CN102516398B - Method for constructing anti-mammitis transgenic mouse model and special vector for method - Google Patents
Method for constructing anti-mammitis transgenic mouse model and special vector for method Download PDFInfo
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Abstract
The invention discloses a method for constructing an anti-mammitis transgenic mouse model and a special vector for the method. An anti-mammitis-related protein composition comprises lysostaphin and peptidoglycan endolysin B30; the peptidoglycan endolysin B30 derives from a Streptococcus agalactiae phage B30, and an amino acid sequence of the peptidoglycan endolysin B30 is sequence 2 in a sequence table; and an amino acid sequence of the lysostaphin is sequence 3 in the sequence table. The experiment proves that a transgenic plasmid for mammary gland-specific expression of the lysostaphin and the peptidoglycan endolysin B30 is constructed by using a mammary gland-specific expression vector; and the anti-mammitis transgenic mouse model is constructed by a microinjection method, a transgenic mouse with an anti-bovine mastitis gene is obtained, and a technical model is provided for further producing anti-mammitis transgenic calves.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of construction process and dedicated carrier thereof of anti-mastitis transgene mouse model.
Background technology
Mammitis of cow as a kind of complexity of milk cow, endanger serious common disease, its sickness rate is very high, controls difficulty and also strengthens thereupon.Although the whole world has years of researches, up to the present in raising dairy cattle, remain one of the highest disease of cost, seriously hindered dairy development.The financial loss that mastitis causes is to have reduced much than normal milk cow owing to suffering from ox milk producing ability; Thereby suffer from ox milk and cause abandoning of milk because the change of composition declines milk matter; Pregnant time and the oestrus cycle of suffering from ox extend; The mortality increase of ox and expense that the treatment of trouble ox is produced etc., and the decline of milk producing ability is the major cause the most that causes the loss of dairy tremendous economic.Every year because affecting cow producing milk ability, mammitis of cow cause the whole world to lose the milk of about 4,000,000 tons.As can be seen here, mammitis of cow is very huge to the economic impact of whole world milk cattle cultivating industry and dairy industry, and it seriously hinders the development of dairy.And for our national mammitis of cow sickness rate generally higher than in external situation, effectively preventing and treating this disease is our current key of doing milk cow development well.
At occurring in nature, nearly all bacterium has corresponding phage, and the total amount of phage is about 1031, thereby lytic enzyme resource is quite abundant.Although, as far back as nineteen fifty-nine Freimer EH etc., found that phage lytic enzyme can kill bacterium.But because antibiotic being widely used makes people not high enough to the antibacterial effect of phage lytic enzyme and application prospect attention rate thereof, until 21 century just gradually the research of restructuring lytic enzyme is become interested after it should be noted that the seriousness of antibiotic resistance.The reported first such as Schuch the application of phage lytic enzyme aspect treatment bacterium infection; The lytic enzyme LysK that has found phage K can suppress activity against staphylococci, effective to large-scale staphylococcus, and research shows that LysK can kill 9 kinds of staphylococcuses, comprising resistance X-1497 streptococcus aureus (MRSA).The many character about streptococcus agalactiae phage B30 peptidoglycan Inner lysin (Peptidoglycan endolysin B30) has been reported in the researchs such as David G.Therefore the research that utilizes in recent years the research of the autotelic transformation lytic enzyme of genetic engineering means (endolysin) and utilize phage lytic enzyme to carry out bacterial infection treatment gets more and more.
Lysostaphin (lysostaphin, lysostaphin) derive from staphylococcus and secrete the protein outside born of the same parents, it is a kind of antibacterial enzyme, this enzyme is found and is separated in the culture of imitation staphylococcus (S.simulan) that is numbered NRRL B22628 for 1964 by Schindler etc. from a strain, it is a kind of metalloprotease containing zinc, hydrolyzable aureus cell wall peptidoglycan glycine pentapeptide key (5 glycine be connected) thus dissolution of bacteria cell walls reaches sterilization object.Lysostaphin mainly has bacteriolyze germicidal action to staphylococcus, and different staphylococcuses is larger to its sensitivity differences, and wherein Yi Jin Portugal bacterium susceptibility is the highest.
Streptococcus aureus and suis are the topmost pathogenic bacterium of mastadenitis of cow, wherein infectious with the tool of streptococcus aureus.At present for the treatment of mastitis, still take microbiotic as main both at home and abroad, but after life-time service microbiotic, can cause bacterial drug resistance, many unfavorable factors such as antibiotic remains.It is fast that phage lytic enzyme has speed of action to bacterium, specificity is high, be difficult for producing the features such as resistance, the concentration that Lysostaphin is extremely low in Ruzhong in addition have antistaphylococcic activity, bacteriolysis is very powerful, and germicidal action is rapid, do not produce resistance yet, restructuring Lysostaphin is combined to use with microbiotic, endolysin etc. be the new approaches of clinical treatment mastadenitis of cow from now on.
Summary of the invention
An object of the present invention is to provide the relevant protein composition of a kind of and anti-mastitis.
The invention provides protein composition, comprise lysostaphin and peptidoglycan Inner lysin; Described peptidoglycan Inner lysin derives from streptococcus agalactiae phage B30, and its aminoacid sequence is the sequence 2 in sequence table; The aminoacid sequence of described lysostaphin is the sequence 3 in sequence table.
Lysostaphin and peptidoglycan Inner lysin are all by genetic modification, can not be by glycosylation in eukaryotic cell.
The gene of above-mentioned protein composition of encoding is also the scope of protection of the invention.
Said gene comprises the encoding gene of described lysostaphin and the encoding gene of described peptidoglycan Inner lysin; Said gene is specifically comprised of with the internal ribosome entry site that is connected the two described lysostaphin encoding gene, described peptidoglycan Inner lysin encoding gene.
Said gene is connected to form successively by the encoding gene of the encoding gene of described lysostaphin, described internal ribosome entry site and described peptidoglycan Inner lysin.
The nucleotides sequence of said gene is classified the sequence 1 in sequence table as.
Above-mentioned sequence 1 is comprised of 2065 Nucleotide, from 5 ' end 1-799 position Nucleotide, it is the encoding gene of lysostaphin (lysostaphin), from 5 ' end 800-1469 position Nucleotide, be internal ribosome entry site (Internal ribosome entry site, IRES), from 5 ' end 1470-2065 position Nucleotide, be the encoding gene of peptidoglycan endolysin (Peptidoglycan endolysin B30).
The recombinant vectors that contains said gene, recombinant bacterium, transgenic cell or expression cassette are also the scope of protection of the invention.
Above-mentioned recombinant vectors, for said gene is inserted between the Sac1 and Apa1 of pBC1+adapter carrier, obtains expressing the recombinant vectors of lysostaphin and peptidoglycan Inner lysin.
Above-mentioned pBC1+adapter carrier is prepared as follows:
1) by the forward sequence (adapter-seq2+3-F of joint, for tcgatagggcccaataccggtattctcgagattgagctcta) and reverse sequence (adapter-seq2+3-R the is tcgatagagctcaatctcgagaataccggtattgggcccta) annealing of joint after annealed after joint;
2) by step 1) joint is inserted into the XhoI site of pBC1 carrier after the annealing that obtains, obtains pBC1+adapter.
The application in building the animal model relevant to anti-mastitis of above-mentioned protein composition, above-mentioned DNA molecular, above-mentioned recombinant vectors, recombinant bacterium, transgenic cell or expression cassette is also the scope of protection of the invention;
The application in the anti-mastitis product of screening animal of above-mentioned protein composition, above-mentioned DNA molecular, above-mentioned recombinant vectors, recombinant bacterium, transgenic cell or expression cassette is also the scope of protection of the invention;
In above-mentioned application, described animal is specially mouse or ox; Above-mentioned anti-mastitis product is anti-Staphylococcus aureus medicine.
Another object of the present invention is to provide a kind of method that builds the animal model relevant to anti-mastitis.
Method provided by the invention, for above-mentioned DNA molecular is imported in animal, obtains transgenic animal, obtains the animal model relevant to anti-mastitis.
In aforesaid method, the method that described DNA molecular imports animal comprises the steps:
1) above-mentioned recombinant vectors is cut with AatII and NarI enzyme, reclaimed the fragment of 18.5kb;
2) by step 1) fragment of the 18.5kb that obtains imports in animal, obtains transgenic animal, obtains the animal model relevant to anti-mastitis;
Described animal is specially mouse or ox.
The 3rd object of the present invention is to provide a kind of protein B.
Protein B provided by the invention, its aminoacid sequence is the sequence 3 in sequence table.
The application of above-mentioned protein B in building the animal model relevant to anti-mastitis or the anti-mastitis product of screening animal is also the scope of protection of the invention.Above-mentioned anti-mastitis product is anti-Staphylococcus aureus medicine.
The present invention of experiment showed, of the present invention utilizes mammary gland specific expression vector to build the transgenosis plasmid of mammary gland specifically expressing lysostaphin (Lysostaphin) and peptidoglycan endolysin (Peptidoglycan endolysin B30); By microinjection, build anti-mastitis transgene mouse model, obtained the transgenic mice with anti-garget gene, for further producing anti-mastitis transgenic cattle, provide technology model, also for treat the research of mastitis by transgenic technology, established solid working foundation.
Accompanying drawing explanation
Fig. 1 is double digestion result
Fig. 2 is that F0 is for mouse PCR qualification result
Fig. 3 is the SDS-PAGE electrophoresis of transgenic mice milk
Fig. 4 is that RT-PCR detects transgenosis transcription product
Fig. 5 is that Spot on lawn analyzes mouse milk
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, transgenosis construction of recombinant vector
Transgenosis recombinant vectors pBC1+adapter+seq2+IRES-seq3 is the carrier obtaining between the Sac1 of the 1 insertion pBC1+adapter of the sequence in sequence table and Apa1, also can called after plasmid pBC1-Lysotaphin-peptidoglycan endolysin B30.
Wherein, sequence 1 is comprised of 2065 Nucleotide, from 5 ' end 1-799 position Nucleotide, be lysostaphin (lysostaphin), from 5 ' end 800-1469 position Nucleotide, be internal ribosome entry site (Internal ribosome entry site, IRES), from 5 ' end 1470-2065 position Nucleotide, be peptidoglycan endolysin (Peptidoglycan endolysin B30).
PBCl carrier utilizes goat β-casnein promotor to instruct recombinant protein efficiently specific expressed in mammary tissue, purchased from Invitrogen company, and article No. K27001.
Internal ribosome entry site (Internal ribosome entry site, IRES), IRES connects two open reading frame, and its transcription product is when translation, and ribose physical efficiency enters simultaneously and starts to translate two transcriptons in IRES upstream and downstream.In view of two different genes that connected by IRES can jointly be transcribed and can translate separately, this element is widely applied in transgenosis is produced.
Therefore utilize IRES that lysostaphin (lysostaphin) is connected with peptidoglycan endolysin (Peptidoglycan endolysin B30), can make two kinds of anti-mammitis of cow genes connect and all obtain expression, concrete steps are as follows:
1, the structure of expression vector pBC1+adapter
PBC1 carrier only contains the single clone's restriction enzyme site of XhoI, carrying out for convenient clone, at the Xho1 of pBC1 cloning site, inserted the joint that contains Apa1, Age1, Xho1, Sac1 restriction enzyme site, simultaneously the sequence at designed joint two ends is to have removed two Xho1 restriction enzyme sites that originally there will be after sticky end that TCGAT makes it to cut out with Xho1 enzyme is connected.
The forward sequence (adapter-seq2+3-F) of joint is tcgatagggcccaataccggtattctcgagattgagctcta; The reverse sequence of joint (adapter-seq2+3-R) is tcgatagagctcaatctcgagaataccggtattgggcccta, the reverse sequence of the forward sequence of joint and joint is annealed and is connected, joint after being annealed, specific as follows: in the reaction system that annealing connects, forward sequence and reverse sequence final concentration are all 100nM, add 10 * annealing buffer (100mM Tris pH 7.5,1M NaCl, 10mM EDTA), with DEPC water, supply remaining volume, 65 ℃ of water-bath incubations are after 10 minutes, cooling 1 to 2 hour of room temperature (25 ℃), joint after being annealed.
Joint after annealing obtained above is connected with the pBC1 carrier of cutting through same enzyme after XhoI enzyme is cut, obtains pBC1+adapter, be the XhoI site that after annealing, joint is inserted into.
2, the structure of recombinant vectors
1) acquisition of pBC1+adapter+seq2
By the lysostaphin gene after modifying, (in sequence table, sequence 1 is from 5 ' end 1-799 position Nucleotide, and amino acid is the sequence 2 in sequence table.) insert between the Apa1 and Xho1 restriction enzyme site of pBC1+adapter, obtain carrier pBC1+adapter+seq2, specific as follows:
(in sequence table, sequence 1 is from 5 ' end 1-799 for synthetic lysostaphin gene, and Apa1 recognition sequence downstream is carried with Xho1 recognition sequence in upstream, called after seq2, with Apa1 and Xho1 enzyme, cut seq2, the enzyme obtaining is cut product and is cut the pBC1+adapter obtaining and be connected through same enzyme, obtains pBC1+adapter+seq2.
2) acquisition of pIRES-seq3
Peptidoglycan endolysin (Peptidoglycan endolysin B30) is inserted to Nco1 and the Sac1 enzyme of pT2KXIG-IRES-EGFP and cut between restriction enzyme site, obtain carrier pIRES-seq3, specific as follows:
(in sequence table, sequence 1 is from 5 ' end 1470-2065 for synthetic peptidoglycan endolysin (Peptidoglycan endolysin B30), and Nco1 recognition sequence downstream is carried with Sac1 recognition sequence in upstream, called after seq3, with Nco1 and Sac1 enzyme, cut seq3, the enzyme obtaining is cut product and is cut through same enzyme the pT2KXIG-IRES-EGFP obtaining and (is documented in Genes Dev.1998Jul 1; 12 (13): 2048-60.Niwa H, Burdon T, Chambers I, in Smith A, the heredity of the public Ke Cong Chinese Academy of Sciences obtains with developmental biology institute.) connect, obtain recombinant plasmid pIRES-seq3, through order-checking, this plasmid is the downstream that sequence in sequence table 1 is inserted to IRES in pT2KXIG-IRES-EGFP from 5 ' end 1470-2065 position Nucleotide, forms pIRES-seq3.
3) acquisition of pIRES-seq3
Xho1 and Sac1 enzyme cut above-mentioned 2) pIRES-seq3 that obtains, reclaim the endonuclease bamhi of 1266bp, with through same enzyme, cut above-mentioned 1) pBC1+adapter+seq2 that obtains is connected, obtain connecting product, above-mentioned connection product is proceeded in e.colistraindh5α, obtain transformant, extract the plasmid of transformant and send to order-checking, result is for this plasmid is for inserting the sequence in sequence table 1 in the carrier obtaining between the Sac1 of pBC1+adapter and Apa1, by this plasmid called after pBC1+adapter+seq2+IRES-seq3, be plasmid pBC1-Lysotaphin-peptidoglycan endolysin B30.
The structure of embodiment 2, anti-mastitis transgene mouse model
One, the acquisition of microinjection DNA fragmentation
Get the plasmid pBC1+adapter+seq2+IRES-seq3 approximately 2 μ L that obtained by embodiment 1, the restriction enzyme A atII adding and NarI, restriction enzyme reaction damping fluid, add water and be settled to 40 μ L, flicking tube wall mixes, put 37 ℃ of thermostat container 1h, then get 3 μ L reaction solution 1% agarose gel electrophoresis observation enzymes and cut result.
The reaction system that transgene carrier enzyme is cut is as table 1:
The reaction system that table 1 transgene carrier enzyme is cut
The result of DNA agarose gel electrophoresis as shown in Figure 1,1:AatII and NarI endonuclease bamhi; M:1000bp DNAladder, the endonuclease bamhi of recovery 18.5kb, obtains microinjection DNA fragmentation, and through order-checking, this microinjection DNA fragmentation fragment comprises the sequence 1 in sequence table, and also having part is the promotor of mammary gland specifically expressing and the afterbody of poly A.
Microinjection DNA fragmentation ultraviolet spectrophotometer after recovery is measured concentration, and being diluted to concentration is 2ng/ μ L, 10 μ L/ pipe packing, and-80 ℃ save backup.Before injection, by the centrifugal 5min of injection DNA fragmentation 12000rpm, get middle portion solution for injection.
Two, the acquisition of anti-mastitis transgene mouse model
The microinjection DNA fragmentation being obtained by embodiment 1 is carried out to microinjection and enter in mouse male pronucleus, obtain transgenic mice, be anti-mastitis transgene mouse model, specific as follows:
1, experiment material
1) laboratory animal
SPF level ICR closed colony mouse is purchased from Test Animal Centre, Academy of Military Medical Sciences, P.L.A, credit number: SCXK-(army) 2007-004, below also referred to as wild-type mice.All experimental rats are all raised and breeding at the soft isolation bag of moulding of SPF level Barrier Facility, and temperature is controlled at 23 ℃ ± 1 ℃, humidity (55 ± 5) %.Light application time (12h bright/12h is dark), free choice feeding and drinking-water, used Beijing section Austria to pull together feed corporation,Ltd through Co60 irradiation sterilization feed.
2) main agents
Pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) and human chorionic gonadotrophin (hCG) (Ningbo three lives Pharma Inc.); M2 (sigma, M7167); M16 (sigma, M4287); Unidasa (sigma, H3506) and paraffin oil (sigma, M8410).
3) key instrument
Constant temperature incubator (Tianjin Stettlen Instrument Ltd.); CO
2incubator (U.S. Forma); Inverted microscope (Japanese NIKON TE2000); Micromanipulation system (German Eppendorf); Microinjection system (German Eppendorf Femtojet); Stereo microscope (Japanese NIKON SMZ-645); Draw pin instrument (David Kopf Instruments, USA); Forge pin instrument (Japanese Nari shi ge MF-400), card grinding instrument (Japanese Nari shigeEG-400); Bechtop (Shanghai San Fa scientific instrument company limited); Glass capillary (U.S. Harvard Apparatus); Various instruments (Beijing medicine limited-liability company); Syringe (Beijing Ke Youjia Bioisystech Co., Ltd); Various culture dish (U.S. Costar).
4) hold the preparation of ovum pin and injection needle
(1) hold the preparation of ovum pin
The Glass tubing of cut-off footpath 1mm, is drawing on pin instrument, and setting and drawing pin parameter is heating power 15, pulling force 25, is broken into forging pin instrument the flat mouth pin that diameter is about 80 μ m after drawing again, then on broken needle instrument, bakes pin after fracture being polished with card grinding instrument, internal diameter is baked to the m to 20-30 μ, save backup.
(2) injection needle preparation
The Glass tubing of cut-off footpath 1mm, at heating power 17, pulling force 35, directly saves backup after pulling into the entry needle that needle point is less than 1 μ m.
(3) capillary glass pipette preparation
Select the glass tubule of external diameter 1.5mm, length 10cm, right-hand man pinches respectively Glass tubing two ends, middle part is placed in to extremely rubescent deliquescing of rotary heating on flame, both hands draw to two ends gently, in the middle of Glass tubing, with emery wheel, row dry, break and be broken into 2 tubules, it the most carefully locates approximately 130 to 160 μ m, and port slips over flame gently makes its heating become smooth.Prepare disposable transfusion device, prolong the about 20cm in filter front and back end on transfusion device and cut short respectively tubing in left and right, its thinner one end connects the thick fracture of Glass tubing, and the other end is a mouthful suction end.
2, the preparation of the male mouse of ligation
Prepare the ICR male mice in 6 week age, 2% vetanarcol are pressed the dosage anesthetized mice of 45mg/kg body weight, Baoding of being lain on the back after anesthesia, with the little arrow of cutting of elbow operation, remove abdomen mouse hair, 75% alcohol disinfecting operative site, the outside of belly between cross sections two hind legs, integumentary musculature otch is large (about 1cm) too, find fat pad to pull out gently with blunt nosed tweezers, testis, epididymis and vas deferens can be pulled out thereupon, with tweezers, peel off vas deferens surrounding tissue, with tweezers, pick up the vas deferens having dissociated and form semicircular ring, with burning the semicircle vas deferens of the rapid pinch off of red ophthalmology pincet, with tweezers, send whole tissue back to intraperitoneal, same method pinch off opposite side vas deferens.By after all organized renewing original positions, suture muscles, skin, put into mouse cage by mouse after sterilization otch, after 2 weeks, gets final product rehabilitation.Afternoon or evening, by mating with the heat mouse in 8 week age after male mice numbering, are tested bolt morning next day, and positive mouse is put to death and rushes ovum, and whether microscopic examination has zygote, if without zygote explanation ligation success, this hero mouse can be used for producing the female mouse of false pregnancy.
3, the preparation of the female mouse of donor
Choose the female ICR of the not oestrusing mouse about 6 week age, every the mouse peritoneal PMSG Injection in 2 left and right that afternoon (10IU), 48h pneumoretroperitoneum injection hCG (10IU), then mates with healthy male ICR mouse at 1: 1, examine bolt morning next day, positive is as the female mouse of donor.
4, the preparation of the female mouse of acceptor
When the female mouse of donor and male mouse mate, select 8 week about age rutting sedson Healthy female ICR mouse and the successful male ICR mouse of ligation mate at 2: 1, examine bolt morning next day, positive is chosen the acceptor mouse as the same day.
5, rush ovum and reclaim embryo
The female ICR mouse of the injection hCG 28h inspection bolt positive, de-cervical vertebra method is put to death mouse, lie on the back in operation plate, 75% alcohol disinfecting skin, in belly mid-way, with eye scissors, skin is cut to an osculum, both hands pinch skin and gently draw along mouse cephlad-caudal, tear skin, with the scissors tweezers of sterilization, open abdominal cavity, can see uterine tube and ovary that fat pad is other, free uterine tube, cut and put into the M2 cultivation droplet filling 37 ℃ of preheatings, under dissecting microscope, tear ampulla of uterine tube, can see that cumulus cell group spills and (or draw M2 and insert fimbriae tubae portions and rinse whole uterine tube with joining blunt nosed No. 4 syringe needle 1mL syringes, liquid flows out by managing another opening), after adding the Unidasa of 500IU/mL to process about 0.5min, can obtain zygote, immediately with glass pipette by discharge second polar body and there are two protokaryons, kytoplasm is even, without the abnormal spilting of an egg, cell is full, zona pellucida high-quality fertilization clearly embryo move in another M2 culture dish, with M2, wash 2-3 time, during the M16 that proceeds to mineral oil covering 37 ℃ of preheatings cultivates and drips, put into the 5%CO of 37 ℃
2incubator is cultivated.
6, microinjection
On slide glass, do the M2 drop of a diameter 2.5mm, drop covers with paraffin oil, do again the DNA drop of the about 2ng/ μ of a concentration L and cover with vaporization prevention with paraffin oil, the unicellular pronuclear-stage embryos of the high-quality of select is proceeded in M2 drop by M16, slide glass moves on on microscopical Stage microscope, to hold ovum pin and entry needle is fixed on motion arm, adjust angle direction, suction is blown liquid and is confirmed entry needle opening, and with glass needle marshalling zygote, then start microinjection, by fixed tube, hold an ovum, pressure is not too big, with syringe, adjust its position, make the protokaryon of zygote high-visible, and make needle point and protokaryon is clear is positioned at same focal plane syringe is thrust to protokaryon, the microinjection DNA fragmentation that injection embodiment 1 obtains, when see protokaryon obviously swell after the fast withdraw of the needle.With holding ovum pin, push zygote to below, again hold a new zygote and carry out same injection operation, so repeat until all zygote injection is complete.The M16 that after injection, zygote is proceeded to 37 ℃ of preheatings of mineral oil covering cultivates about 60min in cultivating and dripping in 5% CO2gas incubator of 37 ℃.After renewal cultivation, be inverted and examine under a microscope embryo record.To there is complete ooecium plasma membrane, normal ovum week gap, be judged as survival embryo, can be used for embryo transfer; Kytoplasm diffusion, what all gaps of ovum disappeared is dead embryo, discards.
7, embryo transfer
By zygote survival number, 2% vetanarcol are pressed the dosage intraperitoneal injection of anesthesia acceptor mouse of 45mg/kg body weight, mouse is lain on the back in operation plate, cut off back two center line both sides, hind leg back side mouse hairs, 75% alcohol disinfecting, in the last rib level of dorsomeson, longitudinally cut 1cm left and right osculum, mouse is moved on under stereoscopic microscope and finds fatty body to press from both sides out with blunt nosed tweezers, ovary, uterine tube etc. are organized also and are drawn out thereupon, with Small clamp, clamp the fatty body of the ovary of ining succession, fixing.With the first interval of capillary glass grafts 3mm air amount bubble and substratum, at front end, suck 12 left and right embryos, grafts is placed on to safe place.Tear cyst membrane and find uterine tube hydraucone and ampulla, by hydraucone, insert grafts, be blown into embryo, examine until the air filled cavity of the inside enters the portion of expanding.Extract grafts out, then the tissues such as ovary are reset into intraperitoneal, suture muscles and skin with 75% alcohol disinfecting operation mouthful, are incubated observation and treat that it revives under 37 ℃ of environment.
Use the microinjection DNA fragmentation being obtained by embodiment 1 to carry out microinjection and enter in mouse male pronucleus, injected altogether 320 pieces of zygotes, wherein survive 205 pieces, be transplanted to 10 acceptor mouse, 5 pregnancies, 29 F0 that have been born are altogether for transgenic mice.
Three, the evaluation of transgenic mice and going down to posterity
1, the evaluation of transgenic mice
PEASY-T1 carrier is from Quan Shi King Company.
Extract 2 weeks F0 of birth for the genomic dna of the about 0.5cm of tail point of transgenic mice, with two couples of PCR, screen and carry out pcr amplification with Auele Specific Primer respectively, take wild-type mice as contrast.Primer sequence is as shown in table 2 below:
Table 2Seq2, Seq3 and contrast primer sequence
Table 3PCR amplification reaction system
PCR reaction conditions is: 95 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, and 72 ℃ are extended 15min, 35 circulations, 4 ℃ of preservations.
As shown in Figure 2, for F0 is for transgenic mice PCR qualification result, Fig. 2 A is for being used the result of seq2 primer amplification for result; Fig. 2 B is for being used the result of seq3 primer amplification; Fig. 2 C is for being used the result of contrast primer Gapdh amplification, C1: wild-type ICR mouse PCR product; C1-1x: wild-type ICR mouse gene group DNA adds the PCR product of positive control plasmid (positive control plasmid is pBC1+adapter+seq2+IRES-seq3) 0.3pg; C1-5x: wild-type ICR mouse gene group DNA adds the PCR product of positive control plasmid 1.5pg, C1-50x: wild-type ICR mouse gene group DNA adds the PCR product of positive control plasmid 15pg;-: negative control; F21-F38:F0 is for transgenic mice PCR product; M:100bp DNA ladder, can find out, Seq2 primer pair obtains 437bp size amplified band, and Seq3 primer pair obtains the positive F0 of 409bp size amplified band for transgenic mice, show to have 3 positive F0 of mouse for transgenic mice, positive rate is 10.3% (3/29), and numbering is respectively F32, F35, F38.
Reclaim the 437bp size pcr amplification product that positive F0 obtains for the Seq2 primer pair amplification of mouse, be connected with pEASY-T1 cloning vector, obtain connecting product, obtain transformant, extract the plasmid of transformant, carry out HindIII and Xho1 double digestion, obtain the positive plasmid of 437bp, this positive plasmid is sent to order-checking, this plasmid contains PCR product and has in sequence table sequence 1 from 5 ' end 75-511 position Nucleotide, and lysostaphin gene, further proves numbering and be respectively F32, F35, the positive F0 of F38 is for mouse.
Illustrate that the numbering obtaining is respectively F32, F35, the positive F0 of F38 is anti-mastitis transgene mouse model for mouse.
2, transgenic mice goes down to posterity
Numbering is respectively F32, F35, the positive F0 of F38 for transgenic mice (primary be F0 generation) about 3 weeks from breast, male and female sub-cage rearing, wait to grow to about 8 weeks, mate with normal ICR mouse the conservation that goes down to posterity respectively, obtain F1 generation transgenic mice (if 32# is F1 generation transgenic mice), for subsequent experimental.Again F1 generation transgenic mice and normal ICR mouse are mated to the conservation that goes down to posterity, obtain F2 for mouse.Wherein F38 when 4 week age because unknown cause is dead.F32 and F35 are in good condition, after it grows to 6 week age, mate with the male mouse of wild-type ICR.
The results are shown in Table 4 and table 5, is F2 generation by the male F1 mouse of 35-9# (the positive F0 of numbering F35 is for the first filial generation of transgenic mice) with the mouse of wild-type ICR female mice mating gained, obtains altogether F2 for 32 of mouse.Wherein, female 12, male 20.The F2 that founder35# mouse is obtained, for mouse and wild-type mice mating, obtains F3 altogether for 35 of mouse.Up to the present, two transgenic lines (32# and 35#) that obtain can be passaged to F4 generation.The stably heredity of two transgenic lines is described.
The F1 generation mouse detected result of founder32# mouse and founder35# mouse is as follows:
Table 4founder 32# and wild-type generation mice that post-coitum obtains
Table 5founder 35# and wild-type generation mice that post-coitum obtains
Four, the expression of goal gene in transgenic mice
1, destination gene expression in milk
PCR is detected to (the newborn sample damping fluid: 125mM/L NaCl, 25mM/L Tris pH7.4,5mM/L KCl, 2mM/L PMSF of newborn sample damping fluid for milk that 32# is F1 generation transgenic mice and wild-type mice.) 1: 4 dilution after, at 4 ℃ of centrifugal 15min of refrigerated centrifuge 12000rpm, remove gently the fat on upper strata, get middle layer whey liquid and save backup in another centrifuge tube-20 ℃, obtain whey liquid.
Whey liquid is carried out to SDS-PAGE electrophoresis, result as shown in Figure 3,1: wild-type mice milk, applied sample amount is 2.5 μ L; 2:32# is F1 generation mouse milk, and applied sample amount is 2.5 μ L; 3: wild-type mice milk, applied sample amount is 5 μ L; 4:32# is F1 generation mouse milk, and applied sample amount is 5 μ L; 5: wild-type mice milk, applied sample amount is 10 μ L; 6:32# is F1 generation mouse milk, and applied sample amount is 10 μ L; 7: wild-type mice milk, applied sample amount is 15 μ L; 8:32# is F1 generation mouse milk, and applied sample amount is 15 μ L; M: albumen marker; Can find out, with respect to wild-type mice, in transgenic mice milk, occurred an obvious protein band, size is about 20KD (shown in black arrow), with the transgene protein lysostaphin inserting and endolysin (size is also 20KD left and right) in the same size.
2, goal gene expression in mouse tissue
The 32# of the clip positive is the liver of F1 generation mouse, each 200 μ g of mammary gland are put in respectively in the homogenizer of different numberings, with Triozl, extract RNA, reverse transcription obtains cDNA, with seq3 primer PCR, increase, using Gapdh primer pair as negative control, result as shown in Figure 4, RT-PCR detects transgenosis transcription product, 1: wild-type mice mammary tissue, 2: wild-type mice spleen tissue, 3:32# is F1 generation mouse spleen tissue, 4:32# is F1 generation mammary gland of mouse tissue, 5:32# is F1 generation mammary gland of mouse tissue, as can be seen from the figure, at 32#, be in F1 generation mammary gland of mouse tissue, to have the fragment of 409bp, at 32#, be in mouse, transgenosis is expressed specifically in mammary tissue, and not at other tissue expression.
3, the function assessment of transgenic mice milk analysis-spot on lawn analyzes
1) picking streptococcus aureus mono-clonal 1 and 2 (is all purchased from Institute of Microorganism, Academia Sinica respectively, 1 bacterium numbering is 1.2386,2 bacterium numbering is 1.2419) be inoculated in 2ml substratum, 220 revs/min, 37 ℃ of shaking table shaking culture are to logarithmic phase.
2) preparation is respectively 1.5% and 0.7% substratum, autoclaving containing agar concentration.
3) in 100mm Micro-Organism Culture Dish, pour the 1.5% nutrient agar 10ml that is cooled to 55 ℃ of left and right into, room temperature (25 ℃) is cooling rear standby.
4) by logarithmic phase bacterium in 1: 200 ratio add be cooled to 55 ℃ containing in the substratum of 0.7% agar, after mixing, pour containing in 1.5% nutrient agar ware of solidifying into, obtain two culture dish of A and B, wherein, A is streptococcus aureus CGMCC1.2386, and B is streptococcus aureus CGMCC1.2419; According to each 100mm culture dish, pour 7ml liquid into, naturally cooling after 30 minutes in Bechtop, setting-out on each culture dish.
5) 32# of extracting degreasing is that the milk 15 μ L of F1 generation mouse drop on the substratum that culture dish solidifies.
6) liquid nutrient medium that adds same volume is as negative control, and concentration is that the lysostaphin (lysostaphin) of 100 μ g/mL is as positive control.
7) culture dish that drips milk is observed after being placed on and proceeding to 37 ℃ of incubator incubated overnight after standing 30 minutes in super clean bench, takes pictures.
As shown in Figure 5, Spot on lawn analyzes mouse milk to result,, the streptococcus aureus in A is CGMCC1.2386, the streptococcus aureus in B is CGMCC1.2419; In A and B figure, 1 is wild-type mice milk; 2 and 3 to be 32# be F1 generation mouse milk; 4 are nutrient solution, as negative contrast; 5 are lysostaphin, as positive control.Can find out, wild-type mice does not produce inhibition zone, and being F1 generation mouse, 32# produces obvious inhibition zone, illustrate that albumen germ resistance in transgenic mice milk is apparently higher than wild-type mice, illustrate in transgenic mice milk and may specific expressedly have lysostaphin (Lysostaphin), and it has antibacterial.
Above description of test, the present invention has obtained the transgenic mice with anti-garget gene, thereby can check rapidly antibiotic protein gene and the transgenic regulation element expression regulation situation in mammary gland, for somatocyte transgenic cattle provides basis and foundation; Can system detect the various unit price antibacterial proteins of transgenic mice expression and the disease-resistant efficiency that multivalence antimicrobial protein resists tiring of artificial inoculation mastitis pathogenic bacterium and whole animal simultaneously.
Claims (5)
1. a gene for the protein composition that coding is relevant to anti-mastitis, is comprised of with the internal ribosome entry site that is connected the two lysostaphin encoding gene, peptidoglycan Inner lysin encoding gene;
Described gene is connected to form successively by the encoding gene of the encoding gene of described lysostaphin, described internal ribosome entry site and described peptidoglycan Inner lysin; The nucleotides sequence of described gene is classified the sequence 1 in sequence table as.
2. the recombinant vectors, recombinant bacterium, transgenic cell or the expression cassette that contain gene described in claim 1.
3. recombinant vectors according to claim 2, is characterized in that: described recombinant vectors, for gene described in claim 1 is inserted in pBC1+adapter carrier, obtains expressing the recombinant vectors of lysostaphin and peptidoglycan Inner lysin.
4. gene, recombinant vectors claimed in claim 2, recombinant bacterium, transgenic cell or the expression cassette application in building the animal model relevant to anti-mastitis described in claim 1;
Described animal is specially mouse or ox.
Described in claim 1 gene, recombinant vectors claimed in claim 2, recombinant bacterium, transgenic cell or expression cassette in the application of screening animal anti-mastitis product;
Described animal is specially mouse or ox.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US7572602B1 (en) * | 2004-12-03 | 2009-08-11 | The United States Of America As Represented By The Secretary Of Agriculture | Nucleic acid encoding endolysin fusion protein |
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| CN101297034A (en) * | 2005-08-24 | 2008-10-29 | 洛克菲勒大学 | PLY-GBS mutant lysins |
Non-Patent Citations (2)
| Title |
|---|
| 重组溶葡萄球菌酶对奶牛乳房炎和子宫内膜炎常见病原菌的体外杀菌试验;黄青山;《中国兽药杂志》;20070831;第41卷(第8期);41-47 * |
| 黄青山.重组溶葡萄球菌酶对奶牛乳房炎和子宫内膜炎常见病原菌的体外杀菌试验.《中国兽药杂志》.2007,第41卷(第8期),41-47. |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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