CN103554265A - Solid-phase synthesis method for gonadotropin-releasing hormone (GnRH) castration vaccine - Google Patents
Solid-phase synthesis method for gonadotropin-releasing hormone (GnRH) castration vaccine Download PDFInfo
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Abstract
The invention provides a method for preparing a gonadotropin-releasing hormone (GnRH) parallel body and a vaccine in solid-phase conditions. After Lys is coupled on carrier resin, rink amide liners are respectively coupled on the two side chains of the Lys, and two same peptide chains are synthesized simultaneously. Disulfide bond bridging is realized on a solid phase, full-protective oxidation is adopted, and a large amount of impurities caused by the reaction involved by the side chains in the process are avoided.
Description
Technical field
The present invention relates to a kind of polypeptide synthesis method, relate in particular to a kind of solid phase synthesis for the synthetic method of the polypeptide of GnRH castration vaccine.
Background technology
For centuries, except planting with male animal, the male animal of most of domestic animals adopts operation castration always, the feed loss causing because of sexuality at fattening stage to reduce animal, reduce and to oestrus and that the Aggression of buck causes is injured and dead, and can improve the quality (as smell of mutton, peculiar smell alleviate) of meat, can also utilize the growth characteristic of castrated animal to make its getting fat.Therefore, castration is that domestic animal is fattened the important technical links in production process.
Yet after the advantage of self-discovery castration, in herding production, castration technology used is operation castration, very large to Animal stress, generally needs within 7-10 days, could recover, and sometimes can cause animal dead because of wound infection always.In addition, to the modus operandi castration of jenny, not only infection risk is larger, and operator's technology and muscle power are all had to very high requirement.In addition,, in a lot of countries and regions, the radical in animal welfare exerts pressure to prevent the castration of performing the operation always.
A kind of selection of alternative operation method of emasculation is to adopt the inoculation of GnRH castration vaccine, eliminate GnRF (GnRH, also referred to as LHRH) impact in vivo, make the animal body can not sexual maturity, can access better feed efficiency, compare safe ready simultaneously with operation castration, reduce infection chance, and partly retain the hormesis of sexual gland to breeding performonce fo animals.The antigen general formula of GnRH castration vaccine is as follows:
Z
1-Glx-His-Trp
1-Ser-Tyr-Gly-Leu-Arg-Pro-(Gly-X-Gln-His-Trp
2-Ser-Tyr-Gly-Leu-Arg-Pro)
n-Gly-Z
Wherein, Trp
1and Trp
2be respectively tryptophane or formylation tryptophane (N-indoles-formyl radical-tryptophane); X is direct connecting key or Gly, Gln; Z
1-Glx is pGlu or the Glu that connects additional amino acid; Glx-Z
1for Gly-NH
2or the Gly of connection additional amino acid.
In the research of early stage GnRH vaccine, adopt GnRH monomer, immune consumption is large, needs multiple injection more, and US4608251 adopts body immunity arranged side by side, and result shows that immunogenicity is higher than monomer, better effects if.US6284733B1 carries out dimerization by GnRH body arranged side by side, forms dipolymer; Sequence is:
Wherein, * can be the amino acid such as Gly or D-Lys, D-Glu.Result of study shows, the immune castration vaccine that GnRH body dipolymer arranged side by side is basic structure is very effective.
In the preparation method of the disclosed described body arranged side by side of above-mentioned patent, the monomer of solid phase synthesis castration vaccine body arranged side by side before this, then by high performance liquid phase technology of preparing, prepare high-purity monomer, finally high-purity monomer is dissolved, under liquid-phase reaction condition, between Cys, connect and obtain body arranged side by side, then high performance liquid chromatography is purified.This method is also the current the most frequently used method of preparing GnRH body arranged side by side.
But in the disclosed preparation method of above-mentioned patent, in the end, in the process of synthetic body arranged side by side, side chain is easy to participate in reaction, thereby introduces a large amount of impurity, for purifying work afterwards brings very large difficulty.
Summary of the invention
For prior art, prepare that foreign matter content in body process arranged side by side is high, the problem of poor product quality, the invention provides the method for a kind of solid phase synthesis GnRH body arranged side by side.
First aspect of the present invention is to provide the method for a kind of solid phase synthesis GnRH body arranged side by side, and step comprises:
Step 1 connects Lys amino acid on solid-phase synthetic peptide vector resin (supporting resin), distinguishes coupling rink amide linker on two side chains of Lys, obtains the intermediate (A) of following structure:
Wherein, PG is side chain protected group; Z is one or more amino acid or connecting key, and wherein, Z is in amino acid whose situation, can be with or without side chain protected group, more preferably, and the amino acid that Z is non-Cys;
Step 3, then according to required synthetic GnRH body peptide chain structure arranged side by side successively other amino acid of coupling, obtains the intermediate (C) of following structure:
Wherein, peptide is peptide chain;
Step 4, utilizes two amino acid whose sulfydryls of Cys shown in intermediate (C), and between two Cys positions, formation-S-S-link, obtains intermediate (D);
But should be noted that, formation-S-S-link between two Cys positions shown in intermediate (C) of the present invention, can be to form between two peptide chains in same intermediate (C), can be also to form between the peptide chain between different intermediates (C);
In the method described in the present invention is aspect first, Z is preferably the sequence that 1-4 amino acid forms, and 1-2 amino acid more preferably, as the sequence of Gly or Lys or the two composition, more preferably Lys.
In the method described in the present invention is aspect first, in described one or more coupling process, be preferably under condensing agent exists and carry out, described condensing agent can be to can be used in arbitrarily the condensing agent that promotes carboxylic acid and amine condensation, as any one or a few the combination in carbodiimide class condensing agent, salt condensing agent, organic phosphates condensing agent and other condensing agent etc.Wherein:
Carbodiimide class condensing agent is as dicyclohexylcarbodiimide (DCC), DIC (DIC), 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDCI) etc.;
Salt condensing agent is as O-(7-azepine benzotriazole-1-yl)-bis-(dimethylin) carbon hexafluorophosphate (HATU), O-(benzotriazole-1-yl)-bis-(dimethylin) carbon hexafluorophosphate (HBTU), O-(5-Chloro-Benzotriazole-1-yl)-bis-(dimethylin) carbon hexafluorophosphate HCTU, O-(7-azepine benzotriazole-1-yl)-bis-(Pyrrolidine base) carbon hexafluorophosphates (HAPyU), O-(benzotriazole-1-yl)-bis-(Pyrrolidine base) carbon hexafluorophosphates (HBPyU), O-(benzotriazole-1-yl)-bis-(dimethylin) carbon a tetrafluoro borate (TBTU), O-(N-succimide base)-bis-(dimethylin) carbon a tetrafluoro borate (TSTU), (down alkene-2 fall to O-in N-endo-5-, 3-bis-carbon imide bases)-bis-(dimethylin) carbon a tetrafluoro borate (TNTU), benzotriazole-1-base oxygen-tri-(dimethylin) Phosphonium hexafluorophosphate (BOP), benzotriazole-1-base oxygen-tri-(Pyrrolidine base) Phosphonium hexafluorophosphate (PyBOP), 7-azepine benzotriazole-1-base oxygen-tri-(Pyrrolidine base) Phosphonium hexafluorophosphate PyAOP etc.,
Organic phosphates condensing agent is as diphenyl phosphoryl chloride (DPP-Cl), diethyl cyanophosphonate (DECP), azide diphenyl phosphate (DPPA), sulfo-solutions of dimethyl phosphoryl base nitrine (MPTA), two (2-oxygen-3-oxazolidinyl) phosphoryl chloride (BOP-Cl) etc.;
Other condensing agent is as triphenyl phosphorus-many methyl halides, triphenyl phosphorus-hexachloroacetone, triphenyl phosphorus-NBS(NBS:N-bromo-succinimide) etc.
Wherein, in described one or more coupling process, can also add activator, the activator that can be used for carbodiimide class condensing agent comprises 4-N, N-lutidine (DMAP), 4-pyrrolidyl pyridine (4-PPY), 1-hydroxy benzo triazole (HOBt), 1-hydroxyl-7-azo benzotriazole (HOAt), (N-hydroxy-succinamide) HOSu, HP (NHPI), N-hydroxyl-5-norbornylene-2, the combination of any one or a few in 3-dicarboximide NHNI, Pentafluorophenol (PFPOH).
Wherein, in described one or more coupling process, can also add alkaline matter, can be organic bases and/or mineral alkali, organic bases more preferably, and described mineral alkali is as NaHCO
3, Na
2cO
3, NaOH, K
2cO
3, KOH, KH
2pO
4deng, described organic bases is preferably organic amine material, as triethylamine, N-methylmorpholine (NMM), diisopropylethylamine (DIEA) etc.
In the method described in the present invention is aspect first, step 1 connects the amino acid whose method of Lys and is preferably: Fmoc-Lys (Boc)-OH is connected on vector resin, and then deprotection group, obtains NH
2-Lys-supporting resin.
In the method described in the present invention is aspect first, the method for step 1 coupling rink amide linker is preferably: under DIC+NMM+HOBT exists, and Fmoc-rink amide linker and NH
2-Lys-supporting resin reaction, then deprotection base Fmoc obtains intermediate (A).
In the method described in the present invention is aspect first, in intermediate A the one or more amino acid of coupling, Cys amino acid and in intermediate B other amino acid whose method of coupling can be any one coupling method that adopts existing solid-phase synthetic peptide.
In the method described in the present invention is aspect first, the concept of described Side chain protective group, purposes and selection are all well-known to those skilled in the art.
In the method described in the present invention is aspect first, described vector resin can be arbitrarily for the resin of solid-phase synthetic peptide, can be the derivative of vinylbenzene-benzene divinyl cross-linked resin, polyacrylamide, polyethylene-ethylene glycol resin and above-mentioned resin; As PAM, MBHA, Merrifield, Wang(king's resin), 2-Cl-Trt etc., the present invention is preferably mbha resin.
In the situation that described vector resin is mbha resin, in step 5, the separated of deprotection base and resin can synchronously carry out.Preferably, step 5 is implemented under strong acidic condition.
Wherein, described strong acid is preferably trifluoroacetic acid (TFA).
More preferably, step 5 is implemented under TFA+ tri isopropyl silane (TIS) exists.
In method described in first aspect of the present invention, step 4 is preferably take iodine and reacts as oxygenant.
More preferably, the operation steps of step 4 comprises: in ice-water bath, and I
2join in intermediate (C) methanol solution and react.Reaction times is preferably 30min.
Wherein, described I
2the form being preferably with solution adds, more preferably I
2methanol solution, its concentration is preferably 0.5-2mol/L, more preferably 1-1.5mol/L.
In method described in first aspect of the present invention, can also comprise step 6, by prepared GnRH body freeze-drying arranged side by side.
In method described in first aspect of the present invention, described GnRH body peptide chain structure arranged side by side is preferably any one or a few the mixture with following structure.
p-Glu-His-Trp-Ser-Tyr-*-Leu-Arg-Pro-(&-X-Gln-His-Trp-Ser-Tyr-#-Leu-Arg-Pro)?
n-Gly-Cys-Z-NH
2
Wherein, the integer that n is >=0, is preferably 0-2, and more preferably 1;
Wherein, Z is one or more amino acid, and is preferably Gly or Lys, more preferably Lys;
Wherein, X is Gly or Gln or connecting key;
Wherein, * and # can be identical or different, and are independently respectively any one D-amino acid or Gly; Wherein, described D-amino acid is preferably any one or a few in D-Ala, D-Lys, D-Glu, D-Orn; Wherein, & is Gly or Lys.
Second aspect of the present invention is to provide a kind of method of the GnRH of preparation castration vaccine, and step comprises:
Step 1, prepares GnRH body arranged side by side according to the method described in first aspect of the present invention;
In method described in aspect second of the present invention, in made GnRH vaccine, can also add immunological adjuvant.
Synthetic method provided by the invention, an amino acid of coupling (as Lys) on vector resin, then coupling rink amide linker on two side chains respectively, has realized two peptide chains simultaneously synthetic.
Disulfide linkage is oxidized formation on solid phase carrier; than traditional liquid phase oxidation technique; what the present invention adopted is full guard oxidation; other amino acid whose side chain in peptide sequence; in sulfydryl bridging process; all in protected state, avoided amino acid side chain in traditional method to participate in a large amount of impurity that reaction brings, for next step purifying work brings very big facility.
At MBHA as vector resin in the situation that; the present invention can also utilize the distinctive acid acceptance of MBHA; the summit that can realize peptide chain discharges, and cracking under the strong acid conditions such as TFA can and realize the cracking of synchronizeing of polypeptide and the separated of resin and amino acid side chain protecting group simultaneously.
Should be understood that, term in the present invention " amino acid " also comprises the amino-acid residue after being connected on peptide chain, and this is apparent for those skilled in the art.
Therefore, compare with traditional technology, synthetic method craft of the present invention quality more simple, the thick product of gained is significantly improved, and is conducive to subsequent purification, contributes to isolate high-quality antigenic peptide.
Accompanying drawing explanation
Fig. 1 is in the synthetic GnRH body method arranged side by side of the present invention, synchronously realizes the schematic diagram of polypeptide and amino acid side chain protecting group cracking separated with resin fixed point; Wherein PG is amino acid side chain protecting group;
Fig. 2 is body TOF-MS mass spectrometric detection result arranged side by side synthetic in the embodiment of the present invention 1;
Fig. 3 is body TOF-MS mass spectrometric detection result arranged side by side synthetic in the embodiment of the present invention 2;
Fig. 4 is body TOF-MS mass spectrometric detection result arranged side by side synthetic in the embodiment of the present invention 3.
Embodiment
Embodiment 1
In the present embodiment, GnRH peptide chain is: p-Glu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-Gln-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-Cys-Lys-NH
2.
The present embodiment GnRH body arranged side by side and vaccine synthetic method are as follows:
synthetic GnRH body arranged side by side
Take mbha resin as vector resin, more than methylene dichloride swelling 3h, carry out linked reaction with Fmoc-Lys (BOC)-OH, Lys is coupled on mbha resin, obtain Fmoc-Lys (BOC)-MBHA Resin.Method is: the Fmoc-Lys of 3eq (Fmoc)-OH and the DIC of 3eq HOBT and 3eq and the NMM of 1.5eq react, and reaction solvent is dry DMF, reaction times 1.5h.
Under 20% piperidines and DMF exist, remove the protecting group Fmoc of Lys, obtain NH
2-Lys-MBHA Resin.
Under catalyzer and coupling agent DIC+NMM+HOBT existence, difference coupling Fmoc-rink amide linker on two side chains of Lys, method is: the Fmoc-Rink amid linker-OH of 6eq and the DIC of 6eqHOBT and 6eq and the NMM of 3eq react, reaction solvent is dry DMF, reaction times 1.5h.
Then remove protecting group, obtain:
Then adopting uses the same method carries out linked reaction with Fmoc-Lys (BOC)-OH, removes Fmoc protecting group, then according to the above-mentioned peptide chain structure of the present embodiment successively other amino acid of coupling, synthetic when realizing two peptide chains.Obtain the polypeptide of coupling resin.
Coupling is had in the polypeptide of resin and add methyl alcohol, under ice-water bath condition, stir 30min, drip the I of 1mol/L
2/ EtOH solution, until reaction system becomes brown color, continues to stir, if brown color disappears, continues to drip I
2/ EtOH solution.After reinforced complete reaction 30min, with in 10% (v/v) Sulfothiorine with excessive I
2.Ether and methanol wash, then vacuum-drying.
Under 95% (v/v) TFA+2.5% (v/v) water+2.5% (v/v) TIS exists; under 25-30 ℃ of condition, react 3h and carry out cracking, make polypeptide separated with resin fixed point, with this understanding; can also realize the synchronous cracking of amino acid side chain protecting group, as shown in Figure 1 simultaneously.
The lysate obtaining after the cracking of Ether precipitation, centrifugal, abandoning supernatant, washing, repeatable operation 2-3 time, throw out is fully dry under vacuum condition.
Preparative high performance liquid chromatography instrument is purified, and collects purity higher than 95% product, freeze-drying.
Fig. 2 has provided the TOF-MS mass spectrometric detection result of the synthetic body arranged side by side of the present embodiment, and spectrogram result is consistent with the present embodiment object product.
the preparation of GnRH vaccine
GnRH liquid solution arranged side by side joins in carrier proteins solution, adds coupling agent to carry out coupling.Make the solution of 400 μ g/ml.
Fully emulsified with French 206 immunological adjuvant equal-volumes, make vaccine.
In the present embodiment, GnRH peptide chain is:
p-Glu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Lys-Gln-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Lys-Cys-Gly-NH
2
The present embodiment GnRH body arranged side by side and vaccine synthetic method are as follows:
synthetic GnRH body arranged side by side
Take mbha resin as vector resin, carry out linked reaction with Fmoc-Lys (BOC)-OH, Lys is coupled on mbha resin, obtain Fmoc-Lys (BOC)-MBHA Resin.Under 20% piperidines and DMF exist, remove the protecting group of Lys, obtain NH
2-Lys-MBHA Resin.
Under catalyzer and coupling agent DIC+NMM+HOBT existence, on two side chains of Lys, difference coupling Fmoc-rink amide linker, then removes protecting group, obtains:
Coupling Gly and Cys, then according to the above-mentioned peptide chain structure of the present embodiment successively other amino acid of coupling, synthetic when realizing two peptide chains successively.
Iodine is catalyzer, carries out oxidizing reaction and between the Cys of two peptide chains amino acid, realizes disulfide linkage bridging.
There is lower cracking in TFA+EDT+Thioanisole, makes polypeptide separated with resin fixed point, with this understanding, can also realize the synchronous cracking of amino acid side chain protecting group simultaneously.
Preparative high performance liquid chromatography instrument is purified, and collects purity higher than 95% product, freeze-drying.
The synthetic GnRH physique spectrogram arranged side by side of the present embodiment as shown in Figure 3.
the preparation of GnRH vaccine
GnRH liquid solution arranged side by side joins in carrier proteins solution, adds coupling agent to carry out coupling.Make the solution of 400 μ g/ml.
Fully emulsified with French 206 immunological adjuvant equal-volumes, make vaccine.
Embodiment 3
In the present embodiment, GnRH peptide chain is:
p-Glu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-Gln-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-Cys-Gly-NH
2
The present embodiment GnRH body arranged side by side and vaccine synthetic method are as follows:
synthetic GnRH body arranged side by side
Take mbha resin as vector resin, carry out linked reaction with Fmoc-Lys (BOC)-OH, Lys is coupled on mbha resin, obtain Fmoc-Lys (BOC)-MBHA Resin.Under 20% piperidines and DMF exist, remove the protecting group of Lys, obtain NH
2-Lys-MBHA Resin.
Under catalyzer and coupling agent DIC+NMM+HOBT existence, on two side chains of Lys, difference coupling Fmoc-rink amide linker, then removes protecting group, obtains:
Coupling Gly and Cys, then according to the above-mentioned peptide chain structure of the present embodiment successively other amino acid of coupling, synthetic when realizing two peptide chains successively.
Iodine is catalyzer, carries out oxidizing reaction and between the Cys of two peptide chains amino acid, realizes disulfide linkage bridging.
TFA+H
2there is lower cracking in O+Thioanisole, makes polypeptide separated with resin fixed point, with this understanding, can also realize the synchronous cracking of amino acid side chain protecting group simultaneously.
Preparative high performance liquid chromatography instrument is purified, and collects purity higher than 95% product, freeze-drying.
The synthetic GnRH physique spectrogram arranged side by side of the present embodiment as shown in Figure 4.
the preparation of GnRH vaccine
GnRH liquid solution arranged side by side joins in carrier proteins solution, adds coupling agent to carry out coupling.Product is dissolved in the DEAE-dextran solution of 5% mass concentration, makes the pharmaceutical solutions of 400 μ g/ml.
Embodiment 4
In the present embodiment, GnRH peptide chain is:
p-Glu-His-Trp-Ser-Tyr-D-Orn-Leu-Arg-Pro-Gly-Gln-His-Trp-Ser-Tyr-D-Orn-Leu-Arg-Pro-Gly-Cys-Lys-NH
2
The present embodiment GnRH body arranged side by side and vaccine synthetic method are as follows:
synthetic GnRH body arranged side by side
Take mbha resin as vector resin, carry out linked reaction with Fmoc-Lys (BOC)-OH, Lys is coupled on mbha resin, obtain Fmoc-Lys (BOC)-MBHA Resin.Under 20% piperidines and DMF exist, remove the protecting group of Lys, obtain NH
2-Lys-MBHA Resin.
Under catalyzer and coupling agent DIC+NMM+HOBT existence, on two side chains of Lys, difference coupling Fmoc-rink amide linker, then removes protecting group, obtains:
Coupling Lys and Cys, then according to the above-mentioned peptide chain structure of the present embodiment successively other amino acid of coupling, synthetic when realizing two peptide chains successively.
Iodine is catalyzer, carries out oxidizing reaction and between the Cys of two peptide chains amino acid, realizes disulfide linkage bridging.
There is lower cracking in TFA+EDT+Thioanisole, makes polypeptide separated with resin fixed point, with this understanding, can also realize the synchronous cracking of amino acid side chain protecting group simultaneously.
Preparative high performance liquid chromatography instrument is purified, and collects purity higher than 95% product, freeze-drying.
the preparation of GnRH vaccine
GnRH liquid solution arranged side by side joins in carrier proteins solution, adds coupling agent to carry out coupling.Product is dissolved in the DEAE-dextran solution of 5% mass concentration, makes the pharmaceutical solutions of 400 μ g/ml.
In the present embodiment, GnRH peptide chain is:
p-Glu-His-Trp-Ser-Tyr-D-Glu-Leu-Arg-Pro-Gly-Gln-His-Trp-Ser-Tyr-D-Glu-Leu-Arg-Pro-Gly-Cys-Gly-NH
2
The present embodiment GnRH body arranged side by side and vaccine synthetic method are as follows:
synthetic GnRH body arranged side by side
Take mbha resin as vector resin, carry out linked reaction with Fmoc-Lys (BOC)-OH, Lys is coupled on mbha resin, obtain Fmoc-Lys (BOC)-MBHA Resin.Under 20% piperidines and DMF exist, remove the protecting group of Lys, obtain NH
2-Lys-MBHA Resin.
Under catalyzer and coupling agent DIC+NMM+HOBT existence, on two side chains of Lys, difference coupling Fmoc-rink amide linker, then removes protecting group, obtains:
Coupling Gly and Cys, then according to the above-mentioned peptide chain structure of the present embodiment successively other amino acid of coupling, synthetic when realizing two peptide chains successively.
Iodine is catalyzer, carries out oxidizing reaction and between the Cys of two peptide chains amino acid, realizes disulfide linkage bridging.
There is lower cracking in TFA+EDT+Thioanisole, makes polypeptide separated with resin fixed point, with this understanding, can also realize the synchronous cracking of amino acid side chain protecting group simultaneously.
Preparative high performance liquid chromatography instrument is purified, and collects purity higher than 95% product, freeze-drying.
the preparation of GnRH vaccine
GnRH liquid solution arranged side by side joins in carrier proteins solution, adds coupling agent to carry out coupling.Product is dissolved in the DEAE-dextran solution of 5% mass concentration, makes the pharmaceutical solutions of 400 μ g/ml.
Embodiment 6
In the present embodiment, GnRH peptide chain is:
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gln-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Cys-NH
2
The present embodiment GnRH body arranged side by side and vaccine synthetic method are as follows:
synthetic GnRH body arranged side by side
Take mbha resin as vector resin, carry out linked reaction with Fmoc-Lys (BOC)-OH, Lys is coupled on mbha resin, obtain Fmoc-Lys (BOC)-MBHA Resin.Under 20% piperidines and DMF exist, remove the protecting group of Lys, obtain NH
2-Lys-MBHA Resin.
Under catalyzer and coupling agent DIC+NMM+HOBT existence, on two side chains of Lys, difference coupling Fmoc-rink amide linker, then removes protecting group, obtains:
Coupling Gys, then according to the above-mentioned peptide chain structure of the present embodiment successively other amino acid of coupling, synthetic when realizing two peptide chains.
Iodine is catalyzer, carries out oxidizing reaction and between the Cys of two peptide chains amino acid, realizes disulfide linkage bridging.There is lower cracking in TFA+EDT+Thioanisole, makes polypeptide separated with resin fixed point, with this understanding, can also realize the synchronous cracking of amino acid side chain protecting group simultaneously.
Preparative high performance liquid chromatography instrument is purified, and collects purity higher than 95% product, freeze-drying.
the preparation of GnRH vaccine
GnRH liquid solution arranged side by side joins in carrier proteins solution, adds coupling agent to carry out coupling.Make the solution of 400 μ g/ml.
Fully emulsified with French 206 immunological adjuvant equal-volumes, make vaccine.
GnRH castration vaccine prepared by the method for the invention, twice of immunity, can effectively excite GnRH antibody to produce, in and body in endogenous GnRH, thereby significantly reduce the size of testis, and reduce the sex hormone levels such as testosterone, reach the object of gentle castration, the weight loss that the castration that prevents from performing the operation causes is hemorrhage, infect and stress cause.
The GnRH castration vaccine that uses the present invention to prepare carries out after immunity, and with respect to the boar of operation castration, the speed of growth is very fast, and after butchering, on trunk, the eye muscle area at same position is obviously larger, and fat thickness is obviously thinner; With respect to uncastrated boar, the speed of growth is basically identical, and the quality of meat is good, there is no the distinctive shy taste of boar.
Above specific embodiments of the invention be have been described in detail, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and alternative also all among category of the present invention.Therefore, equalization conversion and the modification done without departing from the spirit and scope of the invention, all should contain within the scope of the invention.
Claims (10)
1. a method for solid phase synthesis GnRH body arranged side by side, is characterized in that, step comprises:
Step 1 connects Lys amino acid on solid-phase synthetic peptide vector resin, distinguishes coupling rink amide linker on two side chains of Lys, obtains the intermediate (A) of following structure:
Step 2, successively after the one or more amino acid of coupling, coupling Cys amino acid, or direct coupling Cys amino acid in other amino acid whose situation of not coupling, obtain the intermediate (B) of following structure:
Wherein, PG is side chain protected group; Z is one or more amino acid or connecting key;
Step 3, then according to required synthetic GnRH body peptide chain structure arranged side by side successively other amino acid of coupling, obtains the intermediate (C) of following structure:
Wherein, peptide is peptide chain;
Step 4, utilizes the sulfydryl in two Cys shown in intermediate (C), and between Cys, formation-S-S-link, obtains intermediate (D);
Step 5, removes Side chain protective group, and makes polypeptide separated with vector resin, obtains GnRH body arranged side by side (E):
2. method according to claim 1, is characterized in that, Z is the sequence that 1-4 amino acid forms.
3. method according to claim 2, is characterized in that, Z is Gly or Lys or the sequence of the two composition.
4. method according to claim 1, is characterized in that, described vector resin is mbha resin.
5. method according to claim 4, is characterized in that, the process of deprotection base and resin in step 5 is implemented under strong acidic condition.
6. method according to claim 1, is characterized in that, the link process of formation-S-S-described in step 4 is implemented under iodine exists.
7. method according to claim 6, is characterized in that, formation-S-S-link process is implemented under ice-water bath condition described in step 4.
8. method according to claim 1, is characterized in that, also comprises:
Step 6, by prepared GnRH body freeze-drying arranged side by side.
9. a method of preparing GnRH castration vaccine, is characterized in that, step comprises:
Step 1, prepares GnRH body arranged side by side according to the method described in first aspect of the present invention;
Step 2, carries out coupling by described GnRH body arranged side by side and carrier proteins, is dissolved in and in solvent, makes GnRH vaccine.
10. the method for preparing GnRH castration vaccine according to claim 9, is characterized in that, in made GnRH vaccine, also adds immunological adjuvant.
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| CN201310557715.2A CN103554265B (en) | 2013-11-11 | 2013-11-11 | The method of the synthetic GnRH castration vaccine of solid phase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108047328A (en) * | 2017-12-14 | 2018-05-18 | 天津瑞普生物技术股份有限公司 | A kind of GnRH polypeptide antigens and its application for being used to prepare castration vaccine |
| CN110954706A (en) * | 2019-11-20 | 2020-04-03 | 宁波三生生物科技有限公司 | Method for measuring coupling rate of GnRH paralog dimer artificial antigen |
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| US6284733B1 (en) * | 1995-06-07 | 2001-09-04 | Dlo Instituut Voor Dierhouderij En Diergezondheid | Peptide, immunogenic composition and vaccine or medical preparation, a method to immunize animals against the hormone LHRH, and analogs of the LHRH tandem repeat peptide and their use as vaccine |
| CN1455781A (en) * | 2000-09-12 | 2003-11-12 | Id－莱利斯塔德动物保健及动物育种研究所公司 | Discrimination between GnRH-I and GnRH-II |
| US7361349B2 (en) * | 1995-06-07 | 2008-04-22 | Pepscan Systems B.V. | Peptide, immunogenic composition and vaccine or medical preparation, a method to immunize animals against the hormone LHRH, and analogs of the LHRH tandem repeat peptide and their use as vaccine |
| CN102260321A (en) * | 2011-04-02 | 2011-11-30 | 新加坡保健服务集团 | Polymer form of antibacterial peptide |
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| USRE39048E1 (en) * | 1995-06-07 | 2006-03-28 | Pepscan Systems B.V. | Peptide, immunogenic composition and vaccine or medical preparation, a method to immunise animals against the hormone LHRH, and analogs of the LHRH tandem repeat peptide and their use as vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108047328A (en) * | 2017-12-14 | 2018-05-18 | 天津瑞普生物技术股份有限公司 | A kind of GnRH polypeptide antigens and its application for being used to prepare castration vaccine |
| WO2019114123A1 (en) * | 2017-12-14 | 2019-06-20 | 天津瑞普生物技术股份有限公司 | Gnrh polypeptide antigen and use thereof for preparing castration vaccines |
| CN110954706A (en) * | 2019-11-20 | 2020-04-03 | 宁波三生生物科技有限公司 | Method for measuring coupling rate of GnRH paralog dimer artificial antigen |
| CN110954706B (en) * | 2019-11-20 | 2020-10-02 | 宁波三生生物科技有限公司 | Method for measuring coupling rate of GnRH paralog dimer artificial antigen |
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