CN1569230A - Preparation and application of a gene engineering vaccine of muscle growth retarder - Google Patents

Abstract

The invention belongs to the preparation field of vaccine for animals. It adopts the gene engineering method, clones the gene of muscle somatostatin and expresses the recombined confluence protein of the muscle somatostatin in Escherichia coli or yeast as an immunogen or gene engineering vaccine. Active immunity for growing livestock and birds by the muscle somatostatin engeneering vaccine can promote the animal to generate antibody of the muscle somatostatin, which can neutralize and inhibit the muscle somatostatin produced by self muscle of the livestock and birds, thus absolves the muscle growth inhibiting function of the muscle somatostatin and promotes the muscle growth.

Description

The preparation of myostatin gene engineered vaccine and application thereof

Technical field

The present invention relates to herding production new technology and live vaccine field.

Background technology

The muscle growth that promotes agricultural animal is the target that herding production and herding institute are pursued always.Promote the muscle growth of poultry or improve lean meat percentage, not only improve the efficient of animal feeding, can also production high-quality and healthy meat, and improve the economic benefit that herding is produced greatly.

Mainly promote the speed of muscle growth and the growth of raising livestock and poultry muscle at present by breeding work, adjusting nutrient and the trophic level of routine.But up to the present, poultry routine then produce with breeding technology under the muscle growth performance performed to the limit, utilize above conventional method to promote that the space of muscle growth is more and more littler.To further promote the growth of muscle, improve herding production efficiency and production high-quality meat, must seek the technical method that upgrades, regulate and control and promote the growth of muscle.

The method that can significantly improve the muscle growth performance is mainly the endocrine regulation technology, comprises using 1. steroid hormone, 2. receptor, agonist, 3. growth hormone, 4. growth hormone releasing hormone and preparation thereof and 5. active immunity somatostatin etc.

In the above endocrine regulation technology, because steroid hormone, receptor, agonist (being beta-stimulants) and growth hormone are because residual problem of its medicine and potential influence to the human physiological activity are all prohibited use.The technology of active immunity somatostatin then only limits to young animal because of growth enhancing effect and effect is very unstable, can not be advantageously applied to herding production.

Find at present in the muscle growth process of animal particularly later stage at muscle growth, muscle cell produces a kind of the 8th growth and differentiation factor (Growth Differentiation Factor-8 by name, abbreviation GDF-8) endocrine factor, this factor is also referred to as myostatin (Myostatin is called for short MSTN).Myostatin acts on the receptor on muscle cell surface, thereby suppresses the hypertrophy or the undue growth of muscle cell.The problem that the task of the invention maybe will solve, exactly under the hereditary basis that does not influence animal, nutrition and raising requirement or condition, remove or remove the inhibitory action of animal " myostatin " to muscle growth, making muscle cell or organize at later stages can unrestricted continued growth or hypertrophy, thereby increases substantially muscle growth speed.

The U.S. has produced the specific antibody of special combination or neutralization inhibition " myostatin " at present, this antibody make " myostatin " can not with the receptors bind on muscle cell surface, remove its inhibitory action, thereby realize " unrestrictedly " growth of muscle myocyte's growth.Find at present can improve 20～30% to muscular tissue weight to this antibody of injected in mice (being called " passive immunity ").But should " passive immunity " technology only at the experimental stage at present, the way of " passive immunity " also essential every day of injection of antibodies simultaneously, on producing because of complex operation be subjected to animal welfare personage's protest not have the production application prospect abroad.Therefore purpose of the present invention or task are to adopt the approach of " active immunity ", the recombinant vaccine of preparation " myostatin " also carries out immunity with this vaccine to animal, utilize the antibody of the immune system long-term production anti-" myostatin " of animal self then, this antibody has the ability of special combination and inhibition " myostatin " fully, thereby also can remove " myostatin " inhibitory action, realize " unrestrictedly " growth of muscle myocyte's growth.

Summary of the invention

Content of the present invention comprises: the 1) preparation of myostatin gene engineered vaccine and 2) animal or poultry active immunity myostatin gene engineered vaccine are promoted two parts that animal muscle is grown.

One. the preparation of myostatin gene engineered vaccine

Myostatin gene at first is translated as the hormone peptide of one 375 amino acid residues in muscle cell former, there is a protein cleavage site RSRR at the former 263-266 amino acid residue place of this peptide, herein N-terminal hydrophilic secreting signal peptide is downcut the myostatin mature peptide that the back produces 109 amino acid residues of the bioactive c-terminus of tool.Because myostatin (comprises poultry) and is high conservative between different plant species, its gene nucleotide series is closely similar between different plant species, coded mature peptide amino acid residue sequence is all the same pig, chicken and goose, so the gene of any of these animal may be used to prepare the recombinant vaccine of myostatin.

The preparation of myostatin gene engineered vaccine, 1. clone livestock and poultry muscle amicine encoding gene, 2. utilize livestock and poultry muscle amicine encoding gene, 3. the recombination fusion protein of preparation myostatin utilizes the recombinant vaccine of this Myostatin as the immunogen preparing myostatin.

Clone's livestock and poultry muscle amicine encoding gene, 1. utilize according to the chicken muscle amicine gene order of being delivered among the GenBank, design this gene specificity forward primer P1 (sequence is: 5 '-ATGCAAAAGCTAGCAGTCTATG-3 ') and downstream primer P2 (sequence is: 5 '-TCATGAGCACCCGCAACGAT-3 '); 2. utilize guanidinium isothiocyanate-chloroform-isopropyl alcohol method from the livestock and poultry muscle tissue, to extract total RNA and become the first chain cDNA with primer P2 reverse transcription as template; 3. amplify the coded cDNA sequence of the myostatin of chicken, pig or goose by the PCR reaction as template and primer P1, P2 with this first chain cDNA.

One of preparation livestock and poultry muscle amicine recombination fusion protein preparation method is to utilize IPTG to induce escherichia coli E.coli BL21 (DE3) strain to express the preparation recombination fusion protein: 1. according to the cDNA base sequence of myostatin mature peptide and the multiple clone site base sequence of colibacillus expression plasmid pRSET A, (sequence is 5 ' ATT to synthetic forward primer P3 GGATCCGATTTTGGCCTTG ACT-3 ', underscore part is placing restrictions on property of BamHI restriction enzyme site) and downstream primer P4 (sequence is 5 ' AAT GAGCTCAATGAGCACCCGCAACGA-3 ', the underscore part is placing restrictions on property of SacI restriction enzyme site); 2. utilize this myostatin coded cDNA sequence that primer, above-mentioned post transcription cloning are gone out as template, amplify the cDNA cloned sequence of myostatin mature peptide once more by the PCR reaction; Behind the cDNA cloned sequence process BamHI and SacI double digestion of the myostatin mature peptide that 3. amplifies, be cloned between the BamHI and SacI two restriction enzyme sites of expression plasmid pRSET A, be built into recombinant expression plasmid pMSTN SCAU; 4. with recombinant expression plasmid pMSTN SCAU transformed into escherichia coli E.coli BL21 (DE3) strain, the IPTG that adds 0.01mmol/L in culture fluid induces and made recombinant bacteria express Myostatin in 6 hours.

Two of preparation livestock and poultry muscle amicine recombination fusion protein method is to utilize Pichia sp. to express this fusion rotein under methanol induction: 1. according to the base sequence of lion-headed goose myostatin mature peptide code cDNA be used to transform the base sequence of multiple clone site of the shuttle plasmid pPICZ α A of Pichia sp., (sequence is 5 ' AGG to design forward primer YP1 GAATTCGATTTTGGCC TTGACTGC-3 ', underscore partly are the EcoRI restriction enzyme site) and downstream primer YP2 (5 ' AAT GGTACCTTTGAGCACCCGCAACG-3 ', underscore partly are the KpnI restriction enzyme site); 2. utilize this myostatin coded cDNA sequence that primer, above post transcription cloning are gone out as template, amplify the cDNA yeast shuttle plasmid cloned sequence of myostatin gene mature peptide once more by the PCR reaction, behind this cDNA cloned sequence process EcoRI and the KpnI double digestion, after being cloned between the EcoRI of shuttle plasmid pPICZ α A and the KpnI restriction enzyme site, be built into recombinant plasmid pPICZ-MSTN SCAU; 3. with recombinant plasmid pPICZ-MSTN SCAU linearisation and transformed competence colibacillus yeast X-33, and at Zeocin ^TMThe YPDS solid medium cultivate, select white colony to YPDS fluid medium and cultivate, and use forward primer AOX ₁(5 ' GACTG GTTCCAATTGACAAGC-3 ') and downstream primer AOX ₂(5 ' GCAAATGGCATTCT GACATCC-3 ') are reacted by PCR and are identified and select Mut ⁺The phenotype recombination yeast; 4. picking correctly is integrated into genomic single recombinant yeast with myostatin mature peptide fusion gene and is inoculated in the BMGY culture medium culturing, to adding methanol in the culture fluid to final concentration 0.5% abduction delivering Myostatin, expressed Myostatin can be secreted to born of the same parents in the culture fluid by yeast.

The Myostatin that purification is expressed, will be with the above escherichia coli E.coli BL21 (DE3) that expresses Myostatin of 8Mol carbamide cracking, or go out to contain the Yeast Cultivation liquid of Myostatin by centrifugalize, two kinds of solution that contain Myostatin are carried out chromatography by Ni-NTA gel-purified resin, and last eluting is purified into the antigen of Myostatin as immunity usefulness.

With the above Myostatin that is purified into as the immunogen preparing oil emulsion vaccine: 1. Myostatin is dissolved in the aqueous solution that contains 2～4%Tween80 and makes water by the concentration of 2～6mg/mL; 2. with No. 10 lightweight white oils and Si Ben 80 by 94% and 6% mixed, inwardly add 2% aluminium stearate again, mix homogeneously is made oil phase; 3. water and oil phase are pressed 4: 6 mixed, and under blender, make water in oil homogenizing emulsion as the myostatin gene engineered vaccine.

The application of myostatin gene engineered vaccine, it mainly is the muscle growth that promotes animal by the approach of active immunity, pig, cattle, sheep, goat and deer to growth stage, every 3～4 weeks in intramuscular injection myostatin gene engineering oil emulsion vaccine, promote the animal vivo immuning system to produce the antibody of anti-self myostatin, this antibody can neutralize and suppress the myostatin of animal muscle emiocytosis, remove the inhibitory action of myostatin, promote the growth of animal muscle the livestock and poultry muscle growth.

Now in conjunction with example and accompanying drawing this invention is described further, wherein accompanying drawing 1 is the structure flow chart of the colibacillus expression plasmid of myostatin reorganization fusion gene.Accompanying drawing 2 is the structure flow chart that contains the yeast shuttle plasmid of myostatin reorganization fusion gene.Accompanying drawing 1 listed construction step is, 1. amplifies its mature peptide cDNA cloned sequence from lion-headed goose myostatin code cDNA, and with this fragment through BamHI and SacI double digestion; 2. will be used in addition using BamHI and SacI double digestion equally at the plasmid pRSET of expression in escherichia coli recombiant protein A; 3. then the mature peptide cDNA cloned sequence behind the enzyme action is connected with the T4 ligase with pRSET A carrier, is built into expression plasmid pMSTN SCAU, wherein be used for expressing the nucleotide sequence of Myostatin shown in sequence table sequence 2.Accompanying drawing 2 listed construction steps are, 1. amplify its mature peptide cDNA cloned sequence from lion-headed goose myostatin code cDNA, and with this fragment behind EcoRI and KpnI double digestion; 2. will be used in addition using EcoRI and KpnI double digestion equally to the shuttle plasmid pPICZ of yeast genes group integrate foreign genes α A; 3. be connected with the T4 ligase with shuttle plasmid pPICZ α A carrier with the mature peptide cDNA cloned sequence behind the enzyme action then, be built into recombinant shuttle plasmid pPICZ-Mstn SCAU, wherein be used for expressing the nucleotide sequence of Myostatin shown in sequence table sequence 4.

Specific embodiments

Below be to utilize the myostatin code cDNA that from lion-headed goose, clones to be prepared the explanation of the recombinant vaccine of myostatin.Because the mature peptide amino acid residue sequence of the myostatin of the different animals of up to the present being found is very similar, therefore except the myostatin gene that utilizes lion-headed goose, can also prepare the recombinant vaccine of myostatin with the myostatin gene of chicken, pig, cattle and sheep.

Concrete way is: (1) obtains or clones the myostatin encoding gene of poultry, (2) utilize this gene expresses myostatin in escherichia coli or yeast recombination fusion protein as antigen or immunogen, the expressed myostatin fusion protein immunization of (3) purification is former and itself and mineral oil adjuvant be mixed and made into the myostatin vaccine.

1. the clone of animal muscle amicine code cDNA

1. according to the livestock and poultry muscle amicine gene order of being delivered among the GenBank, design the Auele Specific Primer of this gene.Forward primer P1 (5 '-ATGCAAAAGCTAGCAGTCTATG-3 ') corresponding to 1-22 position, myostatin gene coding region nucleotide; Downstream primer P2 (5 '-TCATGAGCACCCGCAACGAT-3 ') corresponding to 1109-1128 position, myostatin gene coding region nucleotide.The nucleotide sequence that has comprised 1128bp between the upstream and downstream primer, the complete sequence that has covered livestock and poultry muscle amicine coding region cDNA reaches TGA termination codon afterwards.

2. get 100mg lion-headed goose skeletal muscle, place homogenate in the 1ml guanidinium isothiocyanate phenol solution (Trizol liquid, Invitrogen company), remove protein with chloroform then, take out supernatant after centrifugal 15 minutes, in supernatant, add the isopropyl alcohol total RNA of centrifugation again.The concentration that total RNA is made into 1 μ g/ μ l is dissolved in the water that contains DEPC, and mixes with downstream primer P2 and dNTP, carries out the synthetic first chain cDNA of reverse transcription under the catalysis of AMV reverse transcription.

3. use the synthetic first chain cDNA of primer P1, P2, dNTP and reverse transcription as template, under the catalysis of archaeal dna polymerase, amplify the code cDNA fragment of the myostatin of lion-headed goose by the PCR reaction.

4. with being connected of the lion-headed goose myostatin cDNA fragment that amplified and cloned plasmids pUCm-T carrier (purchase give birth in Shanghai worker company), and the recombinant plasmid transformed that is built into gone into bacillus coli DH 5 alpha strain (Invitrogen company) is duplicated, forever preserved, nucleotide sequencing.Sequence 1 is the nucleotide sequence of the lion-headed goose myostatin cDNA that goes out with the method post transcription cloning in the sequence table, wherein underscore is partly distinguished corresponding P1 and P2 primer, the 799th～1125 nucleotide is the coded cDNA sequence of myostatin mature peptide, and last tga is a termination codon.The lion-headed goose myostatin gene nucleotide sequence of this amplification and the homology of other birds more than 90% and the homology of domestic animal more than 85%; Coded mature peptide amino acid residue sequence and chicken, pig just the same is with the difference that 2 amino acid residues are only arranged of cattle, with the difference that 3 amino acid residues are only arranged of sheep.Therefore can prepare the myostatin gene engineered vaccine with lion-headed goose myostatin code cDNA fully.

2. the expression of Myostatin

Utilize lion-headed goose myostatin cDNA, can carry out the expression of Myostatin by prokaryotic expression system such as e. coli bl21 DE3 strain (Invitrogen company) or eukaryotic expression system such as Pichia sp. (Invitrogen company).

(1) expresses Myostatin in the e. coli bl21 DE3 strain (Invitrogen company): 1. according to the nucleotide sequence of the resulting lion-headed goose myostatin cDNA of order-checking, cDNA sequence (sequence 1 in the sequence table) by its mature peptide designs forward primer P3 (5 ' ATT GATT TTGGCCTTGACT-3 '), and contains a BamHI restriction enzyme site (double underline part) and downstream primer P4 (5 ' AAT corresponding to the 799th～814 nucleotide sequence of myostatin gene coding region

TGAGCACCCGCAACGA-3 '), corresponding to the 1109th～1125 nucleotide sequence of myostatin gene coding region, contain additional afterwards two adenylic acids (single underscore part) in a SacI restriction enzyme site (double underline part) and this site to keep the reading frame of amplified fragments.2. the myostatin cDNA fragment of utilizing primer P3, P4, amplification to obtain amplifies the cDNA cloned sequence that two ends have the myostatin mature peptide of BamHI and SacI restriction enzyme site through the PCR reaction as template and dNTP once more under the catalysis of Pfu archaeal dna polymerase, be used to insert expression plasmid pRSET A (Invitrogen company).Fig. 1 has shown the structure flow process of the colibacillus expression plasmid that contains myostatin reorganization fusion gene: utilize primer P3 and P4 amplification to obtain myostatin mature peptide cDNA cloned sequence, with this fragment through BamHI and SacI double digestion; To have polyclone enzyme action site, can load and at expression in escherichia coli expression of exogenous gene plasmid pRSETA with BamHI and SacI double digestion; Connect myostatin mature peptide cDNA cloned sequence and expression plasmid pRSET A carrier with the T4 ligase then, be built into expression plasmid pMSTNSCAU.Wherein the nucleotide sequence of myostatin reorganization fusion gene is shown in sequence in the sequence table 2, wherein the underscore part is corresponding with primer P3 and P4 respectively, part between the underscore is a myostatin mature peptide cDNA sequence, and remainder is the nucleotide sequence that comes from plasmid.3. with this recombinant expression plasmid pMSTN SCAU transformed into escherichia coli BL21 DE3 strain, recombinant bacteria is induced under IPTG concentration at 0.01mmol/L after the cultivation and was expressed Myostatin in 6 hours.The amino acid residue sequence of this recombination fusion protein is shown in sequence in the sequence table 3, wherein single underscore partly is 109 amino acid residue sequences of myostatin mature peptide, its the place ahead and rear are respectively 36 and 14 amino acids residue sequence from expression plasmid, and double underline partly is 6 histidine residues labellings (His-Tag).

(2) in finishing red (Pichia) yeast, express Myostatin:, design a pair of Auele Specific Primer 1. according to lion-headed goose myostatin mature peptide cDNA nucleotide sequence be used to transform the multiple clone site nucleotide sequence of the shuttle plasmid pPICZ α A (Invitrogen company) of Pichia sp..Forward primer YP1 (5 ' AGG

GATTTTGGCCTTGACTGC-3 '), corresponding to the 799th～816 nucleotide sequence of myostatin gene coding region, contain EcoRI restriction enzyme site (double underline part); Downstream primer YP2 (5 ' AA

TGAGCACCCGCAACG-3 '), corresponding to the 1110th～1125 nucleotide sequence of myostatin gene coding region, contain a KpnI restriction enzyme site (double underline part) and keep two base TT (single underscore part) of reading frame.2. utilize myostatin cDNA fragment that primer YP1, YP2, dNTP, reverse transcriptional PCR amplification obtain as template, under the catalysis of Pfu archaeal dna polymerase, amplify the cDNA cloned sequence that two ends have the myostatin mature peptide of EcoRI and KpnI restriction enzyme site through the PCR reaction.Fig. 2 has shown the structure flow process of the yeast shuttle plasmid that contains myostatin reorganization fusion gene: utilize primer YP1 and YP2 amplification to obtain myostatin mature peptide cDNA cloned sequence, with this fragment EcoRI and KpnI double digestion; To have polyclone enzyme action site, can load and with the shuttle plasmid pPICZ α A of exogenous origin gene integrator in the Pichia sp. genome with EcoRI and KpnI double digestion; Connect myostatin mature peptide cDNA cloned sequence shuttle plasmid pPICZ α A carrier with the T4 ligase then, be built into recombinant shuttle plasmid pPICZ-Mstn SCAU.The cDNA sequence of the myostatin reorganization fusion gene in this plasmid is shown in sequence in the sequence table 4, wherein underscore partly corresponds respectively to YP1 and YP2, the centre is a myostatin mature peptide code cDNA, it is the alpha factor signal peptide code cDNA before, afterwards for coming from the coded amino acid residue of nucleotide of shuttle plasmid, comprising the labelling (His-tag) of 6 histidine.3. recombinant shuttle plasmid pPICZ-Mstn SCAU transformed into escherichia coli DH5 α strain is duplicated therein, the DH5 α that contains recombinant shuttle plasmid that gets cultivation cultivates bacterium liquid coating less salt LB (10g/L yeast extract, the 20g/L tryptone, 5g/L NaCl)+Zeocin ^TMCultivate on (25 μ g/mL) solid medium, picking list bacterium colony is to less salt LB+Zeocin ^TMFluid medium is cultivated, and carries out evaluation of PCR and enzyme action and order-checking and identifies.4. will identify to make up correct recombinant plasmid pPICZ-Mstn SCAU linearisation, the back electric shock transformed competence colibacillus yeast X-33 that purifies, coating contains Zeocin ^TMYPDS (10g/L yeast extract, 20g/L tryptone, 20g/L D-glucose, 1mol/L sorbitol, 20g/L agar powder) solid medium, be cultured to white colony and occur.Choosing single bacterium colony to YPDS (10g/L yeast extract, 20g/L tryptone, 20g/L D-glucose) fluid medium cultivates.Synthetic simultaneously a pair of primer, forward primer AOX1 (5 ' GACTGGTTCCAATTGACAAGC-3 ') and downstream primer AOX2 (5 ' GCAAAT GGCATTC TGAATCC-3 ').Cultivate bacterium with primer AOX1, AOX2, dNTP, recombination yeast and under archaeal dna polymerase catalysis, carry out the PCR reaction, identify and select Mut+ phenotype recombination yeast.5. select to make up and correct myostatin mature peptide gene integration gone into genomic single recombinant yeast and be inoculated in 25mLBMGY (10g/L yeast extract, the 20g/L tryptone, 13.4g/L yeast nitrogen, 4 * 10-5g/LD-biotin, the 10mL/L glycerol, the 100mmol/L phosphate buffer, pH6.0) culture medium is cultured to OD600=2.6 for 30 ℃, the results recombinant yeast is also used BMMY (10g/L yeast extract, the 20g/L tryptone, 13.4g/L yeast nitrogen, 4 * 10-5g/L D-biotin, 5mL/L methanol, the 100mmol/L phosphate buffer, pH6.0) culture medium is diluted to OD600=1,30 ℃ of shaken cultivation 4d, add methanol to final concentration 0.5% every 24h, every 12h sampling is equipped with SDS-PAGE and analyzes expressed Myostatin.

The aminoacid sequence of more than using the expressed recombiant protein of Pichia sp. is shown in sequence in the sequence table 5.Wherein contain 89 amino acid whose alpha factor signal peptide sequences that come from carrier; the myostatin mature peptide sequence (underscore part) of 109 amino acid residues is from the peptide sequence and the histidine mark that is used for 6 amino acid residues of purification of 29 coded residues of plasmid gene sequence.Yeast can be with this protein excretion to born of the same parents in the culture fluid when expressing, can hold the cracking of N-terminal 89 amino acid whose alpha factor signal peptide sequences this moment, formation comprises the Myostatin of one 144 amino acid residue, 109 amino acid residue sequences that amino acid residue is the myostatin mature peptide wherein, 35 amino acid residues of c-terminus are the coded by said gene from plasmid pPICZ α A, comprise being positioned at last 6 histidine residues labellings (His-tag).

3. the purification of Myostatin

Utilize the aminoterminal of the Myostatin of escherichia coli expression to have the labelling (His-tag) of one 6 continuous histidine, have the labelling of one 6 continuous histidine at the c-terminus of the Myostatin of yeast expression.Special affine the combination can take place with nickel ion in this His-Tag labelling, therefore will express the antibacterial of Myostatin through the cracking of 8M urea liquid, or the Yeast Cultivation expression liquid that will express Myostatin behind methanol induction is loaded into Ni-NTA gel-purified resin chromatographic column, make wherein Myostatin be adsorbed to Ni-NTA gel-purified resin and eluting can not with the thalline foreign protein of nickel ion resin-bonded, change the eluent pH value at last with destination protein Myostatin eluting purification.

4. the preparation of myostatin oil emulsion vaccine

The above expressed and Myostatin that is purified into is dissolved in the aqueous solution that contains 2～4%Tween 80 by the concentration of 2～6mg/mL makes water; Again with No. 10 lightweight white oils and Si Ben 80 by 94% and 6% mixed, make oil phase to the aluminium stearate mix homogeneously that adds 2% again; Water and oil phase are pressed 4: 6 mixed, and under blender, make water in oil homogenizing emulsion as the myostatin gene engineered vaccine.

Two. poultry active immunity " myostatin " recombinant vaccine promotes the method for muscle growth

The genetic engineering oil emulsion vaccine of " myostatin " that pig, cattle, sheep, goat and deer are prepared more than the intramuscular injection of 3-4 week, promptly can effectively promote the animal vivo immuning system to produce the antibody of anti-self myostatin, this antibody can neutralize and suppress the excretory myostatin of animal muscle cell self, remove the inhibitory action of myostatin, promote the growth of livestock and poultry muscle the livestock and poultry muscle growth.

The compared with prior art advantage that is had, characteristics or good effect

" active immunity myostatin gene engineered vaccine promotes the method for livestock and poultry muscle growth " employed material is the recombination fusion protein of " myostatin " recombinant vaccine or " myostatin ", this albumen avirulence does not cause any disease to animal; Making animal produce essence after this albumen uses as recombinant vaccine is to go up the antibody of immunoglobulin, can not cause other drug residual, to the person poultry harmless, can not produce any harmful effect to consumer.Compare with other any technology, this technology promotes that the effectiveness of muscular tissue growth is strong, and the specificity height has incomparable advantage of other any technology and performance.

Sequence table

＜110〉Agricultural University Of South China

＜120〉preparation of myostatin gene engineered vaccine and application thereof

<140>

<141>

<160>5

<210>1

<211>1128

<212>DNA

＜213〉lion-headed goose (Anser anser)

<220>

<222>(1)...(1128)

＜223〉myostatin coding region cDNA sequence

<220>

<221>misc_recomb

<222>(1)...(12)

＜223〉primer P1 corresponding region

<220>

<221>misc_recomb

<222>(799)...(814)

＜223〉primer P3 corresponding region

<220>

<221>mat_peptide

<222>(799)...(1125)

＜223〉myostatin mature peptide cDNA sequence

<220>

<221>misc_recomb

<222>(1109)...(1128)

＜223〉primer P2 corresponding region

<220>

<221>misc_recomb

<222>(1110)...(1125)

＜223〉primer P4 corresponding region

<400>1

atgcaaaagc?tagcagtcta?tgtttatatt?tacctgttca?tgctgatttc?agttgatccg 60

gtggctcttg?atgacggtag?tcaccccaca?gagaatgctg?aaaaagatgg?actgtgcaat 120

gcttgtacat?ggagacagaa?tacaaaatct?tccagaatag?aagccataaa?aattcaaatc 180

ctcagcaaac?tgcgtctgga?acaagctcct?aacattagca?gggatgttat?taaacaactt 240

ttacccaaag?ctcctccact?acaggaactg?gttgatcaat?atgacgtcca?gagagatgac 300

agtagcgatg?gctctttgga?agacgatgac?tatcatgcca?caactgaaac?gattatcaca 360

atgcctacag?agtctgattt?tcttgtacaa?atggagggaa?aaccaaaatg?ttgcttcttt 420

aagtttagct?ctaaaataca?atataacaaa?gtagtaaagg?cacaattgtg?gatatacttg 480

aggcaagtcc?aaaaacctac?aacagtgttt?gtgcagatcc?tgagacttat?taagcccatg 540

aaagacggta?caagatatac?tggaattcga?tctttgaaac?ttgacatgaa?cccaggcact 600

ggtatttggc?agagtattga?tgtgaagaca?gtgttgcaaa?attggctcaa?acagcctgaa 660

tccaatttag?gcgtcgaaat?aaaagctttt?gatgagaatg?gacgagatct?tgctgtaact 720

ttcccaggac?caggtgaaga?tggattgaat?ccatttttag?aggtcagagt?tacagacaca 780

ccgaaacgat?cccgcaga

gcgatg?agcactcgac?agaatccaga 840

tgttgtcgct?acccactaac?tgtggacttt?gaagcttttg?gatgggactg?gattatcgca 900

cctaaaagat?acaaagccaa?ttactgctct?ggagaatgtg?aatttgtatt?tctacagaaa 960

tatccgcaca?ctcatcttgt?acaccaagca?aatcctagag?gctcggcagg?cccctgctgc 1020

acgcccacca?agatgtcccc?cataaatatg?ttgtatttca?atggaaaaga?acaaataata 1080

tacggaaaga?taccagccat?ggttgtag

1128

<210>2

<211>480

<212>DNA

＜213〉artificial sequence

<220>

<222>(1)...(480)

＜223〉myostatin reorganization fusion gene sequence among the plasmid pMSTN SCAU

<220>

<222>(103)...(124)

＜223〉primer P3 corresponding region

<220>

<222>(419)...(443)

＜223〉primer P4 corresponding region

<400>2

atgcggggtt?ctcatcatca?tcatcatcat?ggtatggcta?gcatgactgg?tggacagcaa 60

atgggtcggg?atctgtacga?cgatgacgat?aaggatcgat?gg ggatccga?ttttggcctt120

gactgcgatg?agcactcgac?agaatccaga?tgttgtcgct?acccactaac?tgtggatttt?180

gaagcttttg?gatgggactg?gattatcgca?cctaaaagat?acaaagccaa?ttactgctct 240

ggagaatgtg?aatttgtatt?tctacagaaa?tatccgcaca?ctcatcttgt?acaccaagca 300

aaccctagag?gctcggcagg?cccctgctgc?acgcccacca?agatgtcccc?cataaatatg 360

ttgtatttca?atggaaaaga?acaaataata?tacggaaaga?taccagccat?ggttgtaga t420

cgttgcgggt?gctcattgag?ctcgagatct?gcagctggta?ccatggaatt?cgaagcttga?480

<210>3

<211>159

<212>PRT

＜213〉artificial sequence

<220>

<222>(1)...(159)

＜223〉the Myostatin amino acid residue sequence of expression of recombinant e. coli

<220>

<222>(5)...(10)

＜223〉His-tag labeled amino acid residue sequence

<220>

<222>(27)...(145)

＜223〉myostatin mature peptide amino acid residue sequence

<400>3

Met?Arg?Gly?Ser

1 5 10

Gly?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln

11 15 20

Met?Gly?Arg?Asp?Leu?Tyr?Asp?Asp?Asp?Asp

21 25 30

Lys?Asp?Arg?Trp?Gly?Ser? Asp?Phe?Gly?Leu

31 35 40

Asp?Cys?Asp?Glu?His?Ser?Thr?Glu?Ser?Arg

41 45 50

Cys?Cys?Arg?Tyr?Pro?Leu?Thr?Val?Asp?Phe

51 55 60

Glu?Ala?Phe?Gul?MMT?arp?Asp?Trp?Ile?Ile?Ala

61 65 70

Pro?Lys?Arg?Tyr?Lys?Ala?Asn?Tyr?Cys?Ser

71 75 80

Gly?Glu?Cys?Glu?Phe?Val?Phe?Leu?Gln?Lys

81 85 90

Tyr?Pro?His?Thr?His?Leu?Val?His?Gln?Ala

91 95 100

Asn?Pro?Arg?Gly?Ser?Ala?Gly?Pro?Cys?Cys

101 105 110

Thr?Pro?Thr?Lys?Met?Ser?Pro?Ile?Asn?Met

111 115 120

Leu?Tyr?Phe?Asn?Gly?Lys?Glu?Gln?Ile?Ile

121 125 130

Tyr?Gly?Lys?Ile?Pro?Ala?Met?Val?Val?Asp

131 135 140

Arg?Cys?Gly?Cys?Ser?Leu?Ser?Ser?Arg?Ser

141 145 150

Ala?Ala?Gly?Thr?Met?Glu?Phe?Glu?Ala

151 155 159

<210>4

<211>702

<212>DNA

＜213〉artificial sequence

<220>

<222>(1)...(702)

＜223〉myostatin reorganization fusion gene sequence among the plasmid pPICZ-MSTN SCAU

<220>

<222>(268)...(291)

＜223〉zone of primer YP1 correspondence

<220>

<222>(585)...(608)

＜223〉zone of primer YP2 correspondence

<400>4

atgagatttc?cttcaatttt?tactgctgtt?ttattcgcag?catcctccgc?attagctgct 60

ccagtcaaca?ctacaacaga?agatgaaacg?gcacaaattc?cggctgaagc?tgtcatcggt 120

tactcagatt?tagaagggga?tttcgatgtt?gctgttttgc?cattttccaa?cagcacaaat 180

aacgggttat?tgtttataaa?tactactatt?gccagcattg?ctgctaaaga?agaaggggta 240

tctctcgaga?aaagagaggc?tgaagct gaa?ttcgattttg?gccttgactg?cgatgagcac300

tcgacagaat?ccagatgttg?tcgctaccca?ctaactgtgg?actttgaagc?ttttggatgg 360

gactggatta?tcgcacctaa?aagatacaaa?gccaattact?gctctggaga?atgtgaattt 420

gtatttctac?agaaatatcc?gcacactcat?cttgtacacc?aagcaaatcc?tagaggctcg 480

gcaggcccct?gctgcacgcc?caccaagatg?tcccccataa?atatgttgta?tttcaatgga 540

aaagaacaaa?taatatacgg?aaagatacca?gccatggttg?tagat cgttg?cgggtgctca600

aaggtacctc?gagccgcggc?ggccgccagc?tttctagaac?aaaaactcat?ctcagaagag?660

gatctgaata?gcgccgtcga?ccatcatcat?catcatcatt?ga 702

<210>5

<211>233

<212>PRT

＜213〉artificial sequence

<220>

<222>(1)...(233)

＜223〉the Myostatin amino acid residue sequence of expression of recombinant yeast

<220>

<222>(1)...(89)

＜223〉alpha factor signal peptide amino acid residue sequence

<220>

<222>(92)...(200)

＜223〉myostatin mature peptide amino acid residue sequence

<400>5

Met?Arg?Phe?Pro?Ser?Ile?Phe?Thr?Ala?Val

1 5 10

Leu?Phe?Ala?Ala?Ser?Ser?Ala?Leu?Ala?Ala

11 15 20

Pro?Val?Asn?Thr?Thr?Thr?Glu?Asp?Glu?Thr

21 25 30

Ala?Gln?Ile?Pro?Ala?Glu?Ala?Val?Ile?Gly

31 35 40

Tyr?Ser?Asp?Leu?Glu?Gly?Asp?Phe?Asp?Val

41 45 50

Ala?Val?Leu?Pro?Phe?Ser?Asn?Ser?Thr?Asn

51 55 60

Asn?Gly?Leu?Leu?Phe?Ile?Asn?Thr?Thr?Ile

61 65 70

Ala?Ser?Ile?Ala?Ala?Lys?Glu?Glu?Gly?Val

71 75 80

Ser?Leu?Glu?Lys?Arg?Glu?Ala?Glu?Ala?Glu

81 85 90

Phe? Asp?Phe?Gly?Leu?Asp?Cys?Asp?Glu?His

91 95 100

Ser?Thr?Glu?Ser?Arg?Cys?Cys?Arg?Tyr?Pro

101 105 110

Leu?Thr?Val?Asp?Phe?Glu?Ala?Phe?Gly?Trp

111 115 120

Asp?Trp?Ile?Ile?Ala?Pro?Lys?Arg?Tyr?Lys

121 125 130

Ala?Asn?Tyr?Cys?Ser?Gly?Glu?Cys?Glu?Phe

131 135 140

Val?Phe?Leu?Gln?Lys?Tyr?Pro?His?Thr?His

141 145 150

Leu?Val?His?Gln?Ala?Asn?Pro?Arg?Gly?Ser

151 155 160

Ala?Gly?Pro?Cys?Cys?Thr?Pro?Thr?Lys?Met

161 165 170

Ser?Pro?Ile?Asn?Met?Leu?Tyr?Phe?Asn?Gly

171 175 180

Lys?Glu?Gln?Ile?Ile?Tyr?Gly?Lys?Ile?Pro

181 185 190

Ala?Met?Val?Val?Asp?Arg?Cys?Gly?Cys?Ser

191 195 200

Lys?Val?Pro?Arg?Ala?Ala?Ala?Ala?Ala?Ser

201 205 210

Phe?Leu?Glu?Gln?Lys?Leu?Ile?Ser?Glu?Glu

211 215 220

Asp?Leu?Asn?Ser?Ala?Val?Asp?His?His?His

221 225 230

His?His?His

231 233

Claims (7)

1. the preparation of myostatin gene engineered vaccine, it is characterized in that 1. cloning livestock and poultry muscle amicine encoding gene, 2. utilize livestock and poultry muscle amicine encoding gene, 3. the recombination fusion protein of preparation myostatin utilizes the recombinant vaccine of this Myostatin as the immunogen preparing myostatin.

2. according to the preparation of the said myostatin gene engineered vaccine of claim 1, it is characterized in that cloning livestock and poultry muscle amicine encoding gene, 1. utilize according to the chicken muscle amicine gene order of being delivered among the GenBank, design this gene specificity forward primer P1 (sequence is: 5 '-ATGCAAAAGCTAGCAGTCTATG-3 ') and downstream primer P2 (sequence is: 5 '-TCATGAGCACCCGCAACGAT-3 '); 2. utilize guanidinium isothiocyanate-chloroform-isopropyl alcohol method from the livestock and poultry muscle tissue, to extract total RNA and become the first chain cDNA with primer P2 reverse transcription as template; 3. amplify the coded cDNA sequence of the myostatin of chicken, pig or goose by the PCR reaction as template and primer P1, P2 with this first chain cDNA.

3. according to the preparation of the said myostatin gene engineered vaccine of claim 1, it is characterized in that preparing livestock and poultry muscle amicine recombination fusion protein, one of preparation method is to utilize IPTG to induce escherichia coli E.coli BL21 (DE3) strain to express the preparation recombination fusion protein: 1. according to the cDNA base sequence of myostatin mature peptide and the multiple clone site base sequence of colibacillus expression plasmid pRSET A, (sequence is 5 ' ATT to synthetic forward primer P3 GGATCCGATTTTGGCCTTG ACT-3 ', underscore partly are placing restrictions on property of BamH I restriction enzyme site) and downstream primer P4 (sequence is 5 ' AAT GAGCTCAATGAGCACCCGCAACGA-3 ', underscore partly are placing restrictions on property of Sac I restriction enzyme site); 2. utilize this myostatin coded cDNA sequence that primer, above-mentioned post transcription cloning are gone out as template, amplify the cDNA cloned sequence of myostatin mature peptide once more by the PCR reaction; Behind the cDNA cloned sequence process BamH I and SacI double digestion of the myostatin mature peptide that 3. amplifies, be cloned between the BamH I and Sac I two restriction enzyme sites of expression plasmid pRSET A, be built into recombinant expression plasmid pMSTN SCAU; 4. with recombinant expression plasmid pMSTN SCAU transformed into escherichia coli E.coli BL21 (DE3) strain, the IPTG that adds 0.01mmol/L in culture fluid induces and made recombinant bacteria express Myostatin in 6 hours.

4. according to the preparation of the said myostatin gene engineered vaccine of claim 1, it is characterized in that preparing livestock and poultry muscle amicine recombination fusion protein, two of preparation method is to utilize Pichia sp. to express this fusion rotein under methanol induction: 1. according to the base sequence of lion-headed goose myostatin mature peptide code cDNA be used to transform the base sequence of multiple clone site of the shuttle plasmid pPICZ α A of Pichia sp., (sequence is 5 ' AGG to design forward primer YP1 GAATTCGATTTTGGCC TTGACTGC-3 ', underscore partly are EcoR I restriction enzyme site) and downstream primer YP2 (5 ' AAT GGTACCTTTGAGCACCCGCAACG-3 ', underscore partly are Kpn I restriction enzyme site); 2. utilize this myostatin coded cDNA sequence that primer, above post transcription cloning are gone out as template, amplify the cDNA yeast shuttle plasmid cloned sequence of myostatin gene mature peptide once more by the PCR reaction, behind this cDNA cloned sequence process EcoR I and the KpnI double digestion, after being cloned between the EcoRI of shuttle plasmid pPICZ α A and the KpnI restriction enzyme site, be built into recombinant plasmid pPICZ-MSTN SCAU; 3. with recombinant plasmid pPICZ-MSTN SCAU linearisation and transformed competence colibacillus yeast X-33, and at Zeocin ^TMThe YPDS solid medium cultivate, select white colony to YPDS fluid medium and cultivate, and react by PCR with forward primer AOX1 (5 ' GACTG GTTCCAATTGACAAGC-3 ') and downstream primer AOX2 (5 ' GCAAATGGCATTCT GACATCC-3 ') and to identify and to select Mut ⁺The phenotype recombination yeast; 4. picking correctly is integrated into genomic single recombinant yeast with myostatin mature peptide fusion gene and is inoculated in the BMGY culture medium culturing, to adding methanol in the culture fluid to final concentration 0.5% abduction delivering Myostatin, expressed Myostatin can be secreted to born of the same parents in the culture fluid by yeast.

5. according to the preparation of the said myostatin gene engineered vaccine of claim 1, it is characterized in that the Myostatin that purification is expressed, will be with the above escherichia coli E.coli BL21 (DE3) that expresses Myostatin of 8Mol carbamide cracking, or go out to contain the Yeast Cultivation liquid of Myostatin by centrifugalize, two kinds of solution that contain Myostatin are carried out chromatography by Ni-NTA gel-purified resin, and last eluting is purified into the antigen of Myostatin as immunity usefulness.

6. according to the preparation of the said myostatin gene recombinant vaccine of claim 1, it is characterized in that the above Myostatin that is purified into as the immunogen preparing oil emulsion vaccine: 1. Myostatin is dissolved in the aqueous solution that contains 2～4%Tween 80 and makes water by the concentration of 2～6mg/mL; 2. with No. 10 lightweight white oils and Si Ben 80 by 94% and 6% mixed, inwardly add 2% aluminium stearate again, mix homogeneously is made oil phase; 3. water and oil phase are pressed 4: 6 mixed, and under blender, make water in oil homogenizing emulsion as the myostatin gene engineered vaccine.

7. the application of myostatin gene engineered vaccine, it mainly is approach by active immunity, be used to promote the muscle growth of animal, it is characterized in that: to the pig of growth stage, cattle, sheep, goat and deer, every 3～4 weeks in intramuscular injection myostatin gene engineering oil emulsion vaccine, promote the animal vivo immuning system to produce the antibody of anti-self myostatin, this antibody can neutralize and suppress the myostatin of animal muscle emiocytosis, remove the inhibitory action of myostatin, promote the growth of animal muscle the livestock and poultry muscle growth.