 1 usefulness cell culture fluid of renaturation gained is diluted to 20ug/mL, then by 4 times of one-level stepwise dilutions, get 20ug/mL, 5 μ g/mL, 1.25 μ g/mL, 3.13 * 10
-1μ g/mL, 7.81 * 10
-2μ g/mL, 1.95 * 10
-2μ g/mL, 4.88 * 10
-3μ g/mL, 1.22 * 10
-3μ g/mL Recombinant Swine interferon beta 1 solution.
Every hole adds the Recombinant Swine interferon beta 1 of above-mentioned 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
2Hatched under the condition approximately 24 hours, 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 simultaneously negative control virus group (virus that only adds PK-15 cell and same dose does not add restructuring pig interferon β 1) and blank cell control group (only add PK-15 cell and cell culture fluid, do not add restructuring pig interferon β 1 and virus).37 ℃, 5%CO
2Cultivate 24h under the condition, observations when treating the cytopathy 90%～100% in virus control hole.Take the amount of the cytopathic Interferon, rabbit that causes the half hole as a unit (being designated as " U ").
Experimental result shows, obvious pathology all occurs the cell in the negative control virus group, and Growth of Cells is normal in the blank cell control group.Take the amount of the cytopathic Interferon, rabbit that causes the half hole as a unit (being designated as " U "), by the cell count that PK-15 Growth of Cells under the microscopic examination positive control existence condition is not affected by VSV virus, determine that tiring of positive control Interferon, rabbit is 3.2 * 10
4U/mL.In like manner, the cell count that the PK-15 Growth of Cells is not affected by VSV virus under Recombinant Swine interferon beta 1 existence condition of gained after the microscopic examination renaturation, calculating tiring of Recombinant Swine interferon beta 1 renaturation sample is 4.8 * 10
4U/mL.
This shows, the activity of the Interferon, rabbit positive control that the activity of the Recombinant Swine interferon beta 1 that the present invention obtains after by renaturation is sold on the market, and preparation technology is simple, showing wide application prospect aspect porkling disease prevention and the treatment, making may being achieved of suitability for industrialized production genetically engineered Recombinant Swine interferon beta 1.
Claims (10)
1. Recombinant Swine interferon beta 1, its aminoacid sequence is shown in SEQ ID NO:2.
2. gene of the Recombinant Swine interferon beta 1 described in the claim 1 of encoding, its base sequence is shown in SEQ ID NO:1.
3. carrier, described carrier has the gene of claim 2.
4. carrier as claimed in claim 3, described carrier is pET21b.
5. intestinal bacteria, described intestinal bacteria have the carrier of claim 3.
6. intestinal bacteria as claimed in claim 5, described intestinal bacteria are BL21(DE3) bacterial strain.
7. the expression method of a Recombinant Swine interferon beta 1 comprises the steps:
(1) picking contains the intestinal bacteria bacterium colony described in claim 5 or 6, and access contains antibiotic LB nutrient solution, overnight incubation;
(2) get overnight culture and transfer in containing antibiotic fresh LB nutrient solution, concussion is cultured to mid-log phase A600=1.0;
(3) adding concentration in culture is the IPTG of 0.5-1.5mmol/L, and 37 ℃, after abduction delivering 1-4 hour, centrifugal treating is collected the coli somatic precipitation that contains Recombinant Swine interferon beta 1.
8. the purifying of a Recombinant Swine interferon beta 1 and refolding method is characterized in that, comprise following steps:
(1) with the coli somatic precipitation that contains Recombinant Swine interferon beta 1 described in the claim 7, resuspended with the PBS of precooling, in 4 ℃, with the centrifugal 15min of 12000rpm/min, repeat once;
(2) suck supernatant, add 3-10mL lysis buffer BufferA by every gram (thalline weight in wet base), stir damping fluid thalline is hanged;
(3) every gram (thalline 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, stirs 20min on ice;
(4) broken somatic cells, 4 ℃, 12000rpm/min is centrifugal, abandons supernatant;
(5) the inclusion body precipitation is washed with lavation buffer solution Buffer B, and 4 ℃, the centrifugal 15min of 12000rpm/min abandons supernatant.The inclusion body precipitation repeats this step once;
(6) the inclusion body precipitation is dissolved stirring at room 30-60min with sex change buffer B uffer C;
(7) the abundant centrifugal 15min of 12000rpm/min under the room temperature condition behind the mixing abandons precipitation, gets supernatant, namely obtains Recombinant Swine interferon beta 1 denaturing soln;
(8) get an amount of described Recombinant Swine interferon beta 1 denaturing soln with sex change buffer B uffer C dissolving, survey its concentration, then with renaturation buffer Buffer D protein concentration is diluted to 0.2mg/mL, during 4 ℃ of renaturation to 24 hour, recombinant protein solution after the renaturation is crossed 0.45 μ m filter membrane, namely obtain Recombinant Swine interferon beta 1 renaturation solution.
9. purifying as claimed in claim 8 and refolding method is characterized in that, described Recombinant Swine interferon beta 1 renaturation solution can be further with molecular weight cut-off 10KDa ultrafiltration and concentration, desalination, and low-temperature vacuum drying namely obtains Recombinant Swine interferon beta 1 powder.
10. pharmaceutical composition is characterized in that: described pharmaceutical composition comprises the Recombinant Swine interferon beta 1 that the method by claim 8 or 9 obtains.
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