CN103045623A - Preparation method of tilapia activin receptor IIB recombinant protein and application thereof - Google Patents
Preparation method of tilapia activin receptor IIB recombinant protein and application thereof Download PDFInfo
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- CN103045623A CN103045623A CN2012105608495A CN201210560849A CN103045623A CN 103045623 A CN103045623 A CN 103045623A CN 2012105608495 A CN2012105608495 A CN 2012105608495A CN 201210560849 A CN201210560849 A CN 201210560849A CN 103045623 A CN103045623 A CN 103045623A
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- tilapia
- activin receptor
- receptor iib
- actriib
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Abstract
The invention discloses a tilapia activin receptor IIB extracellular domain gene fragment. The tilapia activin receptor IIB extracellular domain gene fragment is prepared according to the following steps: taking tilapia muscular total RNA as a template, processing the tilapia muscular total RNA by RT-PCR (reverse transcription-polymerase chain reaction) and RACE (rapid-amplification of cDNA ends) methods to obtain a tilapia activin receptor IIB gene, and cloning the tilapia activin receptor IIB gene into a vector pCR2.1 to obtain a cloned vector pCR2.1-ActRIIB; and taking the vector pCR2.1-ActRIIB as a template, designing primers F and R of the tilapia activin receptor IIB extracellular domain gene fragment, and performing PCR reaction to obtain the extracellular domain gene fragment. The invention further discloses an expression vector pQE30-ActRIIB containing the tilapia activin receptor IIB extracellular domain gene fragment. The expression vector pQE30-ActRIIB is guided into escherichia coli and expressed to obtain a tilapia activin receptor IIB recombinant protein; and the tilapia activin receptor IIB recombinant protein can be applied to preparation of a fry growth-promoting agent or additive.
Description
Technical field
The invention belongs to gene engineering technology field, particularly, relate to a kind of preparation method and application thereof of tilapia activin receptor IIB recombinant protein.
Background technology
Activator (activin) belongs to transforming growth factor-beta (TGF-β) superfamily member, be the GDF that a class has several functions, have the effects such as differentiation of regulating pituitary follicular stimulating hormone (FSH), promoting erythron.Activator by with target cell membrane on receptors bind and transmission of signal, it is I type (ActRI) and II type (ActR II) that its acceptor is divided into two types.
Activin receptor ActR(activin receptor) belong to single transmembrane serine/Serineprotein kinase family, by ectodomain, membrane spaning domain and intracellular kinase structural domain three parts form.From the acceptor called after activator II receptor of AtT20 cell clone, in vertebrates, find at present the existence of two kinds of activator II receptors, such as ActRIIA or ACVR2, the second activin receptor ActRIIB perhaps is called ACVR2B.The II receptor need and the I receptor in conjunction with forming the effect of dimer competence exertion, the I receptor is recruited and the SMAD(R-SMAD of phosphorylation receptor modulators), R-SMAD inserts to the effect of bringing into play transcription factor in the nucleus.ActRIIB has multiple ligands, as Myostatin by with its in conjunction with regulating muscle growth.Myostatin belongs to the TGF-beta superfamily member, is the negative regulatory factor of the muscle growth of generally acknowledging.Blocking-up Myostatin-ActRIIB-RSMAD approach is significant to the regulation and control muscle growth.Mouse, ox, in the bony fishes such as the Mammals such as dog and goldfish to this approach regulate and control to receive good clinically with produce upper good effect.
Tilapia (Tilapia) is under the jurisdiction of Perciformes, Percoidei, Callichthyidae, tilapia genus.What cultivated at present has 15 kinds, and growth rapidly, and is especially faster with the young stage growth.China has now become maximum tilapia producing country, and by 2008, China's tilapia output accounted for 49% of global output.Demand to tilapia in the global range is still increasing, and it is the key that solves this situation that tilapia output is increased quickly.
Summary of the invention
Technical problem to be solved by this invention is in order to satisfy the rapid growth of tilapia demand, to disclose a kind of tilapia activin receptor IIB extracellular domain gene fragment.
Another object of the present invention is the extracellular domain that discloses a kind of tilapia activin receptor IIB albumen.
Another object of the present invention is to disclose a kind of tilapia activin receptor IIB gene.
Another object of the present invention is to disclose a kind of tilapia activin receptor IIB albumen.
Another object of the present invention is the primer that discloses amplification tilapia activin receptor IIB gene fragment.
Another object of the present invention is to disclose a kind of carrier that contains tilapia activin receptor IIB extracellular domain gene fragment or tilapia activin receptor IIB gene.
Another object of the present invention is the preparation method who discloses a kind of tilapia activin receptor IIB recombinant protein.
Another object of the present invention is to disclose the application of a kind of tilapia activin receptor IIB recombinant protein in preparation fry seed growth promoter or additive.
To achieve these goals, the present invention is achieved by following technology:
A kind of tilapia activin receptor IIB extracellular domain gene fragment, its nucleotide sequence is shown in SEQ ID NO:1.
A kind of extracellular domain of tilapia activin receptor IIB albumen, the aminoacid sequence of its proteins encoded is shown in SEQ ID NO:2.
A kind of tilapia activin receptor IIB gene, its nucleotide sequence is shown in SEQ ID NO:3.
A kind of tilapia activin receptor IIB albumen, its aminoacid sequence is shown in SEQ ID NO:4.
A pair of primer can be F and R for amplification tilapia activin receptor IIB gene fragment primer, and its primer sequence is shown in SEQ ID NO:5 ~ 6; Perhaps primer is F7 and R7, and its sequence is shown in SEQ ID NO:7 ~ 8; Sequence primers F and R shown in SEQ ID NO:5 ~ 6 are for amplification tilapia activin receptor IIB extracellular domain gene fragment; Sequence shown in SEQ ID NO:7 ~ 8 primers F 7 and R7 for amplification tilapia activin receptor IIB gene.
Further, described primers F contains the KpnI restriction enzyme site, and primer R contains terminator codon and HindIII restriction enzyme site.
A kind of carrier, described carrier comprise the gene fragment of SEQ ID NO:1 or SEQ ID NO:3.For SEQ ID NO:1 fragment, this carrier is escherichia coli cloning carrier pCR2.1-ActRIIB; For SEQ ID NO:3 fragment, carrier is coli expression carrier pQE30-ActRIIB.
Further, the invention provides a kind of recombinant strain, is to change gained in the intestinal bacteria over to by above-mentioned carrier (expression vector) 6.Preferably, be that the expression vector pQE30-ActRIIB of above-mentioned acquisition changes formed intestinal bacteria recombinant strain pQE30-ActRIIB-M15 among the intestinal bacteria M15 over to.
Further, the invention discloses a kind of preparation method of tilapia activin receptor IIB recombinant protein, comprise the steps:
S1. claim 6 or 7 described expression vectors are changed in the intestinal bacteria, utilize ampicillin resistance gene screening positive transformant;
S2. positive transformant is seeded in the liquid nutrient medium that contains penbritin, overnight incubation, be inoculated in the same medium next day, and induce through IPTG, collects thalline after cultivating, through the separation and purification tilapia activin receptor IIB that obtains recombinating.
Preferably, the preparation method is as follows:
S1. described expression vector pQE30-ActRIIB is changed among the intestinal bacteria M15, utilize ampicillin resistance gene screening positive transformant pQE30-ActRIIB-M15;
S2. positive transformant pQE30-ActRIIB-M15 is seeded in the liquid nutrient medium that contains penbritin, overnight incubation, be inoculated in the same medium next day, and induce through IPTG, collect thalline after cultivating, through the separation and purification tilapia activin receptor IIB that obtains recombinating.
More preferably, step S2 is: positive transformant pQE30-ActRIIB-M15 is seeded in the LB liquid nutrient medium that contains penbritin, 37 ℃, 200 rev/mins of overnight incubation, be inoculated in the same medium next day, and induce through IPTG, 37 ℃, 200 rev/mins of cultivations were collected thalline after 10 hours, through the separation and purification tilapia activin receptor IIB that obtains recombinating.
Preferred, the induced concentration of IPTG described in the step S2 is 1mmol/L.
The application of a kind of as mentioned above tilapia activin receptor IIB recombinant protein in preparation fry seed growth promoter or additive.
Amino acid sequence analysis shows that tilapia activin receptor IIB mature peptide is a fragment behind the former excision signal peptide of tilapia activin receptor IIB, both former the 22nd later aminoacid sequences of tilapia activin receptor IIB, the ectodomain of tilapia activin receptor IIB then is equivalent to 23 to 134 amino acids.The ectodomain of the tilapia activin receptor IIB of free form can be competed in conjunction with parts such as Myostatin with membrane-bound acceptor, and then brings into play its growth-promoting activity; Tilapia activin receptor IIB full-length gene of the present invention be the cDNA that obtains take the total RNA reverse transcription of activin receptor IIB tilapia muscle as template, obtain through PCR and the amplification of RACE method.
Generally speaking, the gene order that the present invention is used for expression tilapia ActRIIB extracellular region recombinant protein is as template take tilapia activin receptor IIB full-length gene double-stranded DNA, obtain through the PCR method amplification, it derives from the corresponding gene fragment of tilapia activin receptor IIB ectodomain.
Gene order according to above-mentioned tilapia ActRIIB recombinant protein, it just in time is that tilapia activin receptor IIB full-length gene 67bp to 402bp(is equivalent to 23 to 134 amino acids) the extracellular region section, its nucleotide sequence and coded aminoacid sequence thereof are shown in sequence table.
The present invention has also made up the carrier of the gene order that contains above-mentioned tilapia ActRIIB extracellular region recombinant protein; The escherichia coli cloning carrier and the expression vector that particularly contain this gene.These Vector construction methods are according to a conventional method, will by the synthetic tilapia activin receptor IIB gene of PCR method through enzyme cut with separation and purification after, be linked into the corresponding restriction enzyme site of existing respective carrier (namely
Kpn IWith
HindIII) between, namely be built into the required expression vector that contains tilapia activin receptor IIB gene.
The recombinant expression vector that the tilapia activin receptor IIB gene that the above-mentioned coli expression carrier that contains tilapia activin receptor IIB gene is preferably synthesized by the present invention and coli expression carrier pQE30 are built into, called after pQE30-ActRIIB.
The present invention has also made up respectively the intestinal bacteria recombinant strain that can efficiently express tilapia activin receptor IIB gene with the above-mentioned corresponding expression vectors that contains tilapia activin receptor IIB gene.
Of the present inventionly above-mentionedly can express that tilapia activin receptor IIB intestinal bacteria recombinant bacterial strain preferably transforms intestinal bacteria M15 by the expression vector pQE30-ActRIIB that contains tilapia activin receptor IIB gene and the bacterial strain that obtains, called after pQE30-ActRIIB-M15.
The present invention also provides the method for utilizing above-mentioned intestinal bacteria recombinant strain to produce tilapia activin receptor IIB.Namely just bacterial classification inoculation is cultivated in containing the liquid nutrient medium of penbritin, and induces through IPTG, further cultivates, through the separation and purification tilapia activin receptor IIB product that obtains recombinating.
Tilapia activin receptor IIB has great economic worth, adopt conventional genetic engineering technique, can utilize tilapia activin receptor IIB gene of the present invention and recombinant strain pQE30-ActRIIB-M15, Restruction tilapia activin receptor IIB albumen, and can be further as preparation fry seed growth promoter or additive.
The present invention compared with prior art has the following advantages and effect:
Preparation method by tilapia activin receptor IIB recombinant protein provided by the invention obtain tilapia activin receptor IIB ectodomain can with membrane-bound acceptor competition in conjunction with parts such as Myostatin, and then bring into play its growth-promoting activity, thereby can promote the Fast Growth of tilapia fry.
Description of drawings
Fig. 1. the cDNA that obtains take the total RNA reverse transcription of tilapia muscle is the gel electrophoresis analysis figure of the activin receptor IIB gene intermediate segment pcr amplification product of template; M. molecular weight standard; 1. pcr amplification product.
Fig. 2. the PCR gel electrophoresis analysis figure second time that tilapia activin receptor IIB gene 5 ' end fragment is synthetic; M. molecular weight standard; 1. pcr amplification product.
Fig. 3. the PCR gel electrophoresis analysis figure second time that tilapia activin receptor IIB gene 3 ' end fragment is synthetic; M. molecular weight standard; 1. pcr amplification product.
Fig. 4. the gel electrophoresis analysis figure of the pcr amplification product take tilapia activin receptor IIB cDNA as template (tilapia activin receptor IIB gene ORF); M. molecular weight standard; 1. pcr amplification product.
Fig. 5. the building process synoptic diagram of recombinant expression vector pQE30-ActRIIB.
Fig. 6. the restriction analysis of plasmid pQE30-ActRIIB; M. molecular weight standard; Plasmid:pQE30-ActRIIB/ Kpn I+HindIII enzyme is cut evaluation.
Fig. 7. the SDS-PAGE gel electrophoresis of restructuring tilapia activin receptor IIB gene expression product; M: molecular weight of albumen; 1 is target protein.
Fig. 8. the Western blot of restructuring tilapia activin receptor IIB gene expression product identifies collection of illustrative plates; NC: negative control, 1 is target protein.
Fig. 9. different treatment is on the impact of tilapia body weight gain; Gray line: contrast, green line: ReActRIIB group, red line: antibody group; Control group, the fish in ReActRIIB group and the antibody group carries out respectively abdominal injection PBS, and ReActRIIB and antibody, dosage are the 10mg/kg body weight, process continuously for 4 weeks.Behind second week, the ReActRIIB group, the antibody group significant difference (p＜0.05) occurs with respect to control group, and utmost point significant difference (p＜0.01) appears in 4th week; * represent significant difference, * * represents utmost point significant difference.
Figure 10. different treatment is on the impact of tilapia liver body ratio; Control group, the fish in ReActRIIB group and the antibody group carries out respectively abdominal injection PBS, and ReActRIIB and antibody, dosage are the 10mg/kg body weight, process continuously for 4 weeks.The 4th injection got the full hepatic tissue of tilapia after one week and weighed, and the result shows that liver body ratio does not have significant difference between each treatment group.
Figure 11. the section statining of control group myofiber.
Figure 12. the section statining of reActRIIB treatment group myofiber.
Figure 13. the section statining of antibody treatment group myofiber.
Figure 14. the statistics different treatment is on the impact of tilapia meat fiber; Control group, the fish in ReActRIIB group and the antibody group carries out respectively abdominal injection PBS, and ReActRIIB and antibody, dosage are the 10mg/kg body weight, process continuously for 4 weeks.After the 4th week, get muscle tissue and observe the impact of different treatment tilapia meat fiber through paraffin section and HE dyeing, the result shows that ReActRIIB compares with the control group diameter of muscle fiber with the antibody group and utmost point significant difference (p＜0.01) all occurs, and do not have significant difference (p〉0.05) between ReActRIIB and the antibody group.* represents utmost point significant difference.
Embodiment
Further explain the present invention below in conjunction with drawings and Examples, but embodiment does not do any type of restriction to the present invention.
Synthesizing of tilapia activin receptor IIB gene intermediate segment
Homology comparative result according to the cDNA sequence of the activin receptor IIB of the people who has delivered among the NCBI and various animals, take with the conserved sequence of the activin receptor IIB cDNA of the nearest several fish of tilapia affinity as masterplate, the synthetic two pairs of degenerated primers of design.Wherein the sequence of the upstream primer F1 of first pair of degenerated primer is shown in SEQ ID NO:9.Be 5'-GTAWGWVRKKTTBYKRTGHCKRTA-3', the sequence of downstream primer R1 is shown in SEQ ID NO:10.Be 5'-GCRACDCRGGAGTGYHTBTWCTWYAACG-3'; The sequence of the upstream primer F2 of second pair of degenerated primer is shown in SEQ ID NO:11.Be 5 '-TTBYKRTGHCKRTACAYCCA-3', the sequence of downstream primer R2 is shown in SEQ ID NO:12.Be 5'-AGTGYHTBTWCTWYAACGYAAYTGG-3'.The cDNA that first round PCR obtains take the total RNA reverse transcription of tilapia activin receptor IIB muscle is as template, and the PCR reaction conditions is: 94 ℃ of denaturations 3 minutes.Whenever, take turns in the reaction 94 ℃ of sex change 15 seconds, 60 ℃ of each circulations of annealing 15s(reduce by 0.5 ℃), 72 ℃ are extended and carried out altogether 15 circulations in 20 seconds; Carry out again 94 ℃ of sex change 15 seconds, 52 ℃ of annealing 15s, 72 ℃ were extended 20 seconds, and carried out altogether 13 circulations.Second takes turns PCR take the product of first round PCR as template, through the intermediate segment sequence of two-wheeled PCR method amplification 425bp.Second takes turns reaction conditions is: 94 ℃ of denaturations 3 minutes.Whenever, take turns in the reaction 94 ℃ of sex change 15 seconds, 56 ℃ of annealing 15 seconds, 72 ℃ are extended and carried out altogether 40 circulations in 20 seconds.After the PCR reaction finished, pcr amplification product carried out electrophoresis detection, and its electrophoresis evaluation figure sees Fig. 1.As seen from Figure 1 pcr amplification the dna sequence dna of the about 425bp of one segment length.
The amplified production of 425bp is connected on the pCR2.1 carrier obtains recombinant plasmid.Recombinant plasmid is carried out sequencing, and the sequence of sequencing result and Genebank compares, and the result shows that clone's PCR product is the intermediate segment of tilapia activin receptor IIB gene.
Synthesizing of tilapia activin receptor IIB gene 3 ' end fragment
According to the sequences Design of the ActRIIB intermediate segment that checked order two downstream primer R3 and R4 to carry out nested PCR.The sequence of primer R3 is shown in SEQ ID NO:13; (sequence of R3 primer is 5'-GGAAAACCCTCAGGTCTTCTTC-3'; The sequence of primer R4 is shown in SEQ ID NO:14.The sequence of R4 primer is 5'-GTGCTGTCCCTGGCCCTTGTTC-3').For the first time PCR is take the cDNA of the reverse transcription of carrying out tailing at 5 ' end as template, and through the nucleotide sequence of touchdown PCR method amplification 3 ' end, reaction conditions is: 94 ℃ of denaturations 3 minutes, carry out subsequently the reaction of 30 circulations.94 ℃ of sex change 15 seconds, 64 ℃ of annealing 15 seconds (each circulation reduces by 0.6 ℃), 72 ℃ were extended 1 minute.Last 72 ℃ were extended 7 minutes.
PCR is take 100 times of diluents of primary PCR product as template for the second time.The condition of reaction is identical with the first set reaction condition for the second time.
Through second take turns PCR after, the product that obtains is about 1.3kb.Be connected to and obtain recombinant plasmid pCR2.1-ActRIIB-3 ' on the pCR2.1 carrier.Recombinant plasmid pCR2.1-ActRIIB-3 ' is carried out sequencing with a pair of universal primer of pCR2.1 carrier, the sequence of sequencing result and Genebank compares, the result show clone products be activin receptor IIB tilapia activin receptor IIB 3 ' end group because of.
The electrophoresis evaluation figure of pcr amplification product sees Fig. 2.
Synthesizing of tilapia activin receptor IIB gene 5 ' end fragment
Two downstream primer R5 and R6 have been designed to carry out nested PCR according to the ActRIIB 3 ' terminal sequence that checked order.The sequence of primer R5 is shown in SEQ ID NO:15; The sequence of primer R6 is shown in SEQ ID NO:16; (sequence of R5 primer is 5'-CTCCATGTTGGTTCCGTGCTTCTC-3', the sequence of R6 primer be 5 '-TTGCCATGACTGCTTATCCTGAACTG-3') for the first time PCR take the cDNA of reverse transcription as template, nucleotide sequence through touchdown PCR method amplification 3 ' end, reaction conditions is: 94 ℃ of denaturations 3 minutes, carry out subsequently the reaction of 40 circulations.94 ℃ of sex change 30 seconds, 68 ℃ of annealing 30 seconds (each circulation reduces by 0.5 ℃), 72 ℃ were extended 1 minute.Last 72 ℃ were extended 7 minutes.
PCR is take 100 times of diluents of primary PCR product as template for the second time.The condition of reaction is for to change the annealing temperature in the first set reaction condition into 52 ℃ for the second time, and other condition is identical with the first set reaction condition.
Through second take turns PCR after, the product that obtains is about 1kb.Be connected to and obtain recombinant plasmid pCR2.1-ActRIIB-5 ' on the pCR2.1 carrier.Recombinant plasmid pCR2.1-ActRIIB-5 ' is carried out sequencing with a pair of universal primer of pCR2.1 carrier, the sequence of sequencing result and Genebank compares, the result show clone products be activin receptor IIB tilapia activin receptor IIB 5 ' end group because of.
The electrophoresis evaluation figure of pcr amplification product sees Fig. 3.
Embodiment 5
The splicing of activin receptor IIB tilapia activin receptor IIB full length gene sequence
The ActRIIB intermediate segment, 5 ' RACE fragment and 3 ' the RACE fragment that have checked order are spliced, and the ORF that obtains tilapia activin receptor IIB gene is 1545bp, infers its aminoacid sequence.ActRIIB gene ORF full length sequence is shown in SEQ ID NO:3, and the aminoacid sequence of its proteins encoded is shown in SEQ ID NO:4.
Embodiment 6
Synthesizing of tilapia activin receptor IIB gene
The tilapia activin receptor IIB gene cDNA complete sequence good according to splicing, the synthetic pair of primers F7 of design and R7, its nucleotide sequence is shown in SEQ ID NO:7 ~ 8.Upstream primer F7 is from initiator codon, and its nucleotides sequence is classified 5'-TCAGGTGCTGGACTCTTTGGG-3 ' as; Downstream primer R7 finishes to terminator codon, and its nucleotides sequence is classified 5'-AGCGGAACATGTCTTTTTCATGGC-3' as.Take tilapia receptor II B gene cDNA as template, through PCR method amplification activin receptor IIB total length ORF sequence.The PCR reaction conditions is: 94 ℃ of denaturations 2 minutes, and 98 ℃ of sex change 10 seconds, 50 ℃ of annealing 30 seconds, 68 ℃ were extended 2 minutes.React 40 circulations.Then add Blend Taq-plus-, 72 ℃ were extended 5 minutes.The activin receptor IIB gene of amplification is inserted into structure plasmid pCR2.1-ActRIIB. in the pCR2.1 carrier
The electrophoresis evaluation figure of pcr amplification product sees Fig. 4.
Embodiment 7
The structure that contains the coli expression carrier pQE0-ActRIIB of tilapia activin receptor IIB extracellular domain gene fragment
Design pair of primers F and R, its nucleotide sequence is shown in SEQ ID NO:5 ~ 6.The nucleotides sequence of upstream primer F is classified this primer of 5'-CGGGTACCGAAGCGGAGACCCGGG-3'(as and is contained the KpnI restriction enzyme site); The nucleotides sequence of downstream primer R is classified this primer of 5'-CCAAGCTTTCA (ATG) 6TACTCGGGGTGGAGGCTGG-3'(as and is contained terminator codon and HindIII restriction enzyme site), carry out pcr amplification take carrier pCR2.1-ActRIIB as masterplate, the condition of PCR reaction is 94 ℃ of denaturations 2 minutes, 98 ℃ of sex change 10 seconds, annealed 15 seconds for 55 ℃, 72 ℃ were extended 30 seconds.React 40 circulations.After the PCR reaction finishes, carry out the recovery of PCR product, then PCR being reclaimed product and carrier pQE30 carries out double digestion with Kpn I and HindIII respectively again, and enzyme carries out agarose gel electrophoresis after cutting, and cuts glue and reclaims product.PCR reclaims product, and separation and purification is to the gene fragment of about 336bp behind Kpn I and HindIII double digestion, and this fragment is tilapia activin receptor IIB extracellular domain gene fragment; The large fragment of 3.4kb is arrived in coli expression carrier pQE30 separation and purification behind restriction enzyme Kpn I and HindIII double digestion, with separation and purification to the large fragment of 3.4kb mix by 1:3 with the tilapia activin receptor IIB gene fragment of 336bp, the heat shock method changes in the bacillus coli DH 5 alpha after connecting 16 hours with 14 ℃ of Invitrogen T4 ligase enzymes, screening has the transformant of amicillin resistance, step with reference to E.Z.N.A Plasmid Extraction Kit specification sheets is extracted plasmid, screen big or small recombinant plasmid, with restriction enzyme Kpn I and HindIII double digestion recombinant plasmid, obtain two fragments of 336bp and 3.4kb, size is identical with expression vector pQE30 size with tilapia activin receptor IIB gene respectively, proof tilapia activin receptor IIB gene has been cloned among the coli expression carrier pQE30 recombinant plasmid called after pQE30-ActRIIB.Plasmid construction figure and restriction analysis figure see respectively Fig. 5 and Fig. 6.
Embodiment 8
Efficiently express the structure of the intestinal bacteria recombinant strain pQE0-ActRIIB-M15 of tilapia activin receptor IIB extracellular domain gene fragment
The heat shock method is with the pQE0-ActRIIB Transformed E. and among the coli M15, screen transformant at the LB flat board that contains penbritin and kantlex, carry out with reference to the step of E.Z.N.A Plasmid Extraction Kit specification sheets
The extraction of plasmid, plasmid obtain to contain the sub-pQE0-ActRIIB-M15 of positive recombinant conversion of pQE0-ActRIIB through order-checking and Kpn I and the evaluation of HindIII double digestion.
Embodiment 9
Utilize Recombinant organism pQE0-ActRIIB-M15 Restruction tilapia activin receptor embryonic stage IIB
The single colony inoculation of picking Recombinant organism pQE0-ActRIIB-M15 is in containing the LB liquid nutrient medium of 100ug/ml penbritin, 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 0.5～0.6, adding the IPTG(final concentration is 1mmol/l), receive bacterium in 37 ℃ of inducing culture after 10 hours.Synthetic tilapia activin receptor IIB has obtained to efficiently express in Recombinant organism pQE0-ActRIIB-M15.
The cell that takes a morsel adds 2 * electrophoresis sample-loading buffer, boils after 5 minutes and runs the SDS-PAGE gel electrophoresis by standard method.The results are shown in Figure 7, the pQE0-ActRIIB-M15 of demonstration through inducing a new protein band occurs in the position of about 15kD, this band of the pQE0-ActRIIB-M15 that does not induce is very weak, proves abduction delivering in pQE0-ActRIIB-M15 of tilapia activin receptor IIB.
The tilapia activin receptor IIB that will recombinate adopts immunoblotting (Western blot) method to carry out immunological identification.Primary antibodie is the monoclonal antibody of anti-His label, and two anti-are sheep anti-mouse igg-HRP.The results are shown in Figure 8, show that the monoclonal anti physical efficiency of anti-His label is identified us with the fusion ActRIIB albumen of escherichia coli expression, in conjunction with the dna sequencing result of pQE0-ActRIIB recon, prove that the albumen that obtains is restructuring tilapia activin receptor IIB.
Embodiment 10
Restructuring ActRIIB and polyclonal antibody thereof are to the physiological action of tilapia muscle growth
All experiments are raised and train in the cement pit that injects mobile river with tilapia, raise and train under natural lighting cycle and natural temperature 10 days.During raising and train and testing processing, every afternoon, 4 timings were thrown something and fed once.All fishes are assigned in the different net cages at random, control group, ReActRIIB injection group and antibody injected component are not provided with three repetitions.Abdominal injection is processed: control group, and ReActRIIB group and antibody component are not injected PBS, and ReActRIIB and antibody are weekly, inject continuously for 4 weeks.Dosage such as following table.
With a rear week of last injection, it is long with body weight and the body of fish to measure respectively all experiments were before the per injection processing.A week carry out the tissue sample collection after the 4th injection finishes, get full liver and weigh, get white muscle, this sample is divided into two portions: a part is packed in the EP pipe that fills 1ml Trizol, and temporary transient frozen in liquid nitrogen.Another part is packed into and is filled in the EP pipe of 1ml Bo Enshi stationary liquid, carries out tissue slice.
Experimental result shows, from second week finishes, reActRIIB and antibody group are beginning significance increase (p＜0.05) to occur with respect to control group aspect the body weight, this variation reaches utmost point significance level (p＜0.01) when finishing to 4th week, as shown in Figure 9.After 4th week finished, the liver body between each group did not change than producing significance, showed that the treatment process that adopts does not produce pathology effects to Experimental fish, as shown in figure 10.Aspect diameter of muscle fiber, after the 4th week, get muscle tissue and observe the impact (seeing Figure 11,12,13) of different treatment tilapia meat fiber through paraffin section and HE dyeing.The result shows that ReActRIIB compares with the control group diameter of muscle fiber with the antibody group and utmost point significant difference (p＜0.01) all occurs, and do not have significant difference (p〉0.05) between ReActRIIB and the antibody group (Figure 14).This experimental result shows that abdominal injection restructuring tilapia ReActRIIB and Anti-TNF-α physical efficiency thereof promote the tilapia muscle growth, so recombinant protein can be applicable to prepare fry seed muscle growth promoter or additive.
SEQUENCE LISTING
＜110〉Zhongshan University
＜120〉a kind of preparation method and application thereof of tilapia activin receptor II B recombinant protein
<130>
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 336
<212> DNA
＜213〉tilapia activin receptor IIB extracellular domain gene fragment
<400> 1
gaagcggaga cccgggagtg tgtgtactac aacgataact ggcgttcgga gaggaccaac 60
cagagcggct tcgagcgttg cgagggggaa aaggacaagc gactgcactg ttatgcctcc 120
tggctcaact cctcagggac catcaaactg gtgaagaaag gctgctggct ggatgacttc 180
aactgctatg acaggcaaga gtgcgtctcc atggaggaaa accctcaggt cttcttctgc 240
tgctgcgagg gcaactactg caatgagcga ttcacccacc ttcccgactt gattggtagt 300
ggcaaccgag tgaaaatcca gcctccaccc cgagta 336
<210> 2
<211> 112
<212> PRT
＜213〉tilapia activin receptor IIB extracellular domain
<400> 2
Glu Ala Glu Thr Arg Glu Cys Val Tyr Tyr Asn Asp Asn Trp Arg Ser
1 5 10 15
Glu Arg Thr Asn Gln Ser Gly Phe Glu Arg Cys Glu Gly Glu Lys Asp
20 25 30
Lys Arg Leu His Cys Tyr Ala Ser Trp Leu Asn Ser Ser Gly Thr Ile
35 40 45
Lys Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp
50 55 60
Arg Gln Glu Cys Val Ser Met Glu Glu Asn Pro Gln Val Phe Phe Cys
65 70 75 80
Cys Cys Glu Gly Asn Tyr Cys Asn Glu Arg Phe Thr His Leu Pro Asp
85 90 95
Leu Ile Gly Ser Gly Asn Arg Val Lys Ile Gln Pro Pro Pro Arg Val
100 105 110
<210> 3
<211> 1545
<212> DNA
＜213〉tilapia activin receptor IIB gene
<400> 3
atgtcttttt catggctgac ttgttcactt cttctgggaa ctttatgcgc agggtacagt 60
cttggcgaag cggagacccg ggagtgtgtg tactacaacg ataactggcg ttcggagagg 120
accaaccaga gcggcttcga gcgttgcgag ggggaaaagg acaagcgact gcactgttat 180
gcctcctggc tcaactcctc agggaccatc aaactggtga agaaaggctg ctggctggat 240
gacttcaact gctatgacag gcaagagtgc gtctccatgg aggaaaaccc tcaggtcttc 300
ttctgctgct gcgagggcaa ctactgcaat gagcgattca cccaccttcc cgacttgatt 360
ggtagtggca accgagtgaa aatccagcct ccaccccgag taccgtccct gctcagtgtg 420
ctggtgtact ccctgctgcc tctctgtgtg ctgtccctgg cccttgttct ggccttgtgg 480
atgtaccgcc accgcaaacc tccctacggc catgtagacc tgagcgagga tccagggccg 540
gctcctccat ctcccttggt gggtctgaaa ccattgcagc ttctagaaat caaagctagg 600
gggcgctttg gttgtgtttg gaaggcccag ttaatgagtg aatatgttgc tgttaagatc 660
ttcccagttc aggataagca gtcatggcaa aatgagcgcg acatattctt gactccgggg 720
atgagacatg aaaatatctt gcgttacatc gctgcagaga agcacggaac caacatggag 780
acggagctgt ggcttatcac agagtttcat gaaaggggat ccttgacaga ccacctgaag 840
ggcaacactg tgacctggac agagctgtgt cacatagcag agaccatgtc ccgcggtttg 900
gcttaccttc atgaggacat tcccagttac aagggagagg ggcctaaacc tacaattgca 960
cacagggact tcaagagcaa gaatgtgtta ctacgagatg acttaactgc agtcattggt 1020
gactttgggc ttgctgtaag attcgagcca ggaaaacctc ctggggatac tcatggacag 1080
gtgggtacga ggcgttacat ggccccagag gtgctggagg gagccatcaa ctttcagcga 1140
gactccttcc tgaggataga catgtatgct atgggcttgg tgctgtggga gctggtgtct 1200
cgttgctccg acacagatgg aacagttggt gagtacatgc tgccatttga ggatgaaata 1260
ggccagcatc cctccctgga ggatctgcag gatgtggtgg tgcacaagaa gatgcgtcca 1320
gtcatcaagg actcctggct caagcatcct ggtctgagcc acatgtgtga gaccatcgag 1380
gaatgctggg accacgacgc agaggctcga ctgtctgcag gctgcgtcga ggagcgcatc 1440
ggtcagatct ccaggacgat aggcagcact acctcagaca gccccatctc tttagtcacg 1500
tctctcatca acgaggacct acctcccaaa gagtccagca cctga 1545
<210> 4
<211> 514
<212> PRT
＜213〉tilapia activin receptor IIB albumen
<400> 4
Met Ser Phe Ser Trp Leu Thr Cys Ser Leu Leu Leu Gly Thr Leu Cys
1 5 10 15
Ala Gly Tyr Ser Leu Gly Glu Ala Glu Thr Arg Glu Cys Val Tyr Tyr
20 25 30
Asn Asp Asn Trp Arg Ser Glu Arg Thr Asn Gln Ser Gly Phe Glu Arg
35 40 45
Cys Glu Gly Glu Lys Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Leu
50 55 60
Asn Ser Ser Gly Thr Ile Lys Leu Val Lys Lys Gly Cys Trp Leu Asp
65 70 75 80
Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ser Met Glu Glu Asn
85 90 95
Pro Gln Val Phe Phe Cys Cys Cys Glu Gly Asn Tyr Cys Asn Glu Arg
100 105 110
Phe Thr His Leu Pro Asp Leu Ile Gly Ser Gly Asn Arg Val Lys Ile
115 120 125
Gln Pro Pro Pro Arg Val Pro Ser Leu Leu Ser Val Leu Val Tyr Ser
130 135 140
Leu Leu Pro Leu Cys Val Leu Ser Leu Ala Leu Val Leu Ala Leu Trp
145 150 155 160
Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Leu Ser Glu
165 170 175
Asp Pro Gly Pro Ala Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu
180 185 190
Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys
195 200 205
Ala Gln Leu Met Ser Glu Tyr Val Ala Val Lys Ile Phe Pro Val Gln
210 215 220
Asp Lys Gln Ser Trp Gln Asn Glu Arg Asp Ile Phe Leu Thr Pro Gly
225 230 235 240
Met Arg His Glu Asn Ile Leu Arg Tyr Ile Ala Ala Glu Lys His Gly
245 250 255
Thr Asn Met Glu Thr Glu Leu Trp Leu Ile Thr Glu Phe His Glu Arg
260 265 270
Gly Ser Leu Thr Asp His Leu Lys Gly Asn Thr Val Thr Trp Thr Glu
275 280 285
Leu Cys His Ile Ala Glu Thr Met Ser Arg Gly Leu Ala Tyr Leu His
290 295 300
Glu Asp Ile Pro Ser Tyr Lys Gly Glu Gly Pro Lys Pro Thr Ile Ala
305 310 315 320
His Arg Asp Phe Lys Ser Lys Asn Val Leu Leu Arg Asp Asp Leu Thr
325 330 335
Ala Val Ile Gly Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys
340 345 350
Pro Pro Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala
355 360 365
Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ser Phe Leu
370 375 380
Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser
385 390 395 400
Arg Cys Ser Asp Thr Asp Gly Thr Val Gly Glu Tyr Met Leu Pro Phe
405 410 415
Glu Asp Glu Ile Gly Gln His Pro Ser Leu Glu Asp Leu Gln Asp Val
420 425 430
Val Val His Lys Lys Met Arg Pro Val Ile Lys Asp Ser Trp Leu Lys
435 440 445
His Pro Gly Leu Ser His Met Cys Glu Thr Ile Glu Glu Cys Trp Asp
450 455 460
His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Ile
465 470 475 480
Gly Gln Ile Ser Arg Thr Ile Gly Ser Thr Thr Ser Asp Ser Pro Ile
485 490 495
Ser Leu Val Thr Ser Leu Ile Asn Glu Asp Leu Pro Pro Lys Glu Ser
500 505 510
Ser Thr
<210> 5
<211> 24
<212> DNA
＜213〉primers F
<400> 5
cgggtaccga agcggagacc cggg 24
<210> 6
<211> 48
<212> DNA
＜213〉primer R
<400> 6
ccaagctttc aatgatgatg atgatgatgt actcggggtg gaggctgg 48
<210> 7
<211> 21
<212> DNA
＜213〉primers F 7
<400> 7
tcaggtgctg gactctttgg g 21
<210> 8
<211> 24
<212> DNA
＜213〉primer R7
<400> 8
agcggaacat gtctttttca tggc 24
<210> 9
<211> 24
<212> DNA
＜213〉primers F 1
<400> 9
gtawgwvrkk ttbykrtghc krta 24
<210> 10
<211> 28
<212> DNA
＜213〉primer R1
<400> 10
gcracdcrgg agtgyhtbtw ctwyaacg 28
<210> 11
<211> 20
<212> DNA
＜213〉primers F 2
<400> 11
ttbykrtghc krtacaycca 20
<210> 12
<211> 25
<212> DNA
＜213〉primer R2
<400> 12
agtgyhtbtw ctwyaacgya aytgg 25
<210> 13
<211> 22
<212> DNA
＜213〉primer R3
<400> 13
ggaaaaccct caggtcttct tc 22
<210> 14
<211> 22
<212> DNA
＜213〉primer R4
<400> 14
gtgctgtccc tggcccttgt tc 22
<210> 15
<211> 24
<212> DNA
＜213〉primer R5
<400> 15
ctccatgttg gttccgtgct tctc 24
<210> 16
<211> 26
<212> DNA
＜213〉primer R6
<400> 16
ttgccatgac tgcttatcct gaactg 26
Claims (10)
1. a tilapia activin receptor IIB extracellular domain gene fragment is characterized in that nucleotide sequence is shown in SEQ ID NO:1.
2. a tilapia activin receptor IIB albuminous cell outer structure territory is characterized in that aminoacid sequence is shown in SEQ ID NO:2.
3. a tilapia activin receptor IIB gene is characterized in that nucleotide sequence is shown in SEQ ID NO:3.
4. a tilapia activin receptor IIB albumen is characterized in that aminoacid sequence is shown in SEQ ID NO:4.
5. a pair of for amplification tilapia activin receptor IIB gene fragment primer, it is characterized in that primer sequence is shown in SEQ ID NO:5 ~ 6; Perhaps primer sequence is shown in SEQ ID NO:7 ~ 8.
6. a carrier is characterized in that containing claim 1 or 3 described gene fragments.
7. described carrier according to claim 6 is characterized in that described carrier is escherichia coli cloning carrier pCR2.1-ActRIIB; Perhaps described carrier is coli expression carrier pQE30-ActRIIB.
8. a recombinant strain it is characterized in that changing gained in the intestinal bacteria over to by claim 6 or 7 described expression vectors 6.
9. the preparation method of a tilapia activin receptor IIB recombinant protein is characterized in that, comprises the steps:
S1. claim 6 or 7 described expression vectors are changed in the intestinal bacteria, utilize ampicillin resistance gene screening positive transformant;
S2. positive transformant is seeded in the liquid nutrient medium that contains penbritin, overnight incubation, be inoculated in the same medium next day, and induce through IPTG, collects thalline after cultivating, through the separation and purification tilapia activin receptor IIB that obtains recombinating.
One kind according to claim 2, the application of 4 or 9 arbitrary described tilapia activin receptor IIB recombinant proteins in preparation fry seed growth promoter or additive.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262485A (en) * | 2014-04-15 | 2015-01-07 | 四川农业大学 | Tilapia sIgM truncated heavy-chain constant-region protein polyclonal antibody and preparing method thereof |
| CN109293764A (en) * | 2018-10-26 | 2019-02-01 | 中国农业科学院特产研究所 | Deer subfamily activin A albumen and its preparation and application |
| CN111269943A (en) * | 2019-08-10 | 2020-06-12 | 湖南文理学院 | Method for increasing growth speed of zebra fish through gene knockout technology |
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2012
- 2012-12-21 CN CN2012105608495A patent/CN103045623A/en active Pending
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| NCBI: "Accession No： XM_003448468", 《GENE BANK》 * |
| YAMILA CARPIO ET AL.: "Regulation of body mass growth through activin type IIB receptor in teleost fish", 《GENERAL AND COMPARATIVE ENDOCRINOLOGY》 * |
| 宋春雷: "草鱼激活素Ⅰ型和Ⅱ型受体的cDNA克隆、结构分析及其在垂体细胞中的表达调控的研究", 《中国优秀硕士论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262485A (en) * | 2014-04-15 | 2015-01-07 | 四川农业大学 | Tilapia sIgM truncated heavy-chain constant-region protein polyclonal antibody and preparing method thereof |
| CN104262485B (en) * | 2014-04-15 | 2017-01-18 | 四川农业大学 | Tilapia sIgM truncated heavy-chain constant-region protein polyclonal antibody and preparing method thereof |
| CN109293764A (en) * | 2018-10-26 | 2019-02-01 | 中国农业科学院特产研究所 | Deer subfamily activin A albumen and its preparation and application |
| CN109293764B (en) * | 2018-10-26 | 2021-11-16 | 中国农业科学院特产研究所 | Lu's subfamily activin A protein and preparation and application thereof |
| CN111269943A (en) * | 2019-08-10 | 2020-06-12 | 湖南文理学院 | Method for increasing growth speed of zebra fish through gene knockout technology |
| CN111269943B (en) * | 2019-08-10 | 2023-01-06 | 湖南文理学院 | Method for increasing growth speed of zebra fish through gene knockout technology |
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