CN103243106B - Epinephelus coioides interferon IFNgamma1 and preparation method and application thereof - Google Patents
Epinephelus coioides interferon IFNgamma1 and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an epinephelus coioides interferon IFNgamma1 gene, a protein coded by the gene and an application of the protein in preparing a novel immunopotentiator. According to the invention, an opened reading frame of the IFNgamma1 gene is cloned from head-kidney RNAs (Ribonucleic Acid) of epinephelus coioides through a method of screening a genome database and designing specific primer amplification. The nucleotide sequence of the gene is shown as SEQIDNO:2. The amino acid sequence of the protein coded is shown as SEQIDNO:1. Through initial verification, the epinephelus coioides interferon IFNgamma1 protein can induce associated expression of immunogenes and can be developed to natural immunopotentiators or immunologic adjuvants for fishes.
Description
Technical field
The present invention relates to Interferon, rabbit field, more specifically, relate to Epinephelus coioide interferon IFNgamma 1 and its preparation method and application.
Background technology
Interferon, rabbit (IFN) is one group of active protein (being mainly glycoprotein) with several functions, cell and body is infected by the virus or other inductor effects after the cytokine that produced by monocyte and lymphocyte.According to the difference of homology and receptor-specific, up to now, find 3 interferoids: I type, II type and III type.Wherein IFN-GAMMA (IFN γ) is also referred to as type II interferon, main macrophage stimulation factor and the immunoreactive key signal molecule of regulation and control, energy activation effect cell, improve natural killer cell (NK) and macrophage activity, the conversion of Promote immunity sphaeroprotein, has the effects such as antitumor, antiviral, adjusting and enhancing body immunologic function.Interferon IFNgamma 1 and IFN γ 2 genes are two hypotypes of fish IFN γ gene, the feature of its aminoacid sequence is to contain [IV]-Q-X-[KQ]-A-X2-E-[LF]-X2-[IV] this conserved structure, this with other kinds of having studied in IFN γ similar.
IFN γ has biologic activity widely.IFN γ mainly by with its receptors bind; activation JAK-STAT signal path; activate transcribing of downstream gene; comprise associated transcription factor and immune molecule; as STAT1, IRF1, TLR3 and MHC II etc.; regulate propagation and differentiation and the enhancement antigen effect of offering of T cell, reach protection host's object.
IFN γ is important immune-regulating factor in body, and its immunoregulation effect is realized by following several respects.IFN γ promotes the processing of mhc class i and class Ⅱ antigens to offer, can raise from many aspects the expression of cell surface mhc class i molecule, IFN γ offers to promote CD4+T cell peptide specific activity by raising MHC class Ⅱ antigens increases the susceptibility of cytotoxic T lymphocyte (CTL) to pathogenic agent by raising the approach of offering of mhc class i antigen, and CTL is more effectively removed pathogenic agent; IFN γ is main macrophage activating factor (MAF), and its regulatory function to scavenger cell comprises: the activation of mediation T cell to scavenger cell, and directly induction participates in the synthetic of respiratory burst enzyme, and enhancing scavenger cell kills and wounds the ability of microorganism etc.; In addition, IFN γ is also the primary product of Th1 cell, and can further make immune response to Th1 Phenotypic Change its can to make static CD4+T cytodifferentiation be Th1 cell, suppress the propagation IFN of Th2 cell by innate immune reaction and the acquired immune response bridging simultaneously, it can coordinate innate immune cells identification pathogenic agent, and the immunoreactive generation of inducing specific.The ability that this coordination of IFN γ is transformed to the acquired immune response by inherent immunity reaction is that other Interferon, rabbit are not available.
The broad-spectrum disease resistance toxic action of IFN γ be mainly by with the combination of cell surface receptor, induced viral infection cell produces multiple antiviral protein, thereby makes to produce antiviral state performance antivirus action in cell, its antivirus action is nonspecific.The protein kinase R(PKR that the main effector molecule of IFN γ has double-stranded RNA to rely on), Mx 2 '-5 ' oligoadenylate synthetase (2 '-5 ' OAS), ribonuclease l (RNase L) system etc.PKR is a kind of serine/threonine kinase, after being combined with dsRNA, be activated, the PKR of activation can make albumen synthesis initiator eIF-2 and nf supressor IkB phosphorylation, thereby suppresses the synthetic of virus and cell protein, plays antiviral effect; Mx can be by disturbing some negative strand viruses nucleic acid fragment to suppress viral propagation to the function of intracellular transport and some viral protein; 2 '-5 ' oligoadenylate synthetase can make ATP be polymerized to 2 '-5 ' oligoadenylate, the activator RNA enzyme L ssRNA that degrades, thus viral interference is copied.In inductive effect factor expression, the expression that can improve cell surface MHC molecule due to IFN γ, strengthen the lethal effect of immunologically competent cell to pathogenic agent, promote body killing virus infected cell thereby work in coordination with, although and make the body all can the reaction of mediated cell to virus infection in the various types of Interferon, rabbit of antiviral state, the immunoregulatory activity of IFN γ is coordinating to bring into play even more important effect in the long-term antiviral state of immune response and definite body.
Similar to Mammals, fish IFN γ recombinant protein can affect the propagation of immune stimulatory cell, increases the genetic expression of IFN γ self, and induction downstream immunogene, as the expression of MHC II and Mx gene.Mx is ripe well known antiviral protein in IFN system, and as a kind of GTP enzyme, it mainly suppresses copying of RNA by the polysaccharase of viral interference, thereby brings into play antiviral effect.It is mainly subject to I type IFN induction great expression to bring into play it to act on, and IFN-GAMMA has certain inducing action to it.The IFN γ of restructuring can affect the cell immune response of fish.The result of preliminary study shows, certain density restructuring rainbow trout IFN γ can promote the expression of downstream immunogene IP10 and MHC II β in RTS-11 cells.
Fish diseases occurs frequently in recent years, and wherein some disease causes catastrophic harm to culture fishery, and this just makes immune protection technology in fish disease prevention and cure, present increasingly wide application prospect.IFN γ because it is antitumor, antiviral, that adjusting and enhancing body immunologic function etc. act on application in Mammals is very extensive, also very general to people's clinical application treatment disease aspect.And its research fish just starts starting with application, can predict that it has broad application prospects.
Summary of the invention
The object of the invention is to prevent and treat according to existing fish immunity the defect that there is no interferon protein IFN γ in method, a kind of new fish interferon IFN γ 1 gene and proteins encoded thereof are provided.
First the gene order of Epinephelus coioide interferon IFNgamma 1 is provided, and the nucleotide sequence of described gene is as shown in SEQ ID NO:2.
Further provide Epinephelus coioide interferon IFNgamma 1 recombinant protein, the aminoacid sequence of described albumen is as shown in SEQ ID NO:1.
A kind of Epinephelus coioide interferon IFNgamma 1 recombinant expression vector is provided according to demand again, in the nucleotide sequence of described recombinant expression vector, contains just like the nucleotide sequence shown in SEQ ID NO:2.
The acquisition methods of the gene order of a kind of Epinephelus coioide interferon IFNgamma 1 is provided according to demand again, and taking the total mRNA of Epinephelus coioide head-kidney as template, taking RNA Oligo dT as primer, reverse transcription obtains cDNA; Taking this cDNA as template, design upstream primer and downstream primer carry out pcr amplification, to obtain final product again.
The sequence of described RNA Oligo dT is as shown in SEQ ID NO:3.
The sequence of described upstream primer is as shown in SEQ ID NO:4.
The sequence of described downstream primer is as shown in SEQ ID NO:5.
According to described recombinant protein, then provide a kind of production method of Epinephelus coioide interferon IFNgamma 1 recombinant protein, comprise the following steps:
S1. build the recombinant expression vector containing the gene order of interferon IFNgamma 1;
S2. recombinant expression vector step S1 being obtained, is transformed in engineering bacteria;
S3. culturing engineering bacterium, makes the recombinant expression vector in engineering bacteria express interferon IFNgamma 1 recombinant protein;
S4. extract interferon IFNgamma 1 recombinant protein, purifying;
Structure described in step S1, containing the recombinant expression vector of the gene order of interferon IFNgamma 1, comprises the following steps:
S11. taking the plasmid containing Epinephelus coioide interferon IFNgamma 1 encoding gene as template, design the upstream and downstream primer with restriction enzyme site respectively, pcr amplification, the sequence of the described upstream primer with restriction enzyme site is as shown in SEQ ID NO:6, and the order of the downstream primer of described restriction enzyme site is as shown in SEQ ID NO:7;
S12. step S11 gained pcr amplification product is cloned into prokaryotic expression carrier pET22b upper, obtains the recombinant expression vector containing the gene order of interferon IFNgamma 1.
Described in step S3, the condition of culturing engineering bacterium is: the LB substratum that single colony inoculation is contained to 100 μ g/mL amicillin resistances to 5ml, 37 DEG C, 250rpm shaken overnight is cultivated, getting overnight culture is inoculated in the fresh LB substratum containing amicillin resistance with 1: 100 ratio, 37 DEG C, 250rpm is cultured to OD600 and reaches approximately 0.6, to add induction final concentration be 0.6mmol/L IPTG, 25 DEG C, 250rpm inducing culture centrifugal collection thalline after 7 hours.
Extraction described in step S4 and purifying are by after total thalline Native Binding Buffer washing, use again Native Binding Buffer resuspended, through supersound process, high speed centrifugation obtains cracking supernatant liquor, immobilization metal affinity chromatogra purifying, collect albumen elution peak, through PD-10 desalting column desalting treatment, obtain purifying protein.
Engineering bacteria described in step S2 and S3 is intestinal bacteria, is preferably BL21 bacterial strain.
According to the recombinant protein providing, then provide a kind of containing described Epinephelus coioide interferon IFNgamma 1 recombinant protein native species immunostimulant or immunological adjuvant.
Compared with prior art, the present invention has following benefit:
The present invention utilizes Epinephelus coioide genome database binding molecule biological method to design special primer, pcr amplification clone obtains the ORF sequence of IFN γ 1 gene, and build prokaryotic expression carrier, be transformed in intestinal bacteria and cultivate, albumen that can great expression Epinephelus coioide interferon IFNgamma 1 coded by said gene.In the present invention, Epinephelus coioide restructuring IFN γ 1 albumen can raise MHC II in renal cell and the expression of TLR3 gene, enrich the system signal path theory of fish interferon, can be for the research and development of relevant fish immunity toughener provide certain theoretical foundation, the additive of bait that IFN γ 1 albumen also can be used as enhancing fish immunity is applicable to aquaculture.
Brief description of the drawings
Fig. 1 is the comparison result figure of Epinephelus coioide interferon IFNgamma 1 aminoacid sequence and part vertebrates IFN γ aminoacid sequence.
Fig. 2 is the PCR amplified production electrophoresis result of Epinephelus coioide interferon IFNgamma 1 mature polypeptide coding sequence.
Fig. 3 is the construction of recombinant expression plasmid schematic diagram of gene IFN γ 1.
Fig. 4. the SDS-PAGE (A) of Epinephelus coioide interferon IFNgamma 1 expression of recombinant proteins and purifying and Western hybridization (B) analysis chart.
Fig. 5. the impact of Epinephelus coioide IFN γ 1 on MHC II in Immune cells in head kidney of bastard halibut and TLR3 genetic expression.
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.Unless stated otherwise, reagent, equipment and the method that the present invention adopts is the conventional commercial reagent of the art, equipment and the conventional method using.
The clone of embodiment 1. Epinephelus coioide interferon IFNgammas 1
1. the extraction of the total RNA of Epinephelus coioide head-kidney
Get the full fish of healthy Epinephelus coioide, anaesthetize after approximately 2 min with ice bath, kill fish sampling, separating head nephridial tissue, adopts the extracting of Trizol reagent method to obtain the total RNA of Epinephelus coioide head-kidney, its OD260/280=1.91.
2. cDNA the first chain is synthetic
Get the total RNA sample of 1 μ g Epinephelus coioide head-kidney and carry out the processing of DNA enzyme to remove the pollution of genomic dna, with RNA Oligo dT(sequence as shown in SEQ ID NO:3) mix, carry out reverse transcription, products therefrom is placed in-20 DEG C and saves backup.
3. the clone of Epinephelus coioide IFN γ 1 gene cDNA complete sequence
According to the information in Epinephelus coioide genome database, at IFN γ opening code-reading frame splicing sequence two ends design special primer, upstream primer sequence is as SEQ ID NO:4, downstream primer sequence is as SEQ ID NO:5, the the first chain cDNA synthesizing taking step 2 is as template, carry out pcr amplification, amplified fragments size is 567 bp.Electrophoretic separation DNA fragmentation, cuts glue and reclaims object product.Object product after purifying is connected to pTZ57R/T carrier, transforms DH5 α intestinal bacteria, select positive colony order-checking.Show through BLAST homology analysis, object product is really the cDNA sequence fragment of IFN γ 1 gene.
The expression of embodiment 2. Epinephelus coioide interferon IFNgamma 1 recombinant proteins
1. the structure of recombinant expression plasmid
Taking the pTZ57R/T plasmid that contains IFN γ 1 encoding gene as template, design the upstream primer SEQ ID NO:6 of a pair of NdeI of containing restriction enzyme site and the downstream primer SEQ ID NO:7 that contains XhoI restriction enzyme site, obtain the single band of product size in 550 bp left and right through pcr amplification, electrophoresis result as shown in Figure 2, wherein, M is DNA Marker, and 1 is the IFN γ 1 Nucleotide object fragment with restriction enzyme site.The mature polypeptide coding sequence of Epinephelus coioide interferon IFNgamma 1 is cloned into prokaryotic expression carrier pET-22b upper, builds recombinant expression vector pET22b-IFN γ 1, (its building process as shown in Figure 3).Exogenous gene sequence in expression vector is errorless through order-checking qualification.
2. the prokaryotic expression of Epinephelus coioide interferon IFNgamma 1 gene
The expression plasmid building in step 1 is transformed to e. coli bl21 (DE3), and IPTG induction target protein is expressed.Genetic engineering bacterium ultrasonic degradation after induction obtains supernatant liquor, show through SDS-PAGE electrophoretic analysis, bacterial strain is being subject to there is obvious specifically expressing product band after IPTG induction, close with the IFN γ that the contains His label 1 recombinant protein molecular size range of software estimation, result as shown in Figure 5, wherein A is the impact of IFN γ 1 on the genetic expression of MHC II, and B is the impact of IFN γ 1 on TLR3 genetic expression, and * represents that there were significant differences (P<0.05) with control group.
The optimal culture condition that the optimization of the condition such as inducing temperature and inductive dose is drawn to genetic engineering bacterium is: order bacterium colony contains in the liquid nutrient medium of penbritin LB to 5ml's, 37 DEG C, and 250 rpm, shaking culture is spent the night; Be inoculated into containing in penbritin TB liquid nutrient medium of 37 DEG C of preheatings of 150ml by 1:100 volume ratio, 37 DEG C, 250 rpm, are cultured to OD600 and reach approximately 0.6; Add IPTG to final concentration 0.6mM, under 25 DEG C of conditions, to IFN γ 1 protein expression engineering bacteria induction 7h, can obtain maximum soluble recombinant protein expression amount.
3. extraction and the purifying of Epinephelus coioide interferon IFNgamma 1 recombinant protein
By the Native Binding Buffer washing for total thalline of IFN γ 1 protein expression engineering bacteria, use again Native Binding Buffer resuspended, after supersound process, being fixed of cracking supernatant liquor metal-ligand affinity chromatography purifying, collect albumen elution peak, SDS-PAGE analyzes expression and the purification result of recombinant protein.Epinephelus coioide interferon IFNgamma 1 recombinant protein can be adsorbed by nickel metal affinity chromatography post, with containing different concns imidazoles elution buffer while washing nickel chromatography column, target protein can be washed down, after desalination, obtain Epinephelus coioide interferon IFNgamma 1 recombinant protein, result as shown in Figure 4 wherein, M is protein molecular weight standard, 1: do not induce total bacterium; 2: total bacterium after induction; 3: supernatant lysate; 4: rinsing liquid; 5-7; Be respectively 150/200/250mM imidazoles elutriant; 8: albumen after desalination (A); Western analytical results (B).
Epinephelus coioide interferon IFNgamma 1 recombinant protein of embodiment 3. embodiment 2 gained is expressed the activation analysis of impact on gene involved in immunity
To the experiment fish sampling of rapid frost anesthesia, clip head-kidney tissue, is soaked in appropriate RPMI1640 substratum, and is placed on ice; Head-kidney tissue is placed in Bechtop and is shredded a little, pick away after reticular tissue, grind with the frosted slide frosting having dried, be milled to powder; The cell of milled is crossed together with substratum to Cell strainer(BD Falcon, 70 μ m, Nylon) and be transferred in 50ml centrifuge tube: centrifugal, abandon supernatant liquor, add RPMI 1640 substratum 2ml re-suspended cells, washing, (RPMI 1640 is containing 2 mM L-glutamine for centrifugal rear use perfect medium, 10% FBS and 1% penicillin/streptomycin) 2 ml re-suspended cells, cell count is adjusted to 1 × 10
7/ mL adds the above-mentioned cell suspending liquid of 2 mL respectively in the 6 each holes of well culture plate; Be 1 ng/mL to adding final concentration in each grouping hole respectively, perfect medium 2 mL of 10 ng/mL and 100 ng/mL IFN γ 1 albumen, and negative control group (each 2 mL of cell suspending liquid and perfect medium) is set.Tissue Culture Plate is placed in to 27 DEG C, 5% CO
2in incubator, cultivate, collect each porocyte after hatching 4 h, Real Time-PCR detects the impact of IFN γ 1 albumen on MHC II and TLR3 genetic expression.Wherein MHC II genetic expression the primer upstream primer sequence is as SEQ ID NO:10, and downstream primer sequence is as SEQ ID NO:11; TLR3 genetic expression the primer upstream primer sequence is as SEQ ID NO:12, and downstream primer sequence is as SEQ ID NO:13.18S rRNA proofreaies and correct the initial cDNA amount of each template as reference gene, and the upstream primer sequence of 18S rRNA gene fragment is as SEQ ID NO:8, and downstream primer sequence is as SEQ ID NO:9.Every group of 3 repetitions, data represent with mean value ± standard error, and * represents that there were significant differences (P<0.05) with control group, and result is as shown in Figure 5.Result demonstration, in Epinephelus coioide renal cell, IFN γ 1 albumen of 10 ng/ml is hatched the mRNA level that can significantly raise MHC II and TLR3 after 4 hours, and the albumen of high density does not have a significant impact not transcribing of MHC II and TLR3.Show transcribing of the two do not present between protein concentration significant concn rely on effect, the albumen of high density can not rise MHC II and TLR3 expression level.
<110> Zhongshan University
<120> Epinephelus coioide interferon IFNgamma 1 and its preparation method and application
<130>
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Claims (8)
1. the gene order of Epinephelus coioide interferon IFNgamma 1, is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:2.
2. Epinephelus coioide interferon IFNgamma 1 recombinant protein, is characterized in that, the aminoacid sequence of described albumen is as shown in SEQ ID NO:1.
3. Epinephelus coioide interferon IFNgamma 1 recombinant expression vector, is characterized in that, in the nucleotide sequence of described recombinant expression vector, contains just like the nucleotide sequence shown in SEQ ID NO:2.
4. an acquisition methods for the gene order of Epinephelus coioide interferon IFNgamma 1 according to claim 1, its feature exists, and taking the total mRNA of Epinephelus coioide head-kidney as template, taking RNA Oligo dT as primer, reverse transcription obtains cDNA; Taking this cDNA as template, design upstream primer and downstream primer carry out pcr amplification again, to obtain final product,
The sequence of described RNA Oligo dT as shown in SEQ ID NO:3,
The sequence of described upstream primer as shown in SEQ ID NO:4,
The sequence of described downstream primer is as shown in SEQ ID NO:5.
5. a production method for Epinephelus coioide interferon IFNgamma 1 recombinant protein according to claim 2, is characterized in that, comprises the following steps:
S1. build the recombinant expression vector containing the gene order of interferon IFNgamma 1;
S2. recombinant expression vector step S1 being obtained, is transformed in engineering bacteria;
S3. culturing engineering bacterium, makes the recombinant expression vector in engineering bacteria express interferon IFNgamma 1 recombinant protein;
S4. extract interferon IFNgamma 1 recombinant protein, purifying.
6. the production method of Epinephelus coioide interferon IFNgamma 1 recombinant protein according to claim 5, is characterized in that, the structure described in step S1, containing the recombinant expression vector of the gene order of interferon IFNgamma 1, comprises the following steps:
S11. taking the plasmid containing Epinephelus coioide interferon IFNgamma 1 encoding gene as template, design the upstream and downstream primer with restriction enzyme site respectively, pcr amplification, the sequence of the described upstream primer with restriction enzyme site is as shown in SEQ ID NO:6, and the sequence of the described downstream primer with restriction enzyme site is as shown in SEQ ID NO:7;
S12. step S11 gained pcr amplification product is cloned into prokaryotic expression carrier pET22b upper, obtains the recombinant expression vector containing the gene order of interferon IFNgamma 1.
7. the production method of Epinephelus coioide interferon IFNgamma 1 recombinant protein according to claim 5, it is characterized in that, described in step S3, the condition of culturing engineering bacterium is: the LB substratum that single colony inoculation is contained to 100 μ g/mL penbritins to 5ml, 37 DEG C, 250rpm shaken overnight is cultivated, getting overnight culture is inoculated in the fresh LB substratum containing penbritin with 1: 100 ratio, 37 DEG C, 250rpm is cultured to OD600 and reaches 0.6, to add induction final concentration be 0.6mmol/L IPTG, 25 DEG C, 250rpm inducing culture centrifugal collection thalline after 7 hours.
8. the production method of Epinephelus coioide interferon IFNgamma 1 recombinant protein according to claim 5, is characterized in that, the engineering bacteria described in step S2 and S3 is intestinal bacteria.
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