CN103059123B - Recombinant porcine interferon beta1, encoding gene and expression method thereof - Google Patents
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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 (being mainly glycoprotein) with several functions, is a kind of cytokine being produced by monocyte and lymphocyte.They on allogenic cell, have wide spectrum antiviral, affect Growth of Cells, and differentiation, regulate the multiple biological activitys such as immunologic function.The character that according to the source of IFN is animal species, cell type, inducer is different with Induction Condition, can be divided into tri-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 broad-spectrum disease resistance toxic action, can be used as the alternative preparation of interferon alpha, and interferon beta can have very big-difference because viral kind is different to viral inhibition degree, and for example interferon beta has better anti-SARS-CoV effect than interferon alpha; Interferon beta is also obviously better than interferon-gamma in the tolerance of acid, alkali, heat.Meanwhile, 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 regulatory role, and therefore the application in field of veterinary is more and more extensive.Yet interferon beta exists obvious species variation, only rely on extraction in live hog body and can not meet the requirement in market far away.Genetic engineering interferon β drug residue free for animal, have no side effect, be subject to very much clinical animal doctor and raiser's favor, but on market most similar medicines be still faced with underproduce, levels of audit quality is uneven, the problem such as expensive.Therefore, first this patent provides a kind of expression system and expression method that can great expression Recombinant Swine interferon beta 1 with low cost, stay-in-grade.
Prokaryotic expression system is used and studies the earliest, and this is also to grasp at present the most ripe expression system.The main method of this technology is by the carrier that is cloned into goal gene DNA fragmentation (being generally plasmid) transform bacteria (what conventionally select is intestinal bacteria), by IPTG, induces also final purifying to obtain required target protein.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 adopted coli expression system to carry out restructuring pig interferon β 1, thus a large amount of target proteins obtained.But due to 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, not only closely bound up with renaturation condition and renaturation material, depend on more to a great extent the character of protein self.If renaturation condition is not suitable for, will cause the mispairing of intramolecular disulfide bond, the intermolecular mode with covalent attachment or hydrophobic binding to form polymer, thereby make 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 the inclusion body of the Recombinant Swine interferon beta 1 that suitable condition produces coli expression system to carry out renaturation, obtains having the product compared with higher specific activity.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, by codon optimized mode, provide a kind of can 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 as shown in SEQ ID NO:2.
The gene that the invention provides coding Recombinant Swine interferon beta 1 described above, its base sequence is as shown in SEQ ID NO:1.This sequence is to aim at escherichia expression system to carry out the codon optimized sequence obtaining, 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 escherichia coli expression method, comprises the steps:
Steps of the method are:
1. the intestinal bacteria bacterium colony that one of picking contains Recombinant Swine interferon beta 1 described above, access LB nutrient solution, overnight incubation;
2. get overnight culture and access in LB nutrient solution, concussion is cultured to mid-log phase (A
600=1.0);
3. in culture, add IPTG to 0.5-1.5mmol/L, in 37 ℃, after abduction delivering 1-4h, centrifugal treating is collected the coli somatic precipitation that contains Recombinant Swine interferon beta 1.
In described LB nutrient solution, all contain penbritin 50-100 μ g/mL.
The present invention also provides the inclusion body purification method of Recombinant Swine interferon beta 1, comprises the steps:
1. the above-mentioned induction Recombinant Swine interferon beta 1 coli somatic precipitation that contains collection being obtained, 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, with slicking glass rod, stirs, and thalline is hanged.
3. every gram (thalline weight in wet base) thalline adds the PMSF that 3-10 μ L concentration is 100mmol/L, and the N,O-Diacetylmuramidase that 3-100 μ L concentration is 100mg/mL, in stirring on ice.
4. broken thalline, sample is placed on ice, ultrasonic, and processes in 4 ℃ of high speed centrifugations, abandons supernatant.
5. lavation buffer solution Buffer B washing for precipitation, and process in 4 ℃ of high speed centrifugations, precipitation inclusion body, repeats once.
6. inclusion body precipitation is dissolved with sex change buffer B uffer C, under room temperature, stirs 30-60min.
7. fully mix rear room temperature high speed centrifugation and process, abandon precipitation, get supernatant, obtain Recombinant Swine interferon beta 1 denaturing soln.
This purification process preferred steps is as follows:
1. the above-mentioned induction Recombinant Swine interferon beta 1 coli somatic precipitation that contains collection being obtained, resuspended with the PBS of precooling, in 4 ℃, with the centrifugal 15min of 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, with slicking glass rod, stirs, and thalline is hanged.
3. every gram (thalline weight in wet base) thalline adds the PMSF that 5 μ L concentration are 100mmol/L, and the N,O-Diacetylmuramidase that 5 μ L concentration are 100mg/mL, stirs 20min on ice.
4. with the broken thalline of sonde-type ultrasonoscope, sample is placed on ice, and ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, waits for that sample is cooling.In 4 ℃, the centrifugal 15min of rotating speed with 12000rpm/min, abandons supernatant.
5. lavation buffer solution Buffer B washing for precipitation, in 4 ℃, with the centrifugal 15min of rotating speed of 12000rpm/min, precipitation inclusion body, repeats once.
6. inclusion body precipitation is dissolved with sex change buffer B uffer C, under room temperature, stirs 30min.
7. fully mix under rear room temperature with the centrifugal 15min of rotating speed of 12000rpm/min, abandon precipitation, get supernatant, 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 appropriate Recombinant Swine interferon beta 1 denaturing soln described above dissolving with sex change buffer B uffer C, 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 renaturation is crossed to 0.45 μ m filter membrane, obtain the Recombinant Swine interferon beta 1 renaturation solution of lower concentration.Molecular weight cut-off 10KDa ultrafiltration desalination, concentrated, in vacuum freeze drier low-temperature vacuum drying, obtains Recombinant Swine interferon beta 1 powder.
Expression described above of the present invention, purifying, refolding method are to grope and verify through the repeated multiple times experiment of contriver the effective means the most for escherichia expression system expression Recombinant Swine interferon beta 1 obtaining, 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 the expression amount at escherichia expression system far above pig interferon β 1 natural gene sequence.
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 viral defence capability.Meanwhile, pig interferon β 1 also can combine use with other vaccines, alleviates the untoward reaction of vaccine, strengthens whole antiviral, bacterium, parasitic effect.
Accompanying drawing explanation
Fig. 1 represents the codon optimized front and back of Recombinant Swine interferon beta 1 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 codon optimized.
Fig. 2-a, Fig. 2-b are restructuring pig interferon β 1 codon optimized front and back CAI index in escherichia coli expression host.
Wherein, Fig. 2-a represents that pig interferon β 1 natural gene nucleotides sequence is listed in CAI index in escherichia coli expression host and is calculated as 0.60 through program; Fig. 2-b represents that the Recombinant Swine interferon beta 1 codon of the present invention CAI index in escherichia coli expression host after optimization is calculated as 0.90 through program.
Fig. 3-a, Fig. 3-b are the codon optimized front and back of pig interferon β 1 optimal codon frequency distribution areal maps in 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 in escherichia coli expression host, as can be seen from the figure: the poor efficiency codon of pig interferon β 1 natural gene nucleotide sequence occurs that per-cent is 15%; Fig. 3-b represents the Recombinant Swine interferon beta 1 codon of the present invention optimal codon frequency distribution areal map in escherichia coli expression host after optimization, and the poor efficiency codon of the Recombinant Swine interferon beta of the present invention 1 codon sequence after optimization occurs that per-cent is 0.
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 escherichia coli expression host.
Wherein, Fig. 4-a represents that pig interferon β 1 natural gene nucleotides sequence is listed in average GC base contents in escherichia coli expression host and is: 46.45%; Fig. 4-b represents that the Recombinant Swine interferon beta 1 codon of the present invention average GC base contents in escherichia coli expression host after optimization 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 is 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 pET21b carrier for NdeI and XhoI enzyme; Swimming lane 2 is 500bp DNA Ladder; The Recombinant Swine interferon beta 1 gene PCR product that swimming lane 3 contains NdeI and XhoI restriction enzyme site for two ends.
SDS-PAGE gel electrophoresis figure and corresponding western blot figure that Fig. 8-a, Fig. 8-b are 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 dying in advance of (10-230kDa) wide region; Swimming lane 2 is not for adding the Recombinant Swine interferon beta 1 intestinal bacteria lysate of IPTG induction; Swimming lane 3 is for adding the Recombinant Swine interferon beta 1 intestinal bacteria lysate of IPTG induction.
Fig. 8-b is restructuring pig interferon β 1 western blot figure.
Wherein, swimming lane 1(10-230KDa) the albumen loading Marker dying in advance of wide region, swimming lane 2 is not for adding the Recombinant Swine interferon beta 1 intestinal bacteria lysate of IPTG induction: swimming lane 3 is for adding the Recombinant Swine interferon beta 1 intestinal bacteria lysate of IPTG induction.
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 dying in advance of (10-230kDa) wide region; Swimming lane 2 be 0.5mmol/L IPTG induction 1h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 3 be 0.5mmol/L IPTG induction 2h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 4 be 0.5mmol/L IPTG induction 3h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 5 be 0.5mmol/L IPTG induction 4h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 6 be 1mmol/LIPTG induction 1h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 7 be 1mmol/L IPTG induction 2h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 8 be 1mmol/L IPTG induction 3h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 9 be 1mmol/L IPTG induction 4h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 10 be 1.5mmol/LIPTG induction 1h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 11 be 1.5mmol/L IPTG induction 2h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 12 be 1.5mmol/L IPTG induction 3h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate; Swimming lane 13 be 1.5mmol/L IPTG induction 4h containing Recombinant Swine interferon beta 1 intestinal bacteria lysate.
Figure 10 is the Recombinant Swine interferon beta 1 inclusion body SDS-PAGE electrophorogram after renaturation
Wherein, swimming lane 1 is the albumen loading Marker dying in advance of (10-230kDa) wide region; Swimming lane 2 is for using the rear full bacterium lysate of induction; 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 after dilution refolding
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, should be understood that quoting embodiment is only not used in and limits the scope of the invention for the present invention is described.
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 or that utilization ratio is low (rare orlow-usage codons).In fact, conventional every kind of biology (comprising intestinal bacteria, yeast, mammalian cell, vegetable cell and insect cell) of doing protein expression or production all shows difference or the preference that codon to a certain degree utilizes.In intestinal bacteria, yeast and fruit bat to the expression efficiency containing the gene of best codon the expression efficiency apparently higher than the gene of the codon containing 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 and synthesize, the redesign of this gene is codon optimized.Optimizing process fully takes into account the Various Complex factor that protein expression different steps may run into, as: codon adaptability, mRNA structure and transcribe 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, and it has caused that same codon is between different organisms, the change of utilization ratio between the expression level of albumen and between the different sites of same operon.The major cause that causes this preferences difference is the difference that tRNAs available in different cells measures.Therefore the method for optimizing translation system the best is exactly to keep the frequency of utilization of codon and the balance between homology tRNA.At expression in escherichia coli mammalian genes, be unpredictable and have challenge, as in intestinal bacteria, just seldom, this species diversity clearly can affect the expression of gene to the corresponding tRNA molecule of AGG and AGA.
3. the codon of poor efficiency is replaced to the conventional codon of host
Conventionally the utilization ratio of the codon comprising in gene in specific host is lower, and the expression amount of this kind of albumen is also just fewer, even when between this codon existence and protein clusters or when N-terminal, expression amount can be still less.The codon of poor efficiency is replaced with to the expression level that the conventional codon of host can improve functional protein under the prerequisite that does not change aminoacid sequence.
If the utilization ratio of the codon in any source in host organisms lower than 5% to 10% time, just there will be expression inhibiting, when these poor efficiency codons close on or are connected, larger on the impact of protein expression.The codon of the poor efficiency of cluster has suppressed ribosomal motion, and this is the obvious mechanism that gene can not be expressed with proper level.The rrna translation movement velocity during by nine molecular couriers of password (containing several poor efficiency codons or be all poor efficiency codon) does not contain courier's the speed of same length of poor efficiency codon than translation slow.Even if poor efficiency password submanifold is positioned at 3 ' end, courier finally also can be damaged by rrna " crowded ", 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 low expression or not express.
4. expression vector and transcripting promoter
Although 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 very responsive with the codon AUG that approaches initiation site for poor efficiency codon.Between the stability of translation and mRNA, also exist reciprocal influence, although reduce translation efficiency, can make mRNA more easily by endo-RNAses, be decomposed owing to having lacked ribosomal protection, also there is no the complete explanation affecting between them at present.
Other factors also can affect protein expression, comprise and make mRNA go stable sequence.Stable mRNA secondary structure also has important impact to genetic expression with the molecule that approaches 5 ' end.While utilizing translation, the open reading frame of goal gene upstream can successfully improve the expression efficiency of difficulty gene.
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 after codon optimized obtaining Recombinant Swine interferon beta 1 gene of the present invention, as shown in SEQ ID No:1.
Be restructuring pig interferon β 1 to be carried out codon optimized below, before and after optimizing, each parameter comparison is as follows:
1. codon adaptation indexI (Codon Adaptation Index, CAI)
From Fig. 2-a, before codon is not optimized, pig interferon β 1 natural gene codon adaptation indexI (CAI) in intestinal bacteria is 0.60.From Fig. 2-b, after codon optimized, making Recombinant Swine interferon beta 1 gene of the present invention CAI index in intestinal bacteria is 0.90.During common CAI=1, be considered to this gene is optimal efficient expression status in this expression system, CAI index is lower shows 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 the expression level of Recombinant Swine interferon beta 1 gene in intestinal bacteria.
2. optimal codon frequency of utilization (Frequency of Optimal Codons, FOP)
From Fig. 3-a, based on coli expression carrier, before codon is not optimized, the poor efficiency codon of pig interferon β 1 natural gene sequence occurs that per-cent is 15%.This gene not being optimized contains series connection rare codon, and these codons may reduce translation efficiency, even can dismiss translation assemblage.From Fig. 3-b, after codon optimized, Recombinant Swine interferon beta 1 gene of the present invention occurs that in intestinal bacteria system the frequency of poor efficiency codon is 0.
3.GC base contents (GC curve)
GC content ideal distribution region is 30%-70%, at this any peak of extra-regional appearance, all can affect to some extent and transcribe and translation efficiency.GC base average content distributed areas figure from pig interferon β 1 gene of Fig. 4-a, Fig. 4-b contrasts, in Fig. 4-a, show in pig interferon β 1 natural gene that GC base average content is 46.45% before optimization, the GC content all bases outside 30%-70% region that demonstrated sequence elimination after optimization in Fig. 4-b, after being finally optimized, the GC base average content of Recombinant Swine interferon beta 1 is 47.26%.
3. before and after optimizing, cis-acting elements situation is as follows:
| Cis-acting elements | After optimization | Before 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. before and after optimizing, the palindrome and tumor-necrosis factor glycoproteins situation are as follows:
The secondary structure prediction figure of 5mRNA
At DNA, be transcribed into after mRNA, because mRNA is strand linear molecule, by self inflection, complementary base pair met, the hairpin structure forming by hydrogen bonded (Hairpin).5 ' hairpin structure can play regulating and controlling effect in the translation initiation stage.
But if hairpin structure is very long, the required energy that unwinds is very high, just likely has influence on translation.So need the sequence of expressing, should avoid long and the high hairpin structure of energy as far as possible.After codon optimized, from the secondary structure prediction figure of Fig. 5-a, Fig. 5-b pig interferon β 1 codon optimized front and back mRNA, 5 ' hairpin structure after optimization and the required energy that unwinds are more suitable for the expression of target protein.
embodiment 2: the expression plasmid of Recombinant Swine interferon beta 1 gene builds
The synthetic fragment of the full gene of Recombinant Swine interferon beta 1 (as shown in SEQ ID No:1) by after optimizing, is building up in pUC57 plasmid (Jin Sirui Science and Technology Ltd. provides by Nanjing), obtains a kind of prolonged preservation plasmid, is designated as pUC57-prIFN β 1 plasmid.PUC57-prIFN β 1 plasmid of take is template, and 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 the dNTP that concentration is 10mmol/L adds 1 μ L, and archaeal dna polymerase Phusion High-Fidelity DNA polymerase(used is purchased from Theromo-Fisher scientific), 2U/ μ L, adds 0.5 μ L.Reaction conditions is 98 ℃ of 5s, 55 ℃ of 20s, 72 ℃ of 30s, and after 25 circulations, product is through 1.0% agarose gel electrophoresis analysis, and result shows that product size is consistent with expection size (498bp).(as shown in Figure 7)
The gene product obtaining is reclaimed to test kit (purchased from Beijing Tian Gen biochemical technology company limited) purifying with DNA gel.After purifying, with NdeI and XhoI(purchased from New England Biolabs company) double digestion, with T4 ligase enzyme (purchased from New EnglandBiolabs company), be connected in pET21b plasmid (purchased from Merck company), be transformed in DH5 α competent cell (purchased from Beijing Tian Gen biochemical technology company limited) 37 ℃ of overnight incubation in the LB flat board of the penbritin that contains 100 μ g/mL (purchased from Amresco company).The order-checking of second day screening positive clone bacterium, compares, in full accord with expected sequence, obtains the expression plasmid of 1 one kinds of forms of Recombinant Swine interferon beta, is designated as pET21b-prIFN β 1.
expression and the evaluation of embodiment 3 Recombinant Swine interferon betas 1 in intestinal bacteria
Concrete steps are as follows:
1. correct pET21b-prIFN β 1 plasmid of order-checking comparison in embodiment 2 is transformed in e. coli bl21 (DE3) competence bacterial strain (purchased from Beijing Tian Gen biochemical technology company limited) to 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, the LB nutrient solution that access contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation.
3. get 50 μ L overnight culture access 5mL containing the LB inducing culture liquid of 100 μ g/mL penbritins, 37 ℃ of shaking culture.
4. after inoculation, every 1h, survey bacterium liquid OD600 value, when OD600=1.0, with the IPTG(of 1mmol/L purchased from Amresco company) carry out abduction delivering.Simultaneously not add the intestinal bacteria nutrient solution of IPTG to do negative control.
After 5.4h, collect bacterium liquid, and high speed centrifugation (rotating speed: 12000rpm/min) 3min, the PBS washing and precipitating with precooling, adds 5XSDS gel loading buffer, 100 ℃ of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm/min) 1min, gets supernatant.Do not add the intestinal bacteria nutrient solution of IPTG by this step process yet.
6. respectively get the culture samples that does not add IPTG and add IPTG to induce of 10 μ L step 5 gained, 12%SDS-PAGE gel electrophoresis analysis.
7.8-15V/cm electrophoresis, moves to separation gel bottom to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining and immunoblotting, observe expression product band, sees Fig. 8-a and Fig. 8-b.
the efficient induced expression condition optimizing of embodiment 4 Recombinant Swine interferon beta 1
Much research shows that cell growth rate has a strong impact on the expression of foreign protein, therefore must control to cell density after Growth of Cells time and induction before inoculation amount of bacteria, culture temperature, induction overgrowth or overrun and all can increase the weight of intestinal bacteria and form Recombinant Swine interferon beta 1 inclusion body.Use three factor four levels, set up IPTG concentration and induction time orthogonal table, by
SDS-PAGE gel electrophoresis analysis induction Recombinant Swine interferon beta 1 expression amount.
Concrete steps are as follows:
1. correct pET21b-prIFN β 1 plasmid of order-checking comparison in embodiment 2 is transformed into BL21(DE3) in competence bacterial strain (purchased from Beijing Tian Gen 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, the LB nutrient solution that access contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation.
3. get 50 μ L overnight culture access 5mL containing the LB inducing culture liquid of 100 μ g/mL penbritins, 37 ℃ of shaking culture.
4. after inoculation, survey bacterium liquid OD600 value, when OD600=1.0, according to table 1, add respectively 0.5,1.0,1.5m mol/LIPTG concentration and time to carry out abduction delivering.Simultaneously not add the intestinal bacteria nutrient solution of IPTG to do negative control.
Table 1 is expressed IPTG concentration and time conditions
5.1,2, after 3,4h, collect successively Recombinant Swine interferon beta 1 bacterium liquid, high speed centrifugation (rotating speed: 12000rpm/min) 3min, PBS washing and precipitating with precooling, the intestinal bacteria precipitation that must contain induction Recombinant Swine interferon beta 1, adds 5XSDS gel loading buffer, 100 ℃ of heating 10min, room temperature high speed centrifugation (rotating speed: 12000rpm/min) 1min, gets 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, Recombinant Swine interferon beta 1 sample of expressing under different induction time conditions, 12%SDS-PAGE gel electrophoresis analysis of 10 μ L steps 5 processing.
7.8-15V/cm electrophoresis, moves to separation gel bottom to tetrabromophenol sulfonphthalein.
8. coomassie brilliant blue staining, observes 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 applicable inductive condition of the present invention is 1m mol/L IPTG, and induction time is 4h.
1. by the intestinal bacteria precipitation that contains induction Recombinant Swine interferon beta 1 obtaining through the PBS of precooling washing and precipitating 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, with slicking glass rod, stirs, 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 is placed on ice, and ultrasonic 120 times, each 5s interval 5s, circulates three times, is circulated between cooling sample at every turn and waits for 2min, waits for that sample is cooling.4 ℃, 12000rpm/min, centrifugal 15min.
5. lavation buffer solution Buffer B washing for precipitation, 4 ℃, 12000rpm/min, centrifugal 15min, precipitation inclusion body, repeats once.
6. inclusion body precipitation is dissolved with sex change buffer B uffer C, under room temperature, stirs 30min.
7. fully mix rear room temperature 12000rpm/min, centrifugal 15min, abandons precipitation, gets supernatant, obtains Recombinant Swine interferon beta 1 denaturing soln.
8. adopt dilution refolding method to carry out renaturation to Recombinant Swine interferon beta 1 denaturing soln in step 7.
Get appropriate Recombinant Swine interferon beta 1 denaturing soln dissolving with sex change buffer B uffer C, 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 renaturation is crossed to 0.45 μ m filter membrane (MerckMillipore company), 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, obtains Recombinant Swine interferon beta 1 powder.
Each damping fluid according to the form below preparation:
Each damping fluid preparation of table 2
9. with the product of twice washed product of lavation buffer solution Buffer B and renaturation gained in step 5, carry out SDS-PAGE electrophoretic analysis (as shown in figure 10) respectively, at the visible obviously band of object scope.
Pig vesicular stomatitis virus (VSV, its TCID
50be 5 * 107/100 μ L; academy of agricultural sciences, Guangdong Province veterinary institute provides) can 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 pig vesicular stomatitis virus under pig interferon β 1 existence condition; by detecting the pathology situation of PK-15 cell, obtain the protective effect curve of 1 pair of PK-15 cell of pig interferon β, 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, purchased from Hong Kong Man Pu animal nutrition company limited to get pig interferon positive reference substance.Measuring that it tires is 5 * 10
4u/mL), after by specification redissolves, use the MEM cell culture fluid (Gibico product) that contains 6% foetal calf serum by 10 times of one-level stepwise dilutions.
The 1 sample solution preparation of Recombinant Swine interferon beta: take Recombinant Swine interferon beta 1 sample 1mg after renaturation, after dissolving with 500 μ L cell culture fluids, then with cell culture fluid, do 10 times of one-level stepwise dilutions, 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.
On 96 orifice plates, every hole adds the Recombinant Swine interferon beta 1 of above-mentioned different concns, then (cell concn is about 1.8 * 10 to add the fresh PK-15 cell suspension going down to posterity of 50 μ L
6～2.2 * 10
6individual/mL), total system 100 μ L, 37 ℃, 5%CO
2under condition, hatch approximately 24 hours, PK-15 cell attachment grows to individual layer.Discard cell culture fluid supernatant, every hole adds 100 μ L containing the MEM nutrient solution of 2% foetal calf serum of 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 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) simultaneously.37 ℃, 5%CO
2under condition, cultivate 24h, observations when the cytopathy 90%～100% in virus control hole.To cause Liang Weiyige unit's (being designated as " U ") of the cytopathic Interferon, rabbit in half hole.
Experimental result demonstration, all there is obvious pathology in the cell in negative control virus group, and in blank cell control group, Growth of Cells is normal.Compare with positive control, when the concentration of Recombinant Swine interferon beta 1 is in 20 μ g/mL～2 * 10
-1during μ g/mL, start to produce the phenomenon of elimination VSV virus on the impact of PK-15 cell normal growth.
2. biological activity is sieved experiment again
According to primary dcreening operation result, adjust positive control and Recombinant Swine interferon beta 1 experimental concentration:
Positive reference substance solution preparation: get pig interferon positive reference substance, after by specification redissolves, be diluted to every 1ml approximately containing 1000IU, by 4 times of one-level stepwise dilutions with cell culture fluid.
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 of renaturation gained 1 use cell culture fluid is diluted to 20ug/mL, then by 4 times of one-level stepwise dilutions, obtain 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.
On 96 orifice plates, every hole adds the Recombinant Swine interferon beta 1 of above-mentioned different concns, then (cell concn is about 1.8 * 10 to add the fresh PK-15 cell suspension going down to posterity of 50 μ L
6～2.2 * 10
6individual/mL), total system 100 μ L, 37 ℃, 5%CO
2under condition, hatch approximately 24 hours, PK-15 cell attachment grows to individual layer.Discard cell culture fluid supernatant, every hole adds 100 μ L containing the MEM nutrient solution of 2% foetal calf serum of 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 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) simultaneously.37 ℃, 5%CO
2under condition, cultivate 24h, observations when the cytopathy 90%～100% in virus control hole.To cause Liang Weiyige unit's (being designated as " U ") of the cytopathic Interferon, rabbit in half hole.
Experimental result shows, obvious pathology all occurs the cell in negative control virus group, and in blank cell control group, Growth of Cells is normal.To cause Liang Weiyige unit's (being designated as " U ") of the cytopathic Interferon, rabbit in half hole, by the cell count that under microscopic examination positive control existence condition, PK-15 Growth of Cells 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 after microscopic examination renaturation, under Recombinant Swine interferon beta 1 existence condition of gained, PK-15 Growth of Cells is not affected by VSV virus, calculating tiring of Recombinant Swine interferon beta 1 renaturation sample is 4.8 * 10
4u/mL.
As can be seen here, the activity of the Recombinant Swine interferon beta 1 that the present invention obtains after by renaturation is apparently higher than the activity of the Interferon, rabbit positive control of selling on market, and preparation technology is simple, aspect porkling disease prevention and treatment, showing wide application prospect, making may being achieved of suitability for industrialized production genetically engineered Recombinant Swine interferon beta 1.
Claims (6)
1. an encoding gene for Recombinant Swine interferon beta 1, its base sequence is as shown in SEQ ID NO:1.
2. a carrier, described carrier has gene as claimed in claim 1.
3. carrier as claimed in claim 2, described carrier is pET21b.
4. intestinal bacteria, described intestinal bacteria have carrier claimed in claim 3.
5. intestinal bacteria as claimed in claim 4, described intestinal bacteria are BL21(DE3) bacterial strain.
6. an expression method for Recombinant Swine interferon beta 1, comprises the steps:
(1) the intestinal bacteria bacterium colony described in picking claim 4 or 5, access is containing antibiotic LB nutrient solution, overnight incubation;
(2) get overnight culture and transfer in containing in antibiotic fresh LB nutrient solution, shake and be cultured to mid-log phase A
600=1.0;
(3) in culture, adding concentration is the IPTG of 0.5-1.5m mol/L, and 37 ℃, after abduction delivering 1-4 hour, centrifugal treating is collected the coli somatic precipitation that contains Recombinant Swine interferon beta 1.
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