CN103233013A - Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application - Google Patents
Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application Download PDFInfo
- Publication number
- CN103233013A CN103233013A CN201310183812XA CN201310183812A CN103233013A CN 103233013 A CN103233013 A CN 103233013A CN 201310183812X A CN201310183812X A CN 201310183812XA CN 201310183812 A CN201310183812 A CN 201310183812A CN 103233013 A CN103233013 A CN 103233013A
- Authority
- CN
- China
- Prior art keywords
- bolti
- growth factor
- tgf
- transforming growth
- mature peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a Nile tilapia transforming growth factor TGF-beta 1 gene, a related protein and application, and belongs to the technical field of biological genetic engineering. The nucleotide sequence of the Nile tilapia transforming growth factor TGF-beta 1 gene is expressed by SEQIDNO:1; and the amino acid sequence of the related protein TGF-beta 1 is expressed by SEQIDNO: 2. Prokaryotic expression of the mature peptide gene of the Nile tilapia transforming growth factor TGF-beta 1 is performed so that recombinant mature peptide of the Nile tilapia transforming growth factor beta 1; and after the mice are immunized by the mature peptide, corresponding polyclone antibodies can be obtained. The recombinant mature peptide of the Nile tilapia transforming growth factor beta 1 and the prepared polyclone antibodies can be further used as immune stimulants and used for scientific research.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, be specifically related to a kind of bolti transforming growth factor TGF-β 1 gene, associated protein and application.
Background technology
Transforming growth factor-beta (Transforming Growth Factor beta, TGF-β) superfamily is a class manifold effect cytokine, plays an important role in suppressing various physiological processes such as tumour, cell proliferation, cytodifferentiation, tissue formation, pedigree decision, cell migration and apoptosis.The TGF-beta superfamily member comprises transforming growth factor-beta (TGF-β), activin (Activin) and joint/bone morphogenetic protein (Bone Morphogenetic Protein, BMP) three families.Sense stricto TGF-β is commonly referred to as the TGF-'beta ' family.TGF-β 1 is the form coding with precursor protein, and every kind of TGF-β 1 is by an independent genes encoding.TGF-β 1 precursor protein is single chain protein, and molecular weight is about 13000D.TGF-β 1 amyloid protein precursor just is secreted into the extracellular through a series of course of processing in cell.Wherein a most important step is the hydrolysis of precursor protein, and the N end-grain cutting of precursor protein is fallen, and there is the aminoacid sequence sign of RXXR at the cleavage site place, and remaining C end is TGF-β 1 mature peptide sequence.The primary structure of the TGF-β 1 of many animals is illustrated.People's TGF-β 1 is made up of 390 amino acid, and the TGF-β 1 of Africa xenopus is made up of 382 amino acid, and the TGF-β 1 of zebra fish is made up of 377 amino acid.TGF-β 1 has the conservative property of height, especially in mature peptide sequence part.The function of TGF-β 1 is mainly relevant with immunity.
Bolti (Oreochromis niloticus) belongs to Actinopterygii, Perciformes, and Percoidei, Callichthyidae, tilapia belongs to.Since its have growth fast, rise catch the rate height, dressing percentage is good, the pond raise conditional request not high, better adapt to characteristics such as net cage and approved by numerous raisers, simultaneously because of its meat flavour deliciousness, fine and tender taste, characteristics such as feeding habits are assorted, lower oxygen concentration resistance, breeding are strong, ready-made is a kind of cultured freshwater fish that water industry emphasis in the world's is cultivated, and be described as one of main source of following animal protein, also be China very important class outlet fish, but be subjected to great loss because disease causes tilapia to be cultured now.Common tilapia disease mainly can be divided into following a few class: 1. parasitic diseases: myxosporidiasis, trichodinasis, ichthyophthiriasis, chilodoniasis, bothriocephaliasis, diplostomumiasis, doctylogyrosis and gyrodactyliasis etc.; 2. fungal disease: fish molds, branchiomycosis etc.; 3. bacteriosis: mobility Aeromonas disease, pseudomonas disease, Edwardsiella disease, streptococcicosis, scale protrusion disease, bacteremic septicemia, bacillary erythroderma and bacterial gill rot disease etc.In these tilapia diseases, the most abominable with streptococcicosis again, the loss maximum that causes.Traditional treatment means mainly is to use microbiotic, but microbiotic lacks specific aim, and life-time service can make pathogenic agent produce resistance, and can influence water surrounding and relate to food-safety problem; Also have by in feed, adding and wait to strengthen the immunizing power of fish as probiotic bacterium, Chinese herb feed additive, VITAMIN; Also there is scholar's research to go out disease-resistant vaccine, but finds that vaccine is bad to the mutation effect of pathogenic bacteria.These methods all fail to solve tilapia disease problem breakthroughly.So the immunological reagent that development can significantly improve the bolti resistance against diseases is the problem that current urgent need will be broken through.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of bolti transforming growth factor TGF-β 1 gene is provided.
Another object of the present invention provides a kind of bolti transforming growth factor TGF-β 1.
Another object of the present invention provides a kind of application of bolti transforming growth factor TGF-β 1 gene.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of bolti transforming growth factor TGF-β 1 gene, its nucleotide sequence is shown in SEQ ID NO:1.SEQ ID NO:1 is encoding sequence (being the cDNA sequence, only is exon).
A kind of bolti transforming growth factor TGF-β 1 mature peptide gene, its nucleotide sequence is shown in SEQ ID NO:2, and the nucleotide sequence of mature peptide gene is corresponding to the sequence of the 808bp ~ 1143bp of bolti transforming growth factor TGF-β 1 gene cDNA complete sequence.
A kind of bolti transforming growth factor TGF-β 1, its aminoacid sequence is shown in SEQ ID NO:3.
A kind of bolti transforming growth factor TGF-β 1 mature peptide, its aminoacid sequence is shown in SEQ ID NO:4.The 270th of the ORF of bolti transforminggrowthfactor-to the corresponding sequence of the 381st amino acids sequence be the sequence of bolti transforminggrowthfactor-mature peptide correspondence.
A pair of primer for amplification bolti transforming growth factor TGF-β 1 gene, primer sequence is shown in SEQ ID NO:5 ~ 6.
Bolti transforming growth factor TGF-β 1 full-length gene of the present invention is that the cDNA that obtains with the total RNA reverse transcription of bolti spleen is template, obtain through PCR and the amplification of RACE method, it derives from the full length gene fragment of the transforminggrowthfactor-on the bolti spleen cDNA.From the ORF1143bp of the cDNA that clones the transforminggrowthfactor-that obtains, 381 amino acid of encoding.Analyze and find that sequence has very high conservative property, and is similar to other vertebratess.The transforminggrowthfactor-precursor aminoacid sequence of deriving finds to have nine conservative halfcystines (this also is transforming growth factor-beta member's a symbolic characteristic).8 halfcystines wherein form 4 pairs of intrachain disulfide bonds of molecule central section in paired mode, form interchain disulfide bond in conjunction with the 9th halfcystine, form TGF-β 1 dimer together.Precursor peptide has the initial enzyme recognition site RXXR of characteristic sign mature peptide.And has the plain binding site RGD of integration.
A kind of recombinant expression vector inserts the bolti transforming growth factor TGF-β 1 mature peptide gene of sequence as mentioned above by the multiple clone site of the carrier that sets out.The carrier that contains above-mentioned bolti transforminggrowthfactor-mature peptide gene order that the present invention makes up; The escherichia coli cloning carrier and the expression vector that particularly contain this gene.These construction of carrier are according to a conventional method, will by the synthetic bolti transforminggrowthfactor-mature peptide gene order of PCR method through enzyme cut with separation and purification after, be linked between the corresponding restriction enzyme site (being Sph I and Hind III) of existing respective carrier, namely be built into the required expression vector that contains bolti transforminggrowthfactor-mature peptide gene order.
The recombinant expression vector that the bolti transforminggrowthfactor-gene that the above-mentioned coli expression carrier that contains bolti transforminggrowthfactor-mature peptide gene order is preferably synthesized by the present invention and coli expression carrier PQE30 are built into, called after PQE30-TGF-β 1.
The carrier that sets out of above-mentioned expression vector can be general expression vector, can select as required.For prokaryotic expression system, the PQE30 carrier has the following advantages: at first the PQE30 zero load has only 3.4kb, makes things convenient for plasmid construction; Secondly, recombinant protein output height has the Histidine purification tag, makes things convenient for the purifying of recombinant protein; Again, the PQE30 carrier does not have molecular chaperones, has reduced the step of follow-up removal molecular chaperones, has simplified purge process.
A kind of recombinant strain contains bolti transforming growth factor TGF-β 1 mature peptide gene as mentioned above.The present invention utilizes the above-mentioned corresponding expression vectors that contains bolti transforminggrowthfactor-mature peptide gene order to make up the intestinal bacteria recombinant strain that can efficiently express bolti transforminggrowthfactor-mature peptide.The above-mentioned intestinal bacteria recombinant bacterial strain that can express bolti transforminggrowthfactor-mature peptide of the present invention is preferably by containing that bolti transforminggrowthfactor-mature peptide expression carrier PQE30-TGF-β 1 is converted into intestinal bacteria M15 and the bacterial strain that obtains, called after PQE30-TGF-β 1-M15.
A kind of preparation method of bolti transforming growth factor TGF-β 1 mature peptide comprises the steps:
S1. recombinant expression vector changes in the intestinal bacteria as mentioned above, utilizes ampicillin resistance gene screening positive transformant;
S2. positive transformant is seeded in the liquid nutrient medium that contains penbritin and kantlex, overnight incubation, be inoculated in the same medium next day, and induce through IPTG, cultivate the back and collect thalline, obtain bolti transforminggrowthfactor-mature peptide through sex change and renaturation separation and purification.
The concrete preparation method of described bolti transforming growth factor TGF-β 1 mature peptide is as follows:
S1. recombinant expression vector changes in the intestinal bacteria as mentioned above, utilizes ampicillin resistance gene screening positive transformant;
S2. positive transformant is seeded in the LB liquid nutrient medium that contains penbritin and kantlex, 37 ℃, 200 rev/mins of overnight incubation, be inoculated in next day in the same medium, and induce through IPTG, 30 ℃, 200 rev/mins of cultivations, collect thalline after 6 hours, through the separation and purification bolti transforminggrowthfactor-mature peptide product that obtains recombinating.
A kind of bolti transforming growth factor TGF-β 1 polyclonal antibody, be by behind the bolti transforming growth factor TGF-β 1 mature peptide immune mouse as mentioned above, the polyclonal antibody that obtains, called after mouse-anti bolti transforminggrowthfactor-polyclonal antibody.
The application of bolti transforming growth factor TGF-β 1 polyclonal antibody for preparing in the preparation immunological reagent.
Bolti has great economic worth, adopt conventional genetic engineering technique, can utilize bolti transforminggrowthfactor-gene of the present invention and recombinant strain PQE30-TGF-β 1-M15, produce reorganization bolti transforminggrowthfactor-mature peptide, and the preparation polyclonal antibody, and can further be used as immunological reagent and scientific research.The transforminggrowthfactor-mature peptide can be used as the immunostimulant of fodder additives or injection, transfer body reply pathogen infection, the polyclonal antibody of preparation can be used for the theoretical investigation about the bolti transforming growth factor, as western-blot and immunohistochemical experiment.
Compared with prior art, the present invention has following beneficial effect:
The present invention utilizes the method for prokaryotic expression to obtain reorganization bolti transforminggrowthfactor-mature peptide first.Method by immune mouse has prepared bolti transforminggrowthfactor-polyclonal antibody, and this is the polyclonal antibody that obtains the fish transforminggrowthfactor-first.
Description of drawings
Fig. 1. long segment pcr amplification product gel electrophoresis analysis figure in the middle of the bolti transforminggrowthfactor-gene cDNA; M. DNA molecular weight standard 100bp ladder; 1. pcr amplification product.
Fig. 2. the terminal pcr amplification product gel electrophoresis analysis of bolti transforminggrowthfactor-cDNA3 ' figure; M. DNA molecular weight standard DS2000; 1. pcr amplification product.
Fig. 3. bolti transforminggrowthfactor-cDNA 5 '-RACE pcr amplification product gel electrophoresis analysis figure; M. DNA molecular weight standard DS2000; 1st, 3 swimming lanes are two primer amplification results, and the 2nd, 4 swimming lanes are single primer contrast.
Fig. 4. bolti transforminggrowthfactor-cDNA total length checking pcr amplification product gel electrophoresis analysis figure; M. DNA molecular weight standard DS2000; 1. pcr amplification product.
Fig. 5. bolti transforminggrowthfactor-cDNA total length and deduced amino acid figure; Underscore is expressed as signal peptide sequence, and the square frame place represents the mature peptide cut label, and dash area is the mature peptide sequence.
Fig. 6. bolti transforminggrowthfactor-mature peptide cDNA sequence PCR amplified production gel electrophoresis analysis figure; M. dna molecular amount standard DS2000; 1. pcr amplification product.
Fig. 7. the building process synoptic diagram of recombinant expression vector PQE30-TGF-β 1.
Fig. 8. the SDS-PAGE gel electrophoresis of reorganization bolti transforminggrowthfactor-mature peptide expression product; M. molecular weight of albumen standard; 1. supernatant; 2. precipitate.
Fig. 9. the SDS-PAGE gel electrophoresis behind the reorganization bolti transforminggrowthfactor-mature peptide expression product purifying; M. molecular weight of albumen standard; 1. purifying after product.
Figure 10. the specificity analyses that the bolti transforminggrowthfactor-resists more; 1. bolti head-kidney lysate; 2. Epinephelus coioide head-kidney lysate; 3. reorganization bolti transforminggrowthfactor-mature peptide.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is made further elaborating, but embodiment does not do any type of restriction to the present invention.
Synthesizing of embodiment 1 bolti transforminggrowthfactor-gene intermediate segment
Homology comparative result according to the cDNA sequence of the transforminggrowthfactor-s of the fish of having delivered, the synthetic cover degenerated primer of design, have a upstream primer F1:5 '-GARGCBATTMGRRGHCAGAT-3 ' (shown in the SEQ ID NO:7) and a downstream primer R1:5 '-GCMGAKGCWCCDGGRTTGTG-3 ' (shown in the SEQ ID NO:8), wherein R=A/G; B=C/G/T; M=A/C; H=A/T/C; K=G/T; W=A/T; D=A/T/G.The cDNA that obtains with the total RNA reverse transcription of bolti spleen is template, and through PCR method amplification, reaction conditions is: 94 ℃ of pre-sex change 3 minutes; Bottom is 40 circulations, is divided into two stages: (1) 94 ℃ of sex change 30 seconds, from 65 ℃ to 0.5 ℃ of 50 ℃ of each cycle down, annealed 30 seconds, totally 30 circulations, 72 ℃ were extended 1 minute; (2) 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, totally 10 circulations, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 10 minutes.
The electrophoresis evaluation figure of pcr amplification product sees Fig. 1.From the visible pcr amplification for the first time of Fig. 1 the sequence of the about 920bp of one segment length.
The amplified production of 920bp is connected to obtains recombinant plasmid pTZ57R/T-TGF-β 1-M on the pTZ57R/T carrier.Recombinant plasmid pTZ57R/T-TGF-β 1-M is carried out sequencing with a pair of universal primer of pTZ57R/T, the sequence of sequencing result and Genebank compares, and the result shows that clone's PCR product is the intermediate segment of bolti transforminggrowthfactor-gene.
Embodiment 2: bolti transforminggrowthfactor-gene 3 ' end fragment synthetic
According to intermediate segment design upstream primer the F2:5 '-TTTACCATGTCCATCCCTA-3 ' (shown in the SEQ ID NO:9) that has obtained, comparison according to existing fish transforminggrowthfactor-cDNA is found very conservative at the terminator codon place, so at this design specificity downstream primer R2:5 '-TTAGCTACACTTGCAGGACTT-3 ' (shown in the SEQ ID NO:10).The cDNA that obtains with the total RNA reverse transcription of bolti spleen is template, and through PCR method amplification, reaction conditions is: 94 ℃ of pre-sex change 3 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, totally 30 circulations; 72 ℃ were extended 10 minutes.
The electrophoresis evaluation figure of pcr amplification product sees Fig. 2.From the visible pcr amplification of Fig. 2 the sequence of the about 393bp of one segment length.
The amplified production of 303bp is connected to obtains recombinant plasmid pTZ57R/T-TGF-β 1-3 ' on the pTZ57R/T carrier.Recombinant plasmid pTZ57R/T-TGF-β 1-3 ' is carried out sequencing with a pair of universal primer of pTZ57R/T.
Embodiment 3: bolti transforminggrowthfactor-gene 5 ' end fragment synthetic
According to the sequences Design of the transforminggrowthfactor-intermediate segment of being cloned into three downstream primer 5 '-R1:5 '-GTTGAACCTGTTAAGCACCT-3 ' of 5 ' RACE (shown in the SEQ ID NO:11), 5 '-R2:5 '-TGCTCCTCATCCTGCTTCT-3 ' (shown in the SEQ ID NO:12) and 5 '-R3:5 '-CTGGTGCTGTTGTAGAGTGATAG-3 ' (shown in the SEQ ID NO:13) to carry out nested PCR.PCR is template with the product of the total cDNA of bolti spleen behind the TdT tailing for the first time, 5 '-R1 and universal primer 5 '-CDS:5 '-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3 ' (shown in the SEQ ID NO:14) is primer, through touchdown PCR method amplification, reaction conditions is: 94 ℃ of pre-sex change 3 minutes; Bottom is 40 circulations, is divided into two stages: (1) 94 ℃ of sex change 20 seconds, from 60 ℃ to 0.5 ℃ of 50 ℃ of each cycle down, annealed 20 seconds, totally 20 circulations, 72 ℃ were extended 1 minute; (2) 94 ℃ of sex change 20 seconds, 50 ℃ of annealing 20 seconds, totally 20 circulations, 72 ℃ were extended 1 minute; Last 72 ℃ were extended 10 minutes.PCR is template for 10 times with the PCR product dilution first time for the second time, with NUPA:5 '-AAGCAGTGGTATCAACGCAGAGT-3 ' (shown in the SEQ ID NO:15): be upstream primer, be downstream primer with 5 '-R2 and 5 '-R3 respectively, through PCR method amplification, reaction conditions is: 94 ℃ of pre-sex change 3 minutes; Bottom is 40 circulations: 94 ℃ of sex change 20 seconds, and 61 ℃ to 0.3 ℃ of 55 ℃ of each cycle down, and 72 ℃ were extended 1 minute; Last 72 ℃ were extended 10 minutes.
The electrophoresis evaluation figure of secondary PCR amplified production sees Fig. 3.
Amplified production is connected on the pTZ57R/T carrier obtains recombinant plasmid.Recombinant plasmid is carried out sequencing with a pair of universal primer of pTZ57R/T, and the sequence of sequencing result and Genebank compares, and the result shows that clone's product is bolti transforminggrowthfactor-gene 5 ' terminal sequence.
Embodiment 4: the splicing of bolti transforminggrowthfactor-cDNA full length sequence and checking
The transforminggrowthfactor-intermediate segment, 5 ' RACE fragment and the 3 ' fragment that have checked order are spliced, primer ORF-F:5 '-GATTCACTGTTCCCAAAGG-3 ' (shown in the SEQ ID NO:5) and ORF-R:5 '-TTAGCTACACTTGCAGGACTT-3 ' (shown in the SEQ ID NO:6) according to the sequences Design total length of splicing is verified carry out the sequence that PCR obtains 1258bp with the KOD enzyme of hi-fi.The ORF total length that obtains the cDNA of bolti transforminggrowthfactor-gene through finishing analysis is 1143bp, and sequence is shown in SEQ ID NO:1.And infer and its aminoacid sequence that sequence is shown in SEQ ID NO:3.
The electrophoresis evaluation figure of pcr amplification product sees Fig. 4.From the visible pcr amplification of Fig. 4 the sequence of the about 1258bp of one segment length.Primer herein is that the two ends outside ORF are designed, thus longer than ORF, also be in order to guarantee the exactness of whole ORF.
Bolti transforminggrowthfactor-cDNA full length sequence and aminoacid sequence are seen Fig. 5.
Amplified production is connected to obtains recombinant plasmid pTZ57R/T-TGF-β 1-ORF on the pTZ57R/T carrier.
Embodiment 5: the corresponding sequence of bolti transforminggrowthfactor-mature peptide synthetic
The bolti transforminggrowthfactor-gene cDNA complete sequence good according to splicing, find the sequence of mature peptide correspondence, two sections primers are synthesized in design, add restriction site F:5 '-CGCGGATCCTCCACTGACACAACGGACAG-3 ' (shown in the SEQ ID NO:16) of Sph I before the upstream primer; Add restriction site and terminator codon the R:5 '-CCCAAGCTTTTAGCTACACTTGCAGGACT-3 ' (shown in the SEQ ID NO:17) of Hind III before the downstream primer.Verify that with bolti transforminggrowthfactor-total length recombinant plasmid dna is template, be the sequence of bolti transforminggrowthfactor-mature peptide correspondence through the 270th of PCR method amplification bolti transforminggrowthfactor-precursor to the corresponding cDNA sequence of the 381st amino acids sequence, bolti transforminggrowthfactor-mature peptide sequence is shown in SEQ ID NO:4, and bolti transforminggrowthfactor-mature peptide gene order is shown in SEQ ID NO:2.The PCR reaction conditions is: 94 ℃ of pre-sex change 3 minutes; Bottom is 40 circulations: 94 ℃ of sex change 30 seconds, and 60 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds; Last 72 ℃ were extended 10 minutes.
The electrophoresis evaluation figure of pcr amplification product sees Fig. 6.From the visible pcr amplification of Fig. 6 the sequence of the about 351bp of one segment length.
Amplified production is connected to obtains recombinant plasmid pTZ57R/T-TGF-β 1 on the pTZ57R/T carrier.
Embodiment 6: the structure that contains the coli expression carrier PQE30-TGF-β 1 of bolti transforminggrowthfactor-mature peptide cNDA sequence
The unloaded plasmid of plasmid pTZ57R/T-TGF-β 1-E and PQE is behind restriction enzyme Sph I and Hind III double digestion, carry out agarose gel electrophoresis, the big fragment that the bolti transforminggrowthfactor-mature peptide cDNA sequence of the about 340bp of separation and purification and PQE carrier enzyme are cut, product reclaims with E.Z.N.A. Gel Extraction Kit.The bolti transforminggrowthfactor-mature peptide cDNA fragment of 340bp is mixed by 1:3 with carrier segments, after connecting 16 hours with 4 ℃ of T4 ligase enzymes, change among the intestinal bacteria M15 with the standard calcium chloride transformation, the transformant of the two resistances of screening tool penbritin and kantlex, extract plasmid with standard method, with the universal primer order-checking of recombinant plasmid with PQE30, inserted sieve tilapia transforminggrowthfactor-mature peptide cDNA sequence in the proof plasmid, and entirely true, recombinant plasmid called after PQE30-TGF-β 1.Plasmid construction figure sees Fig. 7.
Embodiment 7: the structure that can efficiently express the intestinal bacteria recombinant strain PQE30-TGF-β 1-M15 of bolti transforminggrowthfactor-mature peptide
CaCl
2Method is with PQE30-TGF-β 1 Transformed E. coli M15, screen transformant at the LB flat board that contains penbritin and kantlex, and detect and the restriction analysis acquisition contains the sub-PQE30-TGF-β of the recombinant conversion 1-M15 of PQE30-TGF-β 1 through plasmid.
Embodiment 8: utilize bacillus coli gene engineering bacteria PQE30-TGF-β 1-M15 to produce reorganization bolti transforminggrowthfactor-mature peptide
The single colony inoculation of picking bacillus coli gene engineering bacteria PQE30-TGF-β 1-M15 is in containing the LB liquid nutrient medium of 100ug/ml penbritin and kantlex, 37 ℃, 200 rev/mins of overnight incubation, be inoculated in the same medium of proper volume next day by the 1:50 inoculum size, 37 ℃ are cultured to A600 is 0.4～0.6, adding the IPTG(final concentration is 0.5mmol/l), receive bacterium in 30 ℃ of inducing culture after 6 hours.Thalline goes cleer and peaceful precipitation to add 2 * electrophoresis sample-loading buffer respectively after ultrasonication, boils after 5 minutes and runs the SDS-PAGE gel electrophoresis by standard method.The result shows that synthetic bolti transforminggrowthfactor-mature peptide has obtained to efficiently express in bacillus coli gene engineering bacteria PQE30-TGF-β 1-M15, but recombinant protein is mainly in precipitation.See Fig. 8.
Behind the enlarged culturing abduction delivering, collect thalline, resuspended with lysate, shaking table concussion cracking thalline, high speed centrifugation obtains the cracking supernatant liquor, through immobilization metal part affinitive layer purification, collect the albumen of wash-out, SDS-PAGE analyzes Recombinant Protein Expression and purification result.Can draw from SDS-PAGE result: it is adsorbed that the ripe Toplink of 6 * His-tilapia transforminggrowthfactor-is immobilized nickel metal affinity chromatography post, when washing the nickel chromatography column with the elution buffer of different pH, can wash target protein, after the dialysis renaturation, obtain tilapia transforminggrowthfactor-mature peptide.See Fig. 9.
Embodiment 9: bolti transforminggrowthfactor-mature peptide Polyclonal Antibody Preparation
Immune mouse: the bolti transforminggrowthfactor-mature peptide albumen 2mg/mL with purifying with equal-volume Freund's complete adjuvant mixing, forms water in oil emulsification; Back of the body subcutaneous abdomen multiple spot immunity Kunming mouse, 0.2mL/ is only; At interval two weeks, use Freund's incomplete adjuvant instead with same dosage and mode booster immunization mouse, so repeat immunity 3 times.
Gather mouse resisting anteserum: behind the 3rd the booster immunization 7d, blood is got in the mouse docking; Behind 37 ℃ of placements of serum, 30 ~ 60min, place more than the 4h again for 4 ℃; Last 4 ℃ of 1500r/min, centrifugal 10min collects serum.10d after the 4th immunity, mouse is plucked the eyeball blood sampling, separation of serum, method is the same.
Indirect ELISA is measured the antibody titer of serum: the tilapia head-kidney organizes lysate to be diluted to 1 μ g/mL with 0.05mM pH 9.6 carbonate buffer solutions, 100 μ L/ holes, and 37 ℃ of bags are by 3h, and PBS-T washs 3 times, 3min/ time; 5% skim-milk, the 37 ℃ of sealings in 200 μ L/ holes 1h; PBS-T washing 3 times; Add test serum and negative control sera, the design blank is hatched 1.5 h for 37 ℃, PBS-T washing 3 times; Add HRP mark sheep anti-mouse igg antibody (1:8000), hatch 1 h for 37 ℃, PBS-T washing 5 times; Add the tmb substrate colour developing, 37 ℃ of reaction 10min; 2M H
2SO
450 μ L/ hole termination reactions are measured the OD value with wavelength 450 nm on the inherent microplate reader of 15min.OD value under the different extension rates sees Table 1, with the OD under the different extension rates
450After value is drawn, observing the variation tendency of curve, should be a S type curve, and mid point value is tired for it.
Show that by the result of table 1 polyclonal antibody of preparation tires about 1:4000.
Table 1 mouse-anti fish IgG antibody titer measurement result
| Extension rate | OD |
| 2000 | 2.631 |
| 4000 | 1.574 |
| 8000 | 1.128 |
| 16000 | 0.743 |
| 32000 | 0.548 |
| 64000 | 0.312 |
| Blank | 0.123 |
Embodiment 10: the application of bolti transforminggrowthfactor-mature peptide polyclonal antibody
Extract tilapia head-kidney lysate, cabrilla head-kidney lysate, reorganization tilapia TGF-β 1 purifying protein with two kinds of lysates and purifying carries out the SDS-PAGE electrophoresis respectively, be primary antibodie with the bolti transforminggrowthfactor-mature peptide polyclonal antibody that obtains, be the two anti-western-blot that carry out with sheep anti-mouse igg, detect the effect of antibody.See Figure 10.
SEQUENCE LISTING
＜110〉Zhongshan University
＜120〉a kind of bolti transforming growth factor TGF-β 1 gene, associated protein and application
<130>
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 1146
<212> DNA
＜213〉bolti transforminggrowthfactor-full length gene cDNA sequence
<400> 1
atgaaattgt ggttcttgat attgatggtc gtgtacatgg tgggcagtgt acataatctg 60
tctacatgta acactgtgga cttggagatg gtgaagaaaa aacgcattga ggccattagg 120
agccagatcc tcaccaaact gcgtttgcaa aaagcgccag atgaggctgg ggagaaggaa 180
gagatccctg ccaacttgct atcactctac aacagcacca gtgagattct gctggagaag 240
caggatgagg agcagaaaat catccccaga gaacaagagg aggaggaata ctttgccaag 300
gtgcttaaca ggttcaacat gaccacaaaa aatgacacaa acatcaacta caagcccaaa 360
gtcatctcaa tgagcttcaa catctctgag atacggagta acgtcgggga tggcctgcta 420
ctcaccagag cagagctacg gatgctcatc aaggagccca tgattttgaa tgaggagaga 480
gtggagctat accatagcca ggggacctcg actcattacc tggcatctcg ctttgtcaca 540
aacacgctga aggacaaatg gctgtccttt ggtgtcactg aacctctgca gacctggctc 600
caagggaatg agaatgaaca gaagtttgaa cttcggcggt actgtgaatg cggcaataac 660
gatgatacgt taagttttag catctctggg actatgagca ggaggggaga cactaaagac 720
ttacagaagc tgaaccagca gctaccctac atctttacca tgtccatccc taaaaccaac 780
cactctaaga gtttacgcac aaaacgttcc actgacacaa cggacagctg cggcacccaa 840
tcacacaact gctgtttgaa gaaactgtac attgacttca ggaaagatct aggatggaag 900
tggatccata agccaacggg ttactatgct aactactgca tggggtcctg cacctatatc 960
tgggatgccg aaaacaaata ttctcagatt ctggcactgt acaaacatca taacccagga 1020
gcttctgccc agccctgctg tgcgccacag acactagagc cactgccaat catctactat 1080
gtggggaggc aacacaaggt cgagcagctg tccaatatga tcgtgaagtc ctgcaagtgt 1140
agctaa 1146
<210> 2
<211> 339
<212> DNA
＜213〉bolti transforminggrowthfactor-mature peptide gene order
<400> 2
tccactgaca caacggacag ctgcggcacc caatcacaca actgctgttt gaagaaactg 60
tacattgact tcaggaaaga tctaggatgg aagtggatcc ataagccaac gggttactat 120
gctaactact gcatggggtc ctgcacctat atctgggatg ccgaaaacaa atattctcag 180
attctggcac tgtacaaaca tcataaccca ggagcttctg cccagccctg ctgtgcgcca 240
cagacactag agccactgcc aatcatctac tatgtgggga ggcaacacaa ggtcgagcag 300
ctgtccaata tgatcgtgaa gtcctgcaag tgtagctaa 339
<210> 3
<211> 381
<212> PRT
＜213〉bolti transforminggrowthfactor-full length gene aminoacid sequence
<400> 3
Met Lys Leu Trp Phe Leu Ile Leu Met Val Val Tyr Met Val Gly Ser
1 5 10 15
Val His Asn Leu Ser Thr Cys Asn Thr Val Asp Leu Glu Met Val Lys
20 25 30
Lys Lys Arg Ile Glu Ala Ile Arg Ser Gln Ile Leu Thr Lys Leu Arg
35 40 45
Leu Gln Lys Ala Pro Asp Glu Ala Gly Glu Lys Glu Glu Ile Pro Ala
50 55 60
Asn Leu Leu Ser Leu Tyr Asn Ser Thr Ser Glu Ile Leu Leu Glu Lys
65 70 75 80
Gln Asp Glu Glu Gln Lys Ile Ile Pro Arg Glu Gln Glu Glu Glu Glu
85 90 95
Tyr Phe Ala Lys Val Leu Asn Arg Phe Asn Met Thr Thr Lys Asn Asp
100 105 110
Thr Asn Ile Asn Tyr Lys Pro Lys Val Ile Ser Met Ser Phe Asn Ile
115 120 125
Ser Glu Ile Arg Ser Asn Val Gly Asp Gly Leu Leu Leu Thr Arg Ala
130 135 140
Glu Leu Arg Met Leu Ile Lys Glu Pro Met Ile Leu Asn Glu Glu Arg
145 150 155 160
Val Glu Leu Tyr His Ser Gln Gly Thr Ser Thr His Tyr Leu Ala Ser
165 170 175
Arg Phe Val Thr Asn Thr Leu Lys Asp Lys Trp Leu Ser Phe Gly Val
180 185 190
Thr Glu Pro Leu Gln Thr Trp Leu Gln Gly Asn Glu Asn Glu Gln Lys
195 200 205
Phe Glu Leu Arg Arg Tyr Cys Glu Cys Gly Asn Asn Asp Asp Thr Leu
210 215 220
Ser Phe Ser Ile Ser Gly Thr Met Ser Arg Arg Gly Asp Thr Lys Asp
225 230 235 240
Leu Gln Lys Leu Asn Gln Gln Leu Pro Tyr Ile Phe Thr Met Ser Ile
245 250 255
Pro Lys Thr Asn His Ser Lys Ser Leu Arg Thr Lys Arg Ser Thr Asp
260 265 270
Thr Thr Asp Ser Cys Gly Thr Gln Ser His Asn Cys Cys Leu Lys Lys
275 280 285
Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Lys
290 295 300
Pro Thr Gly Tyr Tyr Ala Asn Tyr Cys Met Gly Ser Cys Thr Tyr Ile
305 310 315 320
Trp Asp Ala Glu Asn Lys Tyr Ser Gln Ile Leu Ala Leu Tyr Lys His
325 330 335
His Asn Pro Gly Ala Ser Ala Gln Pro Cys Cys Ala Pro Gln Thr Leu
340 345 350
Glu Pro Leu Pro Ile Ile Tyr Tyr Val Gly Arg Gln His Lys Val Glu
355 360 365
Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys Ser
370 375 380
<210> 4
<211> 112
<212> PRT
＜213〉bolti transforminggrowthfactor-mature peptide aminoacid sequence
<400> 4
Ser Thr Asp Thr Thr Asp Ser Cys Gly Thr Gln Ser His Asn Cys Cys
1 5 10 15
Leu Lys Lys Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp
20 25 30
Ile His Lys Pro Thr Gly Tyr Tyr Ala Asn Tyr Cys Met Gly Ser Cys
35 40 45
Thr Tyr Ile Trp Asp Ala Glu Asn Lys Tyr Ser Gln Ile Leu Ala Leu
50 55 60
Tyr Lys His His Asn Pro Gly Ala Ser Ala Gln Pro Cys Cys Ala Pro
65 70 75 80
Gln Thr Leu Glu Pro Leu Pro Ile Ile Tyr Tyr Val Gly Arg Gln His
85 90 95
Lys Val Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys Ser
100 105 110
<210> 5
<211> 19
<212> DNA
<213> ORF-F
<400> 5
gattcactgt tcccaaagg 19
<210> 6
<211> 21
<212> DNA
<213> ORF-R
<400> 6
ttagctacac ttgcaggact t 21
<210> 7
<211> 20
<212> DNA
<213> F1
<400> 7
gargcbattm grrghcagat 20
<210> 8
<211> 20
<212> DNA
<213> R1
<400> 8
gcmgakgcwc cdggrttgtg 20
<210> 9
<211> 19
<212> DNA
<213> F2
<400> 9
tttaccatgt ccatcccta 19
<210> 10
<211> 21
<212> DNA
<213> R2
<400> 10
ttagctacac ttgcaggact t 21
<210> 11
<211> 20
<212> DNA
<213> 5’-R1
<400> 11
gttgaacctg ttaagcacct 20
<210> 12
<211> 19
<212> DNA
<213> 5’-R2
<400> 12
tgctcctcat cctgcttct 19
<210> 13
<211> 23
<212> DNA
<213> 5’-R3
<400> 13
ctggtgctgt tgtagagtga tag 23
<210> 14
<211> 30
<212> DNA
<213> 5’-CDS
<400> 14
aagcagtggt atcaacgcag agtacgcggg 30
<210> 15
<211> 23
<212> DNA
<213> NUPA
<400> 15
aagcagtggt atcaacgcag agt 23
<210> 16
<211> 29
<212> DNA
<213> F
<400> 16
cgcggatcct ccactgacac aacggacag 29
<210> 17
<211> 29
<212> DNA
<213> R
<400> 17
cccaagcttt tagctacact tgcaggact 29
Claims (10)
1. bolti transforming growth factor TGF-β 1 gene is characterized in that nucleotide sequence is shown in SEQ ID NO:1.
2. bolti transforming growth factor TGF-β 1 a mature peptide gene is characterized in that nucleotide sequence is shown in SEQ ID NO:2.
3. a bolti transforming growth factor TGF-β 1 is characterized in that aminoacid sequence is shown in SEQ ID NO:3.
4. bolti transforming growth factor TGF-β 1 mature peptide is characterized in that aminoacid sequence is shown in SEQ ID NO:4.
5. a pair of primer for amplification bolti transforming growth factor TGF-β 1 gene is characterized in that primer sequence is shown in SEQ ID NO5 ~ 6.
6. a recombinant expression vector is characterized in that, inserts the described bolti transforming growth factor of claim 2 TGF-β 1 mature peptide gene by the multiple clone site of the carrier that sets out.
7. according to the described recombinant expression vector of claim 6, it is characterized in that described recombinant expression vector is PQE30-TGF-β 1.
8. a recombinant strain is characterized in that, is to change gained in the intestinal bacteria over to by claim 6 or 7 described expression vectors.
9. the preparation method of bolti transforming growth factor TGF-β 1 mature peptide, it is characterized in that, concrete grammar is for to be seeded in the described recombinant strain of claim 8 in the liquid nutrient medium that contains penbritin and kantlex, overnight incubation, be inoculated in next day in the same medium, and induce through IPTG, cultivate the back and collect thalline, obtain bolti transforminggrowthfactor-mature peptide through sex change and renaturation separation and purification.
10. bolti transforming growth factor TGF-β 1 polyclonal antibody is characterized in that, is prepared by the described bolti transforming growth factor of claim 2 TGF-β 1 mature peptide immune mouse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310183812XA CN103233013A (en) | 2013-05-17 | 2013-05-17 | Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310183812XA CN103233013A (en) | 2013-05-17 | 2013-05-17 | Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103233013A true CN103233013A (en) | 2013-08-07 |
Family
ID=48881079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310183812XA Pending CN103233013A (en) | 2013-05-17 | 2013-05-17 | Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103233013A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642812A (en) * | 2013-09-24 | 2014-03-19 | 中山大学 | Nile tilapia apoptosis factor FasL gene and applications thereof |
| CN106754941A (en) * | 2016-12-01 | 2017-05-31 | 中国水产科学研究院珠江水产研究所 | The preparation method of bolti Ly75 gene clonings, prokaryotic expression and anti-Tilapia mossambica OnLy75 polyclonal antibodies |
| CN107759663A (en) * | 2017-11-02 | 2018-03-06 | 中国海洋大学 | A kind of method for differentiating bolti collagen |
| CN114213522A (en) * | 2021-12-29 | 2022-03-22 | 福建农林大学 | Large yellow croaker TGF-beta 1 recombinant protein and application thereof |
-
2013
- 2013-05-17 CN CN201310183812XA patent/CN103233013A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| DI PALMA F.等: "Accession number: I3JEX4", 《EMBL-EBI》 * |
| GURNEET KOHLI等: "Cloning of Transforming Growth Factor-β1 (TGF-β1) and Its Type II Receptor from Zebrafish Ovary and Role of TGF-β1 in Oocyte Maturation", 《ENDOCRINOLOGY》 * |
| 平海林 等: "斜带石斑鱼转化生长因子β1(TGF-β1)基因的克隆及表达分析", 《中山大学学报(自然科学版)》 * |
| 杨牧: "草鱼转化生长因子β1的免疫调节功能及其作用机制", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642812A (en) * | 2013-09-24 | 2014-03-19 | 中山大学 | Nile tilapia apoptosis factor FasL gene and applications thereof |
| CN103642812B (en) * | 2013-09-24 | 2016-01-20 | 中山大学 | A kind of bolti antiapoptotic factors FasL gene and application thereof |
| CN106754941A (en) * | 2016-12-01 | 2017-05-31 | 中国水产科学研究院珠江水产研究所 | The preparation method of bolti Ly75 gene clonings, prokaryotic expression and anti-Tilapia mossambica OnLy75 polyclonal antibodies |
| CN107759663A (en) * | 2017-11-02 | 2018-03-06 | 中国海洋大学 | A kind of method for differentiating bolti collagen |
| CN107759663B (en) * | 2017-11-02 | 2023-06-30 | 中国海洋大学 | Method for identifying nile tilapia skin collagen |
| CN114213522A (en) * | 2021-12-29 | 2022-03-22 | 福建农林大学 | Large yellow croaker TGF-beta 1 recombinant protein and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107033250B (en) | Bovine coronavirus recombinant multi-epitope antigen and application thereof | |
| CN101624422B (en) | Schistosoma japonicum recombinant multi-epitope antigens, method for expressing and purifying same and application thereof | |
| CN111925432B (en) | Preparation method of cardiac troponin I recombinant antigen and monoclonal antibody thereof | |
| CN103233013A (en) | Nile tilapia transforming growth factor TGF-beta 1 gene, related protein and application | |
| CN102702323B (en) | Application of procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof | |
| CN109207502B (en) | Porcine mycoplasma pneumonia and porcine circovirus type 2 recombinant protein and preparation of bivalent vaccine | |
| CN103724413B (en) | Trichina paramyosin B cell antigen epi-position 8A1 and application thereof | |
| CN102443053A (en) | Application of using streptococcus suis type-2 hy0245 gene encoded proteins as protective antigens | |
| CN102702324A (en) | Application of human procalcitonin B cell epitope peptide fragment and monoclonal antibody thereof | |
| CN103193887A (en) | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein | |
| CN102676568A (en) | Method for producing recombinant dermatophagoides farinae allergen Der f1 and Der f2 fusion protein | |
| CN103880953B (en) | One boar P21 protein antibodies and preparation method thereof and application | |
| CN107505468B (en) | It is a kind of for detecting the detection reagent and its application of Human interleukin-10 | |
| Gao et al. | Preparation of a novel monoclonal antibody against caprine interleukin-17A and its applications in immunofluorescence and immunohistochemistry assays | |
| CN102643332A (en) | Application of B cell epitope peptide of human PCT (Procalcitonin) and monoclonal antibody of B cell epitope peptide | |
| CN102772795B (en) | The application of brucella flagellin BMEII1112 in preparation brucella subunit vaccine | |
| CN113754782A (en) | Helicobacter pylori egg yolk antibody and preparation method and application thereof | |
| CN107362358B (en) | Application of IL-17A protein in preparation of animal vaccine adjuvant | |
| CN108017694B (en) | PORF65 recombinant protein and its preparation method and application | |
| CN107469076B (en) | Application of IL-2 protein in preparation of animal vaccine adjuvant | |
| CN101298611B (en) | Active fragment of chlamydia proteasome sample active factor and expression method thereof | |
| CN105085638A (en) | KSHV virus vIRF4 DNA binding domain and polyclonal antibody thereof, and preparation method of polyclonal antibody | |
| CN103450358A (en) | Swine GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein antibody and preparation method and application thereof | |
| CN110257390A (en) | Stichopus japonicus Bax gene, coding albumen and its cloning process, recombination stichopus japonicus Bax construction of genetic engineering methods and applications | |
| CN105713096A (en) | Preparation and application of ATT fusion protein for preventing infection of staphylococcus aureus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130807 |