CN100998873B - Gone medicine for preventing and treating mammitis of animal - Google Patents
Gone medicine for preventing and treating mammitis of animal Download PDFInfo
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Abstract
A genetic medicine for preventing and treating mammitis of animal with high and durable effect and no toxic by-effect and no generation of drug tolerance is an injection containing the antibacterial peptide ABPS1 gene and eucaryotic expression carrier.
Description
Technical field
The present invention relates to the genomic medicine of disease preventing and treating, particularly relate to the genomic medicine of a kind of prevention and treatment mammitis of animal.
Background technology
Mastitis is a kind of frequently-occurring disease of milch cow, is to cause one of the most serious disease of dairy loss, and according to statistics, China reduces the loss that is caused by the milch cow lactation amount that mastitis causes every year, and every cattle is roughly equal to 1200-3600 unit.Mastitis to the harm of dairy mainly comprise following some:
1) infection rate and sickness rate height: the whole world has 2.12 hundred million cow heads approximately, wherein has 1/3 milch cow to suffer from various types of mastitiss approximately;
2) not only make milk yield reduce (on average reducing milk yield 10%-15%), also prolonged post-partum estrus and pregnant time;
3) nutritional labeling of milk and quality are reduced, influence health: contain a large amount of pathogenic microorganisms and toxin thereof in the sick Lac Bovis seu Bubali juice, can cause symptoms such as fever, vomiting, diarrhoea, allergy after eating;
4) increase medical expense and extra labour, if obstinate, the mortality of increase milch cow is lost bigger.
The cause of disease of mammitis of cow is very complicated, and main cause is to be subjected to cause pathogeny imcrobe infection, and risk factor comprises nonstandard feeding and management, milking operation, rugged environment and wound etc.Studies show that, cause that the pathogen of mammitis of cow reaches kind more than 150, mainly comprise two big classes, a class is an infectious microorganism, comprises gold-coloured staphylococci, streptococcus agalactiae and streptococcus dysgalactiae etc.; One class is an environmental microorganism, as cow breast streptococcus, escherichia coli, klebsiella and bacillus pyocyaneus etc.Based on escherichia coli, staphylococcus aureus and streptococcus, account for more than 90% of whole mammitis of cow cases in the above-mentioned pathogen.
At present, the drug main for the treatment of mastitis both at home and abroad will comprise following a few class: antibiotic (separately or administering drug combinations), Chinese herbal medicine, vaccine and disinfectant (dipping).Wherein, antibiotic is to use the most general treatment mastitis medicine at present.The subject matter of antibiotherapy is that therapeutic effect is certain inadequately, and also residual in the excretory Lac Bovis seu Bubali of milch cow after treatment a large amount of antibiotic arranged, in addition, life-time service easily causes occurring Resistant strain, not only will cause therapeutic effect to descend, and also can jeopardize human health.Though Chinese herbal treatment does not have serious problems such as the residual and persister of antibiotics, but its therapeutic dose is bigger, and residual Chinese medicine all remains further research to quality, taste and the health effects etc. of milk in milk, secondly, the Chinese herbal treatment cycle is long, and the treatment cost is higher.Vaccine immunity prevention is because to be subjected to pathogenic bacterium many, and the Ruzhong lacks effectively defence composition, and antibody will see through blood-newborn barrier during general immunity, has reduced influences such as the content of Ruzhong antibody and activity, and actual effect is not good.Mastitis can not be fundamentally cured in the disinfectant dipping.
Chinese scholars is carried out big quantity research to the medicine of treatment mastitis, and some patents, as:
The Sun Huaichang of domestic Yangzhou University utilizes human lysozyme to carry out the gene therapy research of mammitis of cow, has certain curative effect (patent No.: 02148545.3).
United States Patent (USP) the 7th, 091 has reported that the proteic gene of lysostaphin of will encoding changes in the breast of milch cow, is used for treating the mammitis of cow that is caused by staphylococcus aureus No. 332.
United States Patent (USP) the 5th, 858 No. 962, has been reported with lysostaphin for the 5th, 760, No. 026 and directly have been treated mammitis of cow.
United States Patent (USP) the 7th, 045 has been reported for No. 510 with DEAE-glucosan or glucosan prevention and treatment mastitis.
United States Patent (USP) the 6th, 984 has been reported the mammitis of cow that is caused by staphylococcus with the staphylobacterin prevention for No. 381.
United States Patent (USP) the 6th, 872 has been reported for No. 709 glycyrrhizin is dissolved in and has been treated mastitis in the saline.
United States Patent (USP) the 6th, 787 has been reported for No. 134 with phytase prevention and treatment mastitis.
United States Patent (USP) the 6th, 562 has been reported for No. 820 separately with oxazolidinone or lactoferrin prevention of associating external source and treatment mastitis.
United States Patent (USP) the 4th, 140 has been reported for No. 766 iodine, potassium iodide to be dissolved in the acid solution external curing mastitis.
United States Patent (USP) the 6th, 369 has been reported for No. 049 with fluorine cephalosporin treatment mastitis.
United States Patent (USP) the 6th, 306 has been reported for No. 653 a kind of protein chemokine has been expressed in mammary gland tissue with the treatment mammitis of cow.
United States Patent (USP) the 6th, 187 has reported that Micrococcin. is injected nipple treats mastitis No. 800.
United States Patent (USP) the 5th, 846 has been reported for No. 543 canada yellow-root, Radix Ginseng, RADIX GELSEMII, Radix Phytolaccae, Aconitum carmichjaelii Debx. and Aloe has been formed compound recipe, the treatment mammitis of cow.
United States Patent (USP) the 5th, 260 has been reported diacetyl and 3-hydroxyl-butanone mixing treatment mastitis for No. 341.
United States Patent (USP) the 5th, 195 has been reported for No. 966 with oxychlorosene monomer or oxychlorosene sodium salts for treating mammitis of cow.
United States Patent (USP) the 3rd, 950 has been reported for No. 554 a kind of half-dried oil ﹠ fat acid esters is coated in to form waterproof membrane (also comprising antibacterial, consolidant, hydrating agents and a kind of dyestuff in the composition simultaneously) treatment mastitis on the nipple.
United States Patent (USP) the 5th, 280 has been reported for No. 768 insecticide is sprayed on the facies ventralis and back leg, breast and nipple of cow's tail, prevents that insecticide from will cause that the antibacterial of mastitis passes to cattle.
At present, the medicine of the treatment mastitis of reporting in the patent documentation is more, but really is applied to put into practice seldom.
Antibacterial peptide (antimicrobial peptides) is that amino acid number is less than 100, often positively charged, and the little peptide of a class of tool broad spectrum antibiotic activity, wherein, antibacterial peptide ABPS1 be us according to the synthetic little peptide with antibacterial activity of the artificial design of the preferences of codon (Lu Qiang, Liu Wei congruence, the synthetic of antibacterial peptide Shiva-1 gene and clone [J]. Chinese veterinary's journal, 2000,20 (2): 160-162).
Summary of the invention
The purpose of this invention is to provide a kind of moderate cost, safety, effectively, the genomic medicine that is used to prevent and treat mammitis of animal of no hazard residue.
For achieving the above object, the present invention takes following design: a kind of genomic medicine of preventing and treating mammitis of animal is to be the injection of effective ingredient with the carrier for expression of eukaryon that contains antibacterial peptide ABPS1 gene.
For making curative effect more definite, the upstream of antibacterial peptide ABPS1 gene also is added with the promoter of mammal beta lactoglobulin gene in described carrier for expression of eukaryon, so that ABPS1 gene specifically expressing in mammary glandular cell.
The promoter of described mammal beta lactoglobulin gene can be promoter or other mammiferous beta lactoglobulin gene promoter of bovine beta-lactoglobulin (BLG) gene, can select according to the effective object of medicine; Wherein, the nucleotides sequence of the promoter of described bovine beta-lactoglobulin (BLG) gene is classified the SEQ ID № in the sequence table as: 1.
In addition, 5 ' of antibacterial peptide ABPS1 gene end also closely is connected with human growth hormone's signal coding sequence in described mammalian expression vector, purpose is that the antibacterial peptide that mammary glandular cell gives expression to can pass cell membrane, the pathogen outer to born of the same parents plays a role, and described human growth hormone's signal coding sequence is the SEQ ID № in the sequence table: 2.
Be used to make up the described carrier that sets out that contains the carrier for expression of eukaryon of antibacterial peptide ABPS1 gene can be any one can be at the carrier for expression of eukaryon of mammal expression alien gene, as pVAX1, pSilence1.0-U6 (Ambion, Austin, TX, USA), pEGFP-N1 (ClonTech company), pCMV (Stratagene company), pSV40 (Marker Gene company), pSI, pCI, pCI-neo (Promega company) or pPICZ α, pTEF1, pcDNA3.1, pcDNA (Invitrogen company) etc.
Wherein, be that the set out carrier for expression of eukaryon that contains antibacterial peptide ABPS1 gene of vector construction is pBLG-hABPS1 with pVAX1; The nucleotide sequence of described antibacterial peptide ABPS1 gene is as SEQ ID № in the sequence table: shown in 3.
Above-mentioned recombinant expression carrier all can make up according to conventional method.
The choice of Solvent of described injection is diversified, as TE buffer, normal saline, PBS or the sterile purified water etc. of pH 7.5-8.5, is preferably the TE buffer of pH7.5-8.5.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, absorption enhancer, surfactant of pharmaceutical field routine etc.
The effective object of said medicine is widely, easily suffers from the mammal of mastitis arbitrarily as cattle, goat etc.
The consumption of said medicine is generally 400-1200 μ g/kg body weight/day, is generally the course of treatment 10-20 days.As using by the prevention mammitis of animal, the dosage and the course of treatment all can be adjusted.
For improving curative effect, medicine of the present invention can also carry out combined therapy with antibiotic, immunostimulant etc.
The invention provides a kind of genomic medicine of preventing and treating mammitis of animal.The active component of this medicine is the eukaryon expression plasmid that carries antibacterial peptide ABPS1 gene, with the ill mammary gland of this medicine direct injection, antibacterial peptide ABPS1 gene is expressed in mammary gland, thereby reach the purpose that prevents and/or treats mammitis of animal.Compare with the existing animal milk adenitis medicine of preventing and treating, genomic medicine of the present invention has the following advantages:
1) the antibacterial peptide ABPS1 that expresses in mammary gland has killing action to multiple mastitis pathogenic bacterium, ABPS1 causes and plasmicly in the born of the same parents oozes out in a large number by destroying cell membrane, cause the inside and outside osmotic pressure of cell unbalance, finally cause antibacterial death, but ABPS1 does not have effect to normal mammalian cell, therefore this medicine has higher specific aim to the mammitis of animal of treatment pathogen complexity, and can produce the better prevention effect;
2) Zhu She genomic medicine can be kept expression and the treatment time of growing (at least 10 days) after being absorbed by mammary glandular cell, does not need medication repeatedly;
3) this genomic medicine if be prepared and purification in strict accordance with the production standard of genomic medicine, will have higher safety in utilization, and animal test results shows that this medicine does not have any toxicity, side effect according to the requirement design of people's gene treatment;
4) the antibacterial peptide ABPS1 that is secreted in the milk is natural non-special defense factor, and is useful and harmless to animal, and the residence time of this genomic medicine in milk is of short duration, even there is minimal residue also to be degraded fully by digestive tract very soon, so there is not the hazard residue problem;
5) mechanism of action of antibacterial peptide is different from existing antibiotic and chemicals, so this medicine is difficult for producing drug resistance;
6) this genomic medicine can be cultivated through engineering bacterium fermentation and obtain, and fermentation all can be adopted conventional method with culture medium and recombinant vector purification process, thereby the preparation method of this medicine is simple, is convenient to batch production production, produces instinct and is accepted by veterinary clinic.
The present invention will have a extensive future playing a significant role aspect the prevention of mammitis of animal and treatment and animal cultivation and the raising livestock products quality.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the physical map of carrier for expression of eukaryon pBLG-hABPS1
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3
RdEdition, 2001, NY, Cold SpringHarbor).It is synthetic that the primer and DNA sequence are given birth to the worker by Shanghai.
The genomic medicine of embodiment 1, preparation control mammitis of animal
Genomic medicine with following method preparation control mammitis of animal may further comprise the steps:
One, makes up the carrier for expression of eukaryon that contains antibacterial peptide ABPS1 gene
1, the amplification of the promoter of bovine beta-lactoglobulin (BLG) gene
Genomic DNA with cattle is a template, under the guiding of primer p1:5 '-tcccacgtgactgcattagccc-3 ' and p2:5 '-tgcagggagtggagggctgag-3 ', and the promoter of pcr amplification bovine beta-lactoglobulin gene.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, it is the purpose fragment of 659bp that the result has obtained length through pcr amplification, and sequencing result shows that sequence is correct, has SEQ ID № in the sequence table: 1 nucleotide sequence.Reclaim and this dna fragmentation of purification.
2, the amplification of human growth hormone's signal coding sequence
With human growth hormone gene group gene for being template, under the guiding of primer p3:5 '-ggatccatggctacaggctccc-3 ' and p4:5 '-ggccactgccctcttgaagcc-3 ', pcr amplification human growth hormone's signal coding sequence.After reaction finishes, pcr amplification product is carried out 2.5% agarose gel electrophoresis to be detected, it is the purpose fragment of 85bp that the result has obtained length through pcr amplification, sequencing result shows the signal coding sequence that has obtained the correct human growth hormone of sequence, have SEQ ID № in the sequence table: 2 nucleotide sequence, reclaim and this dna fragmentation of purification.
3, the amplification of antibacterial peptide ABPS1 gene
Preferences according to e. coli codon, sequence (the Lu Qiang of antibacterial peptide ABPS1 has been synthesized in artificial design, the Liu Wei congruence, the synthetic of antibacterial peptide Shiva-1 gene and clone [J]. Chinese veterinary's journal, 2000,20 (2): 160-162) and as template, under the guiding of primer p5:5 '-atggttaaccgctggcgcctg-3 ' and p6:5 '-gttaacgcccacggcgcgagcg-3 ', pcr amplification antibacterial peptide ABPS1 gene.After reaction finishes, pcr amplification product is carried out 2% agarose gel electrophoresis to be detected, it is the purpose fragment of 123bp that the result has obtained length through pcr amplification, sequencing result shows and has obtained the correct antibacterial peptide ABPS1 gene of sequence, has SEQ ID № in the sequence table: 3 nucleotide sequence, SEQ ID № in the code sequence tabulation: 4 amino acid residue sequence, reclaim and this dna fragmentation of purification.
4, the structure of carrier for expression of eukaryon pBLG-hABPS1
(Invitrogen, CA is USA) for setting out the carrier for expression of eukaryon of vector construction antibacterial peptide ABPS1 gene with pVAX1.Concrete grammar is: the CMV promoter that pVAX1 itself contains is excised with restricted enzyme Nru I and BamH I double digestion, reclaim big fragment, again that an end is flat for benefit after using Hind III enzyme action, one end is inserted in the linearizing pVAX1 carrier that does not contain the CMV promoter through the BLG of BamH I enzyme action, obtains containing the intermediate carrier of BLG; Then with this intermediate carrier BamH I and Xba I double digestion, reclaim big fragment, hold the antibacterial peptide ABPS1 gene of the signal coding sequence that has merged human growth hormone gene behind BamH I and Xba I double digestion with 5 ' again, be inserted in the intermediate carrier of BamH I and Xba I double digestion, obtain a recombinant vector, this recombinant vector is checked order, sequencing result shows that the sequence of exogenous gene in the carrier and on position are all correct, obtained containing the promoter of bovine beta-lactoglobulin gene, the carrier for expression of eukaryon of human growth hormone's signal coding sequence and antibacterial peptide ABPS1 gene, called after pBLG-hABPS1, its physical map is seen Fig. 1.
Two, the acquisition of the genomic medicine of control mammitis of animal
With the carrier for expression of eukaryon pBLG-hABPS1 transformed into escherichia coli DH5 α competent cell that contains antibacterial peptide ABPS1 gene that step 1 makes up, screening positive clone was inoculated in positive colony in the LB culture medium, at 37 ℃ of bottom fermentation 14-18 hours; After the fermentation ends, collect bacterium liquid, centrifugal, abandon supernatant, the upgrading grain carries out purification with polyethylene glycol precipitation, detects OD
260/ OD
280Ratio, the result is 1: 1.9, conformance with standard; Again with plasmid DNA and pH8.0 TE buffer (solution that contains final concentration 10mmol/L TrisCl (pH8.0) and 1mmol/L EDTA (pH8.0)) mix homogeneously, the genomic medicine that obtains preventing and treating mammitis of animal (can be adjusted the mixed proportion of pBLG-hABPS1 plasmid DNA and TE buffer as required, just obtain the genomic medicine of the control mammitis of animal of variable concentrations, as with 10mgpBLG-hABPS1 plasmid DNA and 40mL pH8.0 TE buffer mix homogeneously, promptly obtaining concentration is the genomic medicine of 0.25mg/mL control mammitis of animal).
Embodiment 2, effect experiment
3 of the milch cows of giving milk that select to suffer from mastitis, have 4 breast districts mastitis takes place, detect (collection 2mL milk with mastitis diagnosticum (available from Beijing Milk Cow Center), in plate, drip equivalent mastitis diagnosticum, rock 10s, whether the coagulation degree appears reaching according to milk coagulation situation, judge whether ill and the state of an illness), the result is ++ ++ (being in a bad way), adopt the genomic medicine breast pond drug administration by injection of embodiment 1 preparation, medication is: earlier nipple is carried out disinfection, and squeeze the milk in clean ill breast district, draw 50mL PBS liquid (or normal saline) with sterilizing syringe then, inject newborn pond through milk duct, extrude after massaging breast gently, so repeat to wash newborn pond, at last with 4mL (genomic medicine of 0.25mg/mL control mammitis of animal).Genomic medicine injects newborn pond with milk duct, massages breast gently and makes it to be evenly distributed, duplicate injection next day 1 time), observe the curative effect.
After the result treated 14 days, mastitis was clearly better, and gathered 4 milk of suffering from mastitis breast district simultaneously and detected with the mastitis diagnosticum, and the result is respectively-,+, ++, +++, show that genomic medicine of the present invention has curative effect preferably to mastitis.
Embodiment 3, safety testing
Detect the safety of the genomic medicine of embodiment 1 preparation with following test:
1) mouse test
Give 5 healthy experiment mice tail vein injection embodiment each 2mL of genomic medicine (containing 500 μ g plasmid pBLG-hABPS1 altogether) of 1 preparation, the injection back was observed 5 days continuously.The untoward reaction and the phenomena of mortality do not appear in mice as a result.
2) nascent calf test
Give each 8mL of genomic medicine (containing 2mg plasmid pBLG-hABPS1 altogether) of nascent calf injection muscles injection embodiment 1 preparation of 5 health, the injection back was observed 5 days continuously.Unusual clinical manifestations such as fervescence do not appear in 5 calves as a result.
3) milch cow test
Give 5 each 20mL of genomic medicine (containing 20mg plasmid pBLG-hABPS1 altogether) that suffer from mammary gland injection embodiment 1 preparation of mastitis milch cow, the injecting method of breast base portion is: earlier with iodine tincture cotton balls sterilization breast district base portion skin, after taking off iodine with cotton ball soaked in alcohol, with the puncture needle that is about 10 centimetres, genomic medicine is expelled in the interlayer between each breast district and the stomach wall, should avoid trunks such as milk vein during inserting needle, in order to avoid it is hemorrhage, massaging breast messenger drug thing at last gently is evenly distributed, duplicate injection next day 1 time, the injection back was observed for 1 week continuously.Unusual clinical manifestations such as fervescence all do not appear in 5 breast districts as a result.
4) drug residue detects
Give the genomic medicine 20mL (containing 20mg plasmid pBLG-hABPS1) of mammary gland injection embodiment 1 preparation of suffering from the mastitis milch cow, get and carry out drug residue detection, continuous detecting 14 days under the guiding for the treatment of forward and backward milk primer P1:5 '-atggttaaccgctggcgcctg-3 ' and p2:5 '-gttaacgcccacggcgcgagcg-3 '.The result only injects the 2nd day PCR testing result in back and is positive, and proves that the residual quantity of medicine of the present invention is lower.
5) mice is irritated and feeds test
Irritate the genomic medicine 1mL (containing 0.1mg plasmid pBLG-hABPS1) that hello embodiment 1 prepares to mice, use 2% agarose gel electrophoresis and pcr amplification method (the same step 4) of the primer) degraded of detection of drugs in digestive tract dynamic then.The result is at the gastric of mice, and genomic medicine of the present invention is promptly degraded in 1 minute fully.
Above-mentioned result of the test shows that injecting genomic medicine of the present invention is safe to animal, and residual in Lac Bovis seu Bubali is of short duration, even there is minimal residue also can be degraded fast by digestive tract.
Embodiment 4, clinical trial
One, at the curative effect contrast test of latent mammitis
California mastitis algoscopy (CMT) (Middleton JR with international standard, Hardin D, Steevens B, Randle R, Tyler JW.Use of somatic cell counts and California mastitis testresults from individual quarter milk samples to detect subclinicalintramammary infection in dairy cattle from a herd with a high bulk tank somaticcell count.J Am Vet Med Assoc.2004 Feb 1; 224 (3): 419-423.; Sargeant JM, LeslieKE, Shirley JE, Pulkrabek BJ, Lim GH.Sensitivity and specificity of somaticcell count and California Mastitis Test for identifying intramammary infectionin early lactation.J Dairy Sci.2001 Sep; 84 (9): 2018-2024.; Shen Meiying, Sun Haizhou, Lu Dexun, Hou Zhijun utilizes the CMT method to pass judgment on Ruzhong somatic number and mammitis of cow herding and 2005 the 2nd phase 48-49 of forage science) the detection dairy cow milk, the milk Lac Bovis seu Bubali that reaction is judged to " +++" is distinguished into four groups (8 every group breast districts), inject the genomic medicine 1mL (containing 0.4mg plasmid BLG-hABPS1) that embodiment 1 prepares in first group of breast pond, the genomic medicine 1mL (containing 0.4mg plasmid BLG-hABPS1) of second group of breast base portion injection embodiment, 1 preparation, injection 1,000,000 unit penicillins and 250,000 unit streptomycins in the 3rd group of breast pond, inject 20mL Chinese medicine swelling of the breast disappear (available from Harbin Hua Nongweipu animal health product company limited) in the 4th group of breast pond, inject every day 1 time, inject altogether 3 times.With CMT reaction descend more than 1 "+" number be basis for estimation, the effective percentage that the effective percentage of one all back four the treatment groups of medication as a result was respectively after 8/8,7/8,7/8 and 5/8, ten day is respectively 7/8,7/8,7/8 and 4/8; The total number of bacteria of four treatment group milk, staphylococcus aureus number, streptococcus number and escherichia coli number average have obvious decline before the treatment; In 7 days during treating, the average milk yield of four treatment groups increases by 3.83,2.94,2.69 and 1.99 kilograms/head/sky before the treatment respectively.Above-mentioned result of the test shows that the effect and the antibiotic mixture of genomic medicine treatment mammitis of cow of the present invention are approaching.
Two, at treatment through the invalid mastitis of antibiotic therapy
1, test injection in the newborn pond
With the antibiotic mixture in the step 1 bovine mastitis breast district is treated, select 16 in the invalid clinical type garget milk district of antibiotic therapy, the genomic medicine 10mL (containing 0.8mg plasmid pBLG-hABPS1) of injection embodiment 1 preparation injects 1 every day in the breast pond, injects altogether 3 times.The result is after injection the 7th day the time, and except that the clinical symptoms in 1 breast district does not have the improvement, the clinical manifestations in all the other breast districts all have alleviating in various degree, comprise that red, swollen, hot, pain disappears, and milk color and pH recovery are normally; The CMT testing result shows that the treatment effective percentage is 8 7.5% (14/16), and cure rate is 37.5%.
2, breast district base portion test injection
With the antibiotic mixture in the step 1 bovine mastitis breast district is treated, 32 breasts that antibiotic therapy is invalid are divided into two groups, one group (16 breast districts) is the clinic mastitis group, the milk somatic number is between 500-999 ten thousand, another group (16 breast districts) is the latent mammitis group, the milk somatic number is less than 2,000,000, and every breast district base portion is injected the genomic medicine 10mL (containing 0.8mg plasmid pBLG-hABPS1) of embodiment 1 preparation, duplicate injection next day 1 time.Result the 2nd, 5,10 day after injection, the breast district that the somatic cell of clinic mastitis group descends more than 1,000,000 is respectively 61.1% (11/18), 83.3% (15/18) and 77.8% (14/18), the breast district that transfers latent mammitis to is respectively 33.3% (6/18), 33.3% (6/18) and 55.6% (10/18), and cure rate is 44.4% (8/18); In the identical time, the treatment effective percentage of latent mammitis group is respectively 55.6% (10/18), 94.4% (17/18) and 94.4% (17/18).
Above-mentioned result of the test shows that genomic medicine of the present invention is to also having certain therapeutic effect through the invalid intractable mastitis of antibiotic therapy.
Claims (4)
1. genomic medicine of preventing and treating mammitis of animal is to be the injection of effective ingredient with the carrier for expression of eukaryon that contains antibacterial peptide ABPS1 gene;
The upstream of antibacterial peptide ABPS1 gene is added with the promoter of mammal beta lactoglobulin gene in the described carrier for expression of eukaryon; The promoter of described mammal beta lactoglobulin gene is the promoter of bovine beta-lactoglobulin gene; The promoter of described bovine beta-lactoglobulin gene is the SEQ ID № in the sequence table: 1;
5 ' of antibacterial peptide ABPS1 gene end closely is connected with human growth hormone's signal coding sequence in the described carrier for expression of eukaryon; Described human growth hormone's signal coding sequence is the SEQ ID № in the sequence table: 2.
2. genomic medicine according to claim 1 is characterized in that: being used to make up the described carrier that sets out that contains the carrier for expression of eukaryon of antibacterial peptide ABPS1 gene is pVAX1, pSilence1.0-U6, pEGFP-N1, pCMV, pSV40, pSI, pCI, pCI-neo or pPICZ α, pTEF1, pcDNA3.1 or pcDNA.
3. genomic medicine according to claim 2 is characterized in that: the described carrier for expression of eukaryon that contains antibacterial peptide ABPS1 gene is pBLG-hABPS1; The nucleotide sequence of described antibacterial peptide ABPS1 gene is as SEQ ID №: shown in 3.
4. genomic medicine according to claim 1 is characterized in that: the medium of described injection is TE buffer, normal saline or the PBS of pH 7.5-8.5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5548075A (en) * | 1993-08-30 | 1996-08-20 | Utah State University | DNA cassette and transgenic organisms carrying lytic peptide-encoding genes |
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| 张利芳;刘维全;王吉贵;杨淑艳;孙绍光;李全;张丽;刘.抗菌肽ABPS1真核表达载体的构建及其杀灭肿瘤细胞的效应研究.全国反刍动物营养生理生化第三次学术研讨会论文摘要汇编.2004,176. * |
| 李全 |
| 杨淑艳 |
| 王吉贵 |
| 王铁东，网络出版投稿时间.抗菌肽乳腺组织特异性暂态表达及特性研究.吉林大学 |
| 王铁东,网络出版投稿时间.抗菌肽乳腺组织特异性暂态表达及特性研究.吉林大学硕士论文,2005,34-56. * |
| 硕士论文,2005,34-56. |
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