CN1436789A - livestock castration vaccine gene and recombinant gene vector - Google Patents
livestock castration vaccine gene and recombinant gene vector Download PDFInfo
- Publication number
- CN1436789A CN1436789A CN02155220A CN02155220A CN1436789A CN 1436789 A CN1436789 A CN 1436789A CN 02155220 A CN02155220 A CN 02155220A CN 02155220 A CN02155220 A CN 02155220A CN 1436789 A CN1436789 A CN 1436789A
- Authority
- CN
- China
- Prior art keywords
- gene
- lhrh
- plasmid
- castration
- pet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 37
- 229960005486 vaccine Drugs 0.000 title claims abstract description 34
- 244000144972 livestock Species 0.000 title abstract description 5
- 241001465754 Metazoa Species 0.000 claims abstract description 44
- 239000013612 plasmid Substances 0.000 claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 101000904173 Homo sapiens Progonadoliberin-1 Proteins 0.000 claims abstract 5
- 101000996723 Sus scrofa Gonadotropin-releasing hormone receptor Proteins 0.000 claims abstract 5
- 241000894006 Bacteria Species 0.000 claims description 12
- 230000029087 digestion Effects 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 235000013372 meat Nutrition 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 235000014347 soups Nutrition 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 239000006142 Luria-Bertani Agar Substances 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 108010088350 Lactate Dehydrogenase 5 Proteins 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims 1
- 230000003053 immunization Effects 0.000 abstract description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 6
- 108020001507 fusion proteins Proteins 0.000 abstract description 5
- 102000037865 fusion proteins Human genes 0.000 abstract description 5
- 241000287828 Gallus gallus Species 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 229940031626 subunit vaccine Drugs 0.000 abstract description 4
- 241000282326 Felis catus Species 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241001494479 Pecora Species 0.000 abstract 1
- 241000009328 Perro Species 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 108700012941 GNRH1 Proteins 0.000 description 54
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 37
- 239000000047 product Substances 0.000 description 24
- 230000004927 fusion Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 244000144977 poultry Species 0.000 description 10
- 235000013594 poultry meat Nutrition 0.000 description 10
- 108091008146 restriction endonucleases Proteins 0.000 description 9
- 230000001568 sexual effect Effects 0.000 description 9
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 8
- 238000005215 recombination Methods 0.000 description 7
- 230000006798 recombination Effects 0.000 description 7
- 210000001550 testis Anatomy 0.000 description 7
- 241000271566 Aves Species 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 230000036299 sexual function Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 108010077805 Bacterial Proteins Proteins 0.000 description 3
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 3
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 3
- 241000272496 Galliformes Species 0.000 description 3
- 235000013330 chicken meat Nutrition 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 229940028334 follicle stimulating hormone Drugs 0.000 description 3
- 210000004907 gland Anatomy 0.000 description 3
- 239000003163 gonadal steroid hormone Substances 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000009329 sexual behaviour Effects 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 101000904177 Clupea pallasii Gonadoliberin-1 Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 108010008038 Synthetic Vaccines Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 230000002381 testicular Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229960003604 testosterone Drugs 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- -1 Nco I Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000000739 chaotic effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000007235 immunity generation Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The livestock farm animal castration vaccine gene and recombinant gene vector is used for producing farm animal castration gene engineering subunit vaccine and belongs to biological technology. The production process includes artificial synthesis of livestock, fowl or universal LHRH gene segment with NcoI and XhoI enzyme incision sites separately in its 5' end and 3' end; inserting pET-32c plasmid to constitute four kinds of recombinant pET-32c LHRHn-N/X plasmid to express LHRH-Trx fusion protein; and converting colibacillus BL21(DE3) with the recombinant plasmid to produce expression product soluble protein. The farm animal castration gene engineering subunit vaccine is easy to purify, universal for livestock and fowl, and may be used for immunizing chicken, pig, sheep, rabbit, dog, cat and other animal for effective castration.
Description
The application is the applying date: 2000.6.23, application number: 00107831.3, and title: the dividing an application of animal's castration vaccine gene and engineering body.
(1) technical field
Animal's castration vaccine gene of the present invention and recombination carrier are used to produce the domestic animals and fowls castration with luteinizing hormone releasing hormone (LHRH) genetic engineering subunit vaccine, belong to the biotechnology high-tech area.
(2) technical background
Luteinizing hormone releasing hormone (Luteinizing hormore releasing hormore, LHRH) be by a kind of decapeptide hormone of animal hypothalamus excretory, its main biological function is synthetic and secretion metakentrin (LH) and a follicle stimulating hormone (FSH) of control anterior lobe of hypophysis cell, these hormones can promote the normal development of sexual gland and sexual organ, keep the animal reproduction function.But the property associated hormone level of castration vaccine immunoregulation animal endocrine system, suppress the animality allelotaxis, make animal forfeiture sexual behaviour and sexual function, thereby prevent chaotic mating (causing the drove degeneration) and fight, promote growth of animal, improve that trunk is formed and meat (make Fresh ﹠ Tender in Texture and prevent smell of mutton), the raising price of deed.Its mechanism is after doing the vaccine immunity animal by LHRH synthetic antigen or recombinant antigen, to form LHRH antibody in vivo, in and endogenous LH RH, thereby significantly reduce sex hormone levels such as testosterone and oestrogenic hormon, reach the purpose of gentle castration.Compare with the traditional operation castration; the vaccine castration is not only simple and easy to do; give in the management and bring great convenience; be particularly suitable for large-scale cultivation and herd drove; and the castration that can prevent to perform the operation cause hemorrhage, infect and stress cause subtract food and weight loss; the vaccine castration is applicable to large animal or animalcule animalcule operations such as (difficulty) chickens, female or buck, children's age and adult animals, livestock and poultry and other animals in addition, moreover the vaccine castration can realize reversibility control by supporting technology.
Impel animal to produce the antibody of anti-LHRH by immunization method, with in and the physiological action of LHRH, thereby the growth that suppresses sexual gland is to reach the purpose of castration, its feasibility is confirmed by domestic and international many scholars, but great majority research all concentrates on synthetic peptide vaccine, though immune effect is remarkable, can't enter practical application because of cost is too high eventually.The color equality of king is expressed the LHRH/HBsAg fusion rotein in silkworm baculovirus, but also because of expression yields poorly, and purifying technique is loaded down with trivial details and fail to apply.
(3) summary of the invention
Technical problem the objective of the invention is the designing animal castration vaccine gene, structure can efficiently express the animal's castration vaccine recombination carrier of LHRH fusion rotein, can be used to develop high yield, high-quality, efficient, low-cost, technology is easy, be fit to scale production, easy to use, and safe, noresidue, the animal's castration recombinant vaccine of no potentially dangerous, immune animal substitutes traditional operation castration.
Technical scheme animal's castration vaccine gene provided by the present invention, its general formula is:
5 '------framework guarantees base, and------restriction enzyme site--protectiveness base--3 ' wherein, protectiveness base can be that A, T, G, C are optional to terminator to the LHRH gene to restriction enzyme site to the protectiveness base;
Restriction enzyme site is meant the point of contact of Restriction Enzymes such as Nco I, EcoR I, HindIII, Xho I, BamH I;
Framework guarantees that base can be that A, T, G, C are optional;
The LHRH gene is meant domestic animal LHRH gene, or poultry LHRH gene, or LHRH gene and the series connection of poultry LHRH gene.
Above-mentioned animal's castration vaccine gene, it is as follows to exemplify concrete gene order:
Domestic animal is used castration vaccine LHRH gene order---L1:
5′-GG
CCATGGCTGAGCATTGGTCTTATGGCCTGCGCCCAGGT
NcoI
TAATGAA
CTCGAGCA-3′
Terminator Xho I
Poultry is used castration vaccine LHRH gene order---L2:
5′-GG
CCATGGCTGAGCACTGGTCTTACGGCCTGCAGCCGGGT
Nco?I
TAATGAA
CTCGAGCA-3′
Terminator Xho I
1 of the general castration vaccine LHRH of poultry/fowl gene order---L3:
5′-GG
CCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGGGT
Nco?I
GGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACATTG
GTCTTACGGTCTGCGCCCGGGT
TAATGAA
CTCGAGCA-3′
Terminator Xho I
2 of the general castration vaccine LHRH of poultry/fowl gene order---L4:
5′-GG
CCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGGGT
Nco?IGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGGGTGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACACTGGTCCTACGGTCTGCGTCCGGGTGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGGGT
TAATGAA
CTCGAGCA-3′
Terminator Xho I
With the recombination carrier that above-mentioned animal's castration vaccine gene makes up, pET-32c LHRHn-N/X recombinant expression plasmid is to make up acquisition by the following method:
1.LHRH the annealing of gene fragment
With segmental normal chain of the animal's castration vaccine gene of synthetic and minus strand, being dissolved into concentration with the sterilization ultrapure water respectively is the 100pmol/ μ l solution of (being equivalent to 1.82 μ g/ μ l), respectively get 2 μ l and add mixing in the 72 μ l ultra-pure water, put and slowly cool to room temperature in the boiling water bath behind the 2min ,-20 ℃ frozen standby;
2.LHRH the enzyme of gene is cut
With NcoI and XhoI the LHRH gene after annealing is carried out double digestion, enzyme is cut product and is directly used in ligation;
3.pET-32c the extraction of plasmid, enzyme are cut and are reclaimed
After the extraction of pET-32c plasmid, the pET-32c plasmid is carried out double digestion with NcoI and XcoI.Get 10 μ l enzymes and cut product and carry out 1% agarose gel electrophoresis, reclaim enzyme and cut big fragment, put and freeze 5min in the liquid nitrogen, and melt in rearmounted 37 ℃ of water-baths, the centrifugal 10min of 10000r/min, it is ℃ frozen standby to draw supernatant-20;
4. connect
DNA ligation system is as follows:
The PET-32c double digestion reclaims product 2.5 μ l
The LHRH gene enzyme is cut product 7.5 μ l
Be used for immediately transforming behind 16 ℃ of reactions of DNA Ligation Kit Solution I 10 μ l said mixtures 30min;
5. the preparation of bacillus coli DH 5 alpha competent cell;
6. transform
Get 10 μ l connection product and transform 200 μ l bacillus coli DH 5 alpha competent cells, get the LB agar plate that the coating of 200 μ l converted products contains Amp 50 μ g/ml, flat board is put in 37 ℃ of incubators and was cultivated 18 hours, single bacterium colony after picking transforms, extract plasmid after inoculating the LB meat soup enlarged culturing that contains Amp, be recombination carrier pET-32c LHRHn-N/X recombinant plasmid.
Beneficial effect animal's castration vaccine gene provided by the present invention and recombination carrier thereof, can efficiently express LHRH-Trx fusion rotein (expression amount is more than 30%), be used to produce domestic animals and fowls castration luteinizing hormone releasing hormone (LHRH) genetic engineering subunit vaccine.Because birds have an amino acid different with mammiferous LHRH, made up the recombinant expression plasmid (Ll, L2, L3, L4) of expressing birds, mammals and the alternate placed in-line LHRH gene of different quantities birds/mammals respectively.Through verification test, recombination carrier provided by the present invention conforms in every respect to industrialized requirement at expression amount, immunogenicity, production simplicity, castration validity, security, cost etc.Make vaccine with it, produce simple and easy, with low cost, safe and effective.In 4 kinds of plasmids, the L1 expression product is effective to domestic animal, and L2 is effective to poultry, and L3 and L4 domestic animals and fowls are general, and the effectiveness of aspects such as L4 immunity generation phase and immune duration is better than L3 again, can be selected according to various application need.
Adopt this project body to produce each 3 batches in L1, L2, L3 and L4 seedling sample under laboratory condition, it expresses stable yield, and the expression product protein content all reaches more than 30% of bacterial protein amount.Expression product is all stable to have good immunogenicity, shows: the LHRH standard antibody generation specific reaction of (1) expression product and Sigma company; (2) can effectively excite the high LHRH antibody of tiring of generation behind the immune animal, specific reaction takes place with LHRH in this antibody capable in vitro tests.Immune mouse is 210 altogether, 50 of rabbits, 30 of native chickens, 640 of pigs, 6 of cats, 4 of dogs all can effectively excite antibody behind the immune animal, observation in sexual maturing period, in each side Comprehensive Assessments such as sexual behaviour, sexual function, sex hormone level, sexual organ development's inhibition, effectively castration is obviously improved meat, and can obviously promote weightening finish and improve the price of deed.With the buck is example, and the average testicular weight of immune group is 1.32 ± 0.38g, and the average testis of control group heavily is 3.18 ± 0.47g, and histological observation confirms, the atrophy of immune group parenchyma of testis does not have sperm more and forms ability (sperm is arranged individually, but do not have vigor during biopsy).
(4) description of drawings
Fig. 1. recombinant plasmid pET-32c LHRH
nThe structure of (L1, L2, L3, L4)-N/X
Fig. 2. different IP TG concentration is induced LHRH
1The influence of-Trx (L1) expressing fusion protein output
1.?0mM?IPTG 2.?0.05mM?IPTG 3.?0.1mM?IPTG
4.?0.5mM?IPTG 5.?1.0mMIPTG 6.?2.0mM?IPTG
7,8,9. 2.0mM IPTG (different induction time) M. protein molecular weight sign
Fig. 3. repeatedly transform and express LHRH
3-Trx (L3) the equal efficient stable of fusion rotein output (1mMIPTG induces 4h)
Rabbit testis size variation behind Fig. 4 .LHRH1-Trx (L1) fusion protein immunization
1,2.LHRH-Trx (L1) fusion rotein intradermal injection group 3,4.LHRH-Trx fusion rotein intramuscular injection group
5,6.PET-32c carrier proteins immune group 7,8. blank group
Fig. 5 .LHRH
1Rabbit testicular histology behind-Trx (L1) fusion protein immunization changes
A. normal control rabbit testis tissue section B. expression product immunize rabbit testis tissue section
(5) embodiment
Embodiment:
1.LHRH the design of gene and synthetic
Aminoacid sequence design gene according to birds, mammals LHRH adds respectively at 5 ' end and 3 ' end
Two restriction enzyme sites of last NcoI and XhoI and protectiveness base are used the automatic dna synthesizer synthetic gene.
1.1. domestic animal is used vaccine LHRH gene order---L1:
5′-GG
CCATGGCTGAGCATTGGTCTTATGGCCTGCGCCCAGGT
NcoI
TAATGAA
CTCGAGCA-3′
Terminator XhoI
1.2. poultry is used vaccine LHRH gene order---L2:
5′-GG
CCATGGCTGAGCACTGGTCTTACGGCCTGCAGCCGGGT
NcoI
TAATGAA
C?TCGAGCA-3′
Terminator XhoI
1.3. 1 of the general vaccine LHRH of poultry/fowl gene order---L3:
5′-GG
CCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGGGT
NcoIGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGGGT
TAATGAA
CTCGAGCA-3′
Terminator XhoI
1.4. 2 of the general vaccine LHRH of poultry/fowl gene order---L4:
5′-GG
CCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGGGT
NcoIGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGGGTGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACACTGGTCCTACGGTCTGCGTCCGGGTGGCGGTGAGCACTGGTCTTACGGCCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGGGT
TAATGAA
CTCGAGCA-3′
Terminator XhoI
2.LHRH gene clone (Fig. 1)
2.1.LHRH the annealing of gene fragment
With segmental normal chain of the animal's castration vaccine gene of synthetic and minus strand, being dissolved into concentration with the sterilization ultrapure water respectively is the 100pmol/ μ l solution of (being equivalent to 1.82 μ g/ μ l), respectively get 2 μ l and add mixing in the 72 μ l ultrapure waters, put and slowly cool to room temperature in the boiling water bath behind the 2min ,-20 ℃ frozen standby.
2.2.LHRH the enzyme of gene is cut
With NcoI and XhoI the LHRH gene after annealing is carried out double digestion.Enzyme is cut product and is directly used in ligation.
2.3.pET-32c the extraction of plasmid, enzyme are cut and are reclaimed
The extraction alkaline lysis of pET-32c plasmid, work such as reference literature [U.S.] J. Sa nurse Brooker, the method that " molecular cloning experiment guide " introduced.With NcoI and XhoI the pET-32c plasmid is carried out double digestion.Get 10 μ l enzymes and cut product and carry out 1% agarose gel electrophoresis, reclaim enzyme and cut big fragment, put and freeze 5min in the liquid nitrogen, and melt in rearmounted 37 ℃ of water-baths, the centrifugal 10min of 10000r/min, it is ℃ frozen standby to draw supernatant-20.
2.4. connect
DNA ligation system is as follows:
The pET-32c double digestion reclaims product 2.5 μ l
The LHRH gene enzyme is cut product 7.5 μ l
Be used for immediately transforming behind 16 ℃ of reactions of DNA Ligation Kit Solution I 10 μ l said mixtures 30min.
2.5. the preparation of competent cell
Work such as reference literature [U.S.] J. Sa nurse Brooker, the method that " molecular cloning experiment guide " introduced prepares the bacillus coli DH 5 alpha competent cell.
2.6. transform
Get 10 μ l connection product and transform 200 μ lDH5 α competent cells, work such as concrete operations reference literature [U.S.] J. Sa nurse Brooker, the method that " molecular cloning experiment guide " introduced is carried out.Get the LB agar plate that the coating of 200 μ l converted products contains Amp 50 μ g/ml, establish the competent cell contrast (positive control) that unconverted competent cell contrast (negative control) and pET-32c plasmid transform simultaneously.Flat board is put in 37 ℃ of incubators and was cultivated 18 hours.
2.7. the enzyme of recombinant plasmid is cut screening
PET-32c plasmid multiple clone site has EcoRI and HandIII restriction enzyme site, and these two restriction enzyme sites should disappear after inserting the LHRH gene, and NcoI and XhoI restriction enzyme site still exist.Principle in view of the above, single bacterium colony after picking transforms, inoculation is extracted plasmid after containing the LB meat soup enlarged culturing of Amp, carries out single endonuclease digestion with HindIII, EcoRI, NcoI and XhoI respectively, selects the disappearance of HindIII and EcoRI restriction enzyme site and positive colony that NcoI and XhoI restriction enzyme site still exist.
2.8. the sequencing of recombinant plasmid
Select the recombinant plasmid of cutting evaluation through enzyme, as sequencing primer, company limited carries out sequencing by precious biotechnology (Dalian) with T7 Terminater primer.
3.pET-32c LHRH
nThe expression of-N/X recombinant plasmid in e. coli bl21 (DE3)
3.1.BL21 (DE3) preparation of competent cell
The same 1.2.5 of method.
3.2. transform
Extract in a small amount through the recombinant plasmid of sequencing and pET-32c plasmid transformed into escherichia coli BL21 (DE3) competent cell respectively with alkaline lysis, method is with 2.6.
3.3. the expression of recombinant plasmid in BL21 (DE3)
Picking recombinant plasmid transformed colony inoculation 5ml contains the LB meat soup of Amp 50 μ g/ml, and 37 ℃ of shaking overnight incubation (rotating speed is 250rpm, down together) are inoculated in 30ml by 1% inoculum size to contain in the LB meat soup of Amp 50 μ g/min next day, are cultured to OD in 37 ℃ of shaking tables
600Reach about 0.5, take out 1ml bacterium liquid and put the centrifugal 1min of 2000r/min in the Eppendorf pipe, it is frozen standby in-20 ℃ to abandon supernatant, as inducing preceding contrast, all the other cultures adding final concentrations are that the IPTG of 1mM carried out abduction delivering 3-4 hour, get 1ml and induce the bacterium liquid same centrifugal supernatant of abandoning in back to keep precipitation.Simultaneously pET-32c plasmid transformed bacteria is operated in contrast equally.
3.4.SDS polyacrylamide gel electrophoresis (SDS-PAGE)
To induce preceding thalline, induce back recombinant plasmid transformed bacterium and induce back pET-32c vector plasmid transformed bacteria precipitation to add isopyknic 2 * carrier buffer with distilled water is resuspended, boil 5min, respectively get 10 μ l and carry out discontinuous SDS-PAGE (concentrating glue 5%, separation gel 13%).After electrophoresis finished, gel dyeed with Xylene Brilliant Cyanine G (R250).Carry out the band gray scale scanning with the gel imaging treatment system, measure the ratio of expressed fusion protein and bacterial protein.
3.5. the solubility of expressing protein
By 1% inoculum size inoculation 100ml LB meat soup, 37 ℃ of shaking tables are cultured to OD with LHRH-pET-32c plasmid transformed bacteria overnight culture
600During=0.5 left and right sides, add IPTG to final concentration 1mM, after continuing to cultivate 4h, the centrifugal supernatant of abandoning, all bacterial sediment is resuspended with 10ml PBS (pH7.4), ultrasonic disruption (ice bath) 5min, ultrasonic power is 240W, supernatant is transferred in another container after centrifugal, and precipitation is resuspended with 10ml PBS, respectively get 50 μ l supernatants and the precipitation suspension add isopyknic 2 * load sample buffer, carry out SDS-PAGE; Simultaneously, pET-32c plasmid transformed bacteria being carried out above-mentioned same operation compares.Establish the full bacterium contrast of not using ultrasonic treatment in addition.Gel is carried out the content that gray scale scanning is determined target protein.
4.LHRH-Trx the immunogenicity of fusion rotein is identified
4.1. experimental animal
200 of mouse, 50 of rabbits, 30 of native chickens, 600 of pigs, 6 of cats, 4 of dogs.
4.2. immunization protocol:
With preparation such as freund's adjuvant and white-oil adjuvant vaccine, by immunization routes such as intradermal injection and intramuscular injection injection animal, immunizing dose 0.2-2ml does not wait with expression product, and the apparent motion object is great little and decide.Generally immunity before the animality maturation.
4.3. immune animal sexual function and sexual organ inspection
4.3.1. sexual function inspection
After sexual maturing period, experimental animal and different in nature healthy adult animal of the same race are closed situations such as foster, observation sexual behaviour, sex character and fecundity.
4.3.2. sexual gland, sexual organ inspection
Analyse inspection, gather testis, ovary, one is put in the neutral formalin fixing immediately, does the tissue slice analysis, and it two is weighed, measures size and check that testis has or not sperm and motility of sperm etc.
Interpretation of result:
1. the four kinds of recombinant plasmids that can stablize, efficiently express the LHRH-Trx fusion rotein
4 kinds of recombinant expression plasmids of expressing birds, mammals and the alternate placed in-line LHRH fusion rotein of different quantities birds/mammals have been made up.Sequential analysis shows that gene is synthetic and it is correct to insert.Plasmid transformed into escherichia coli repeatedly after preserving, the immunogenicity of expression amount and expression product is all very stable, and expression level is all the time at more than 30% of bacterial protein.
2. the expression product of solubility
Realized the solubility expression of LHRH-Trx fusion rotein, almost completely there be (but not inclusion body) in expression product with soluble form.With the osmotic shock method LHRH fusion rotein and pET-32c carrier proteins are extracted, bacterium can discharge the LHRH-Trx fusion rotein after height oozes processing singlely in LHRH reorganization as a result, and purity reaches more than 90%, and pET-32c empty carrier albumen still is present in the thalline and can not discharges.For extracting and the purifying expression product prepares vaccine great convenience is provided.
3. expression product has very strong LHRH antigenicity
Expression product and LHRH antibody have good reactivity, and this reaction can be blocked by the LHRH standard substance; Immune animal can effectively excite LHRH antibody, in and endogenous LH RH, thereby significantly reduce sex hormone levels such as testosterone and FSH, reach the purpose of gentle castration, and vaccine immunity can prevent to perform the operation that castration causes is hemorrhage, infects and stress cause subtracts food and weight loss.
Claims (2)
1. domestic animal is used castration vaccine LHRH gene, and its concrete gene order is as follows:
5′-GG
CCATGGCTGAGCATTGGTCTTATGGCCTGCGCCCAGGT
NcoI
TAATGAA
CTCGAGCA-3′
Terminator XhoI
2. containing right and require the recombinant expression vector of 1 described domestic animal with castration vaccine gene, is to make up acquisition by the following method:
2.1LHRH the annealing of gene fragment
The domestic animal of synthetic is used castration vaccine gene fragment L1:
5′-GG
CCATGGCTGAGCATTGGTCTTATGGCCTGCGCCCAGGT
NcoI
TAATGA
ACTCGAGCA-3′
Terminator XhoI
The domestic animal of synthetic is used segmental normal chain of castration vaccine gene and minus strand, respectively with the ultrapure solution that concentration is 100pmol/l of being dissolved into of sterilizing, respectively get 2 μ l and add mixing in the 72 μ l ultrapure waters, put and slowly cool to room temperature in the boiling water bath behind the 2min ,-20 ℃ frozen standby;
2.2 the enzyme of LHRH gene is cut
With NcoI and XhoI the LHRH gene after annealing is carried out double digestion, enzyme is cut product and is directly used in ligation;
2.3 the extraction of pET-32c plasmid, enzyme are cut and are reclaimed
After the extraction of pET--32c plasmid, the PET-32c plasmid is carried out double digestion with NcoI and XhoI.Get 10 μ l enzymes and cut product and carry out 1% agarose gel electrophoresis, reclaim enzyme and cut big fragment, put and freeze 5min in the liquid nitrogen, and rearmounted 37 ℃ of water-baths are melted, the centrifugal 10min of 10000r/min, it is ℃ frozen standby to draw supernatant-20;
2.4 connect
DNA ligation system is as follows:
The PET-32c double digestion reclaims product 2.5 μ l
The LHRH gene enzyme is cut product 7.5 μ l
Be used for immediately transforming behind 16 ℃ of reactions of DNA Ligation Kit Solution I 10 μ l said mixtures 30min;
2.5. the preparation of bacillus coli DH 5 alpha competent cell;
2.6 transform
Get 10 μ l connection product and transform 200 μ lDH5 α competent cells, get the LB agar plate that the coating of 200 μ l converted products contains Amp 50 μ g/ml, flat board is put in 37 ℃ of incubators and was cultivated 18 hours, single bacterium colony after picking transforms, extract plasmid after inoculating the LB meat soup enlarged culturing that contains Amp, be recombinant plasmid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021552207A CN1200102C (en) | 2002-12-10 | 2002-12-10 | Livestock castration vaccine gene and recombinant gene vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021552207A CN1200102C (en) | 2002-12-10 | 2002-12-10 | Livestock castration vaccine gene and recombinant gene vector |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00107831 Division CN1116414C (en) | 2000-06-23 | 2000-06-23 | Animal's castration vaccine gene and gene engineering body |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1436789A true CN1436789A (en) | 2003-08-20 |
| CN1200102C CN1200102C (en) | 2005-05-04 |
Family
ID=27628790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021552207A Expired - Fee Related CN1200102C (en) | 2002-12-10 | 2002-12-10 | Livestock castration vaccine gene and recombinant gene vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1200102C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107880135A (en) * | 2017-11-17 | 2018-04-06 | 华派生物工程集团有限公司 | One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof |
-
2002
- 2002-12-10 CN CNB021552207A patent/CN1200102C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107880135A (en) * | 2017-11-17 | 2018-04-06 | 华派生物工程集团有限公司 | One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1200102C (en) | 2005-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bonneau et al. | Biotechnology in animal nutrition, physiology and health | |
| CN1127352C (en) | CCK antibodies used to improve feed efficiency | |
| CN1226896A (en) | Modulating activity of hormones or their receptors-peptides, antibodies, vaccines and uses thereof | |
| CN1159338C (en) | An improved peptide immunogenic composition and vaccine or medical preparation. Method to immunise animals against hormons LHRH and alalogs of LHRH tandem repeat peptide | |
| CN1974775A (en) | Recombinant plasmid and engineering bacterium containing grass carp interferon gene and their application | |
| RU2501565C2 (en) | Chloramphenicol acetyl transferase (cat) defective somatostatin fused protein and using it | |
| CN101356191B (en) | Neuropeptides for the culture of aquatic organisms | |
| CN1116414C (en) | Animal's castration vaccine gene and gene engineering body | |
| WO2013184024A1 (en) | Preparation for increasing sperm production in stud livestock and roosters and method for the use thereof | |
| CN1200103C (en) | Fowl castration vaccine gene and recombinant gene vector | |
| CN1200102C (en) | Livestock castration vaccine gene and recombinant gene vector | |
| CN1200101C (en) | Universal farm animal castration vaccine gene and recombinant gene vector | |
| CN1149258A (en) | Inhibin compositions and methods of use thereof | |
| CN1958781A (en) | Reagent peptone, and preparation method | |
| CN1218962C (en) | Somatostatin yolk antibody and its preparing process | |
| CN1063109A (en) | Improvement to hormone and associated materials | |
| CN1194008C (en) | Recombination main outer membrane protein of parrot fever chlaydozoan, and preparation process and use thereof | |
| CN101050448A (en) | Oral recombined DNA vaccine for accelerating growth of animal, and application | |
| CN1236059C (en) | Grouper insulin-like growth factor II gene, carrier and recombinant strain containing the gene and application thereof | |
| CN104789514B (en) | A kind of amicine overexpression DNA vaccination and construction method and application | |
| CN1467291A (en) | Avian interleukin-2 (IL-2) gene and eukaryon expressing plasmid and immunity reinforcing agent of bird vaccine | |
| CN1309824C (en) | Antibiotic peptide gene converted viscons bacillus engineering bacterium and its preparing method and use | |
| CN1300307C (en) | Recombined T4 bacteriophage of expressing cholecystokinin gene | |
| CN1209841A (en) | Biologically active and particularly peptide molecules having potentiating effect on growth hormone biological activity | |
| CN1366061A (en) | Genetically engineered body of somatostatin gene vaccine (DNA vaccine) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: CHENGDU RUISHENG NEUTRALIZATION BIOTECHNOLOGY CO., Free format text: FORMER OWNER: CHENGDU RUISHENG HI-TECH CO., LTD. Effective date: 20140317 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 610041 CHENGDU, SICHUAN PROVINCE TO: 611830 CHENGDU, SICHUAN PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20140317 Address after: 611830 Sichuan Economic Development Zone, Dujiangyan, Sichuan, Chengdu, Dujiangyan Patentee after: Chengdu Rui Zhonghe biological science and Technology Co Ltd Address before: High tech Zone Chengdu city Sichuan province 610041 Gaopeng Road No. 3 21D Patentee before: Chengdu Ruisheng Hi-Tech Co., Ltd. |
|
| ASS | Succession or assignment of patent right |
Owner name: CHENGDU RUISHENG HI-TECH CO., LTD. Free format text: FORMER OWNER: CHENGDU RUISHENG NEUTRALIZATION BIOTECHNOLOGY CO., LTD. Effective date: 20150708 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20150708 Address after: High tech Zone Chengdu city Sichuan province 610041 Gaopeng Road No. 3 21D Patentee after: Chengdu Ruisheng Hi-Tech Co., Ltd. Address before: 611830 Sichuan Economic Development Zone, Dujiangyan, Sichuan, Chengdu, Dujiangyan Patentee before: Chengdu Rui Zhonghe biological science and Technology Co Ltd |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20050504 Termination date: 20190623 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |