CN102516398A - Method for constructing anti-mammitis transgenic mouse model and special vector for method - Google Patents

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

The construction process of anti-mastitis transgene mouse model and dedicated carrier thereof

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 complicacy of milk cow, the serious common disease of harm, its sickness rate is very high, the control difficulty 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 the dairy development.The financial loss that mastitis causes is because trouble ox milk producing ability has reduced much than normal milk cow; Thereby suffer from abandoning that ox milk causes because of the change of composition descends milk matter suckling; The pregnant time and the oestrus cycle of suffering from ox prolong; The mortality of ox increases and expense that the treatment of suffering from ox is produced or the like, and the decline of milk producing ability is the major cause the most that causes the loss of dairy tremendous economic.Cause whole world to lose about 400 ten thousand ton milk because of mammitis of cow influences the cow producing milk ability every year.This shows that mammitis of cow is very huge to the economic impact of whole world milk cattle cultivating industry and dairy industry, it seriously hinders the development of dairy.And generally be higher than under the external situation for our national mammitis of cow sickness rate, effectively preventing and treating this disease is our current milk cow development key of doing well.

At occurring in nature, nearly all bacterium all has corresponding phage, and the total amount of phage is about 1031, thereby the lytic enzyme resource is quite abundant.Though, found that as far back as nineteen fifty-nine Freimer EH etc. the phage lytic enzyme can killing bacteria.But make people not high enough because of antibiotic, just gradually the research of reorganization lytic enzyme is become interested it should be noted that the seriousness of antibiotic resistance up to 21 century after the antibacterial effect and the application prospect attention rate thereof of phage lytic enzyme.Reported first such as Schuch the application of phage lytic enzyme aspect the treatment infectation of bacteria; The lytic enzyme LysK that has found phage K can suppress activity against staphylococci, and is 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 Streptococcusagalactiae bacteriophage B30 peptide glycan endolysin (Peptidoglycan endolysin B30) has been reported in researchs such as David G.Therefore the research that utilizes the research of the autotelic transformation lytic enzyme of genetic engineering means (endolysin) in recent years and utilize the phage lytic enzyme to carry out the bacterial infection treatment gets more and more.

Dissolve the plain (lysostaphin of golden yellow Portugal coccus; Lysostaphin) derive from staphylococcus and secrete the protein outside born of the same parents; Be a kind of antibiotic enzyme, this enzyme is found in the culture of the imitation staphylococcus (S.simulan) that was numbered NRRL B22628 in 1964 by Schindler etc. from a strain and is separated, is a kind of zinciferous metalloprotease; Hydrolyzable aureus cell wall Polysaccharides, peptide complexes glycocoll pentapeptide key (5 glycocoll link to each other) thus the dissolution of bacteria cell walls reaches the sterilization purpose.Dissolve golden yellow Portugal coccus element and mainly staphylococcus is had the bacteriolyze germicidal action, different staphylococcuses is bigger to its sensitivity differences, and is wherein the highest with golden Portugal bacterium susceptibility.

Streptococcus aureus and suis are the topmost pathogenic bacterium of mastadenitis of cow, and be wherein infectious with the tool of streptococcus aureus.The domestic and international at present treatment for mastitis is main with microbiotic still, but can cause bacterial drug resistance behind the life-time service microbiotic, many unfavorable factors such as antibiotic remains.It is fast that the phage lytic enzyme has a speed of action to bacterium; Specificity is high, is difficult for producing characteristics such as resistance, dissolves the golden yellow Portugal coccus element concentration extremely low in the Ruzhong in addition and promptly has antistaphylococcic activity; Bacteriolysis is very powerful; And germicidal action is rapid, does not also produce resistance, reorganization is dissolved golden yellow Portugal coccus is plain unites with microbiotic, endolysin etc. that to use 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 present invention provides a protein composition comprising lysostaphin hormone and endolysin peptidoglycan; said endolysin peptidoglycan from Streptococcus agalactiae phage B30, the amino acid sequence of the sequence in the Sequence 2; wherein lysostaphin amino acid sequence of the sequence elements in the Sequence 3.

Lysostaphin and peptide glycan endolysin all are by genetic modification, can not be by glycosylation in eukaryotic.

The gene of above-mentioned protein composition of encoding also is the scope that the present invention protects.

Lysostaphin gene comprising the gene encoding the hormone and the peptidoglycan endolysin encoding gene; the lysostaphin gene specific element by said gene encoding the peptidoglycan endolysin encoding gene and the connection internal ribosome entry between the two loci.

Said gene is connected to form by the encoding gene of the encoding gene of said lysostaphin, said internal ribosome entry site and said peptide glycan endolysin successively.

The nucleotides sequence of said gene is classified the sequence 1 in the sequence table as.

Above-mentioned sequence 1 is made up of 2065 Nucleotide; From 5 ' terminal 1-799 position Nucleotide is the encoding sox of lysostaphin (lysostaphin); From 5 ' terminal 800-1469 position Nucleotide is internal ribosome entry site (Internal ribosome entry site; IRES), be the encoding sox of Polysaccharides, peptide complexes endolysin (Peptidoglycan endolysin B30) from 5 ' terminal 1470-2065 position Nucleotide.

The recombinant vectors, reorganization bacterium, transgenic cell or the expression cassette that contain said gene also are the scopes that the present invention protects.

Above-mentioned recombinant vector obtains expressing the recombinant vector of lysostaphin and peptide glycan endolysin for said gene is inserted between the Sac1 and Apa1 of pBC1+adapter carrier.

Above-mentioned pBC1+adapter carrier prepares according to following method:

1) with the forward sequence (adapter-seq2+3-F of joint; Be tcgatagggcccaataccggtattctcgagattgagctcta) and the annealing of the reverse sequence (adapter-seq2+3-R is tcgatagagctcaatctcgagaataccggtattgggcccta) of joint after the back joint that obtains annealing;

2) joint is inserted into the XhoI site of pBC1 carrier after the annealing that step 1) is obtained, and obtains pBC1+adapter.

Above-mentioned protein composition, above-mentioned dna molecular, above-mentioned recombinant vectors, reorganization bacterium, transgenic cell or the application of expression cassette in making up the animal model relevant with anti-mastitis also are the scopes that the present invention protects;

Above-mentioned protein composition, above-mentioned dna molecular, above-mentioned recombinant vectors, reorganization bacterium, transgenic cell or the application of expression cassette in the anti-mastitis product of screening animal also are the scopes that the present invention protects;

In above-mentioned application, said animal is specially mouse or ox; Above-mentioned anti-mastitis product is anti-streptococcus aureus medicine.

Another object of the present invention provides a kind of method that makes up the animal model relevant with anti-mastitis.

Method provided by the invention for above-mentioned dna molecular is imported in the animal, obtains transgenic animal, promptly obtains the animal model relevant with anti-mastitis.

In the aforesaid method, the method that said 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) fragment of the 18.5kb that step 1) is obtained imports in the animal, obtains transgenic animal, promptly obtains the animal model relevant with anti-mastitis;

Said animal is specially mouse or ox.

The 3rd purpose of the present invention provides a kind of protein B.

Protein B provided by the invention, its aminoacid sequence are the sequence 3 in the sequence table.

The application of above-mentioned protein B in making up animal model relevant with anti-mastitis or the anti-mastitis product of screening animal also is the scope that the present invention protects.Above-mentioned anti-mastitis product is anti-streptococcus aureus medicine.

The present invention of experiment showed, of the present invention utilizes mammary gland specific expression vector to make up the transgenic plasmid of mammary gland specifically expressing lysostaphin (Lysostaphin) and Polysaccharides, peptide complexes endolysin (Peptidoglycan endolysin B30); Make up anti-mastitis transgene mouse model through microinjection; Obtained to have the transgenic mice of anti-Mammitis of cattle gene; For further producing anti-mastitis transgenic cattle technology model is provided, also for having established solid working foundation through the research of transgenic technology treatment mastitis.

Description of drawings

Fig. 1 is the 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 detects the transgenic transcription product for RT-PCR

Fig. 5 is that Spot on lawn analyzes mouse milk

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Embodiment 1, transgenic construction of recombinant vector

The carrier of transgenic recombinant vectors pBC1+adapter+seq2+IRES-seq3 for obtaining between the Sac1 that the sequence in the sequence table 1 inserted pBC1+adapter and Apa1, but also called after plasmid pBC1-Lysotaphin-peptidoglycan endolysin B30.

Wherein, Sequence 1 is made up of 2065 Nucleotide; From 5 ' terminal 1-799 position Nucleotide is lysostaphin (lysostaphin); From 5 ' terminal 800-1469 position Nucleotide is that (Internal ribosome entry site IRES), is Polysaccharides, peptide complexes endolysin (Peptidoglycan endolysin B30) from 5 ' terminal 1470-2065 position Nucleotide to internal ribosome entry site.

The pBCl carrier utilizes goat β-casnein promotor to instruct recombinant protein efficiently specific expressed in mammary tissue, available from Invitrogen company, and article No. K27001.

Internal ribosome entry site (Internal ribosome entry site, IRES), IRES connects two ORFs, and its transcription product is when translation, and the ribose physical efficiency gets into and begins to translate two transcriptons in the IRES upper reaches and downstream simultaneously.Can transcribe jointly and can translate separately in view of two different gene that connected by IRES, this element has obtained widespread use in transgenic is produced.

Therefore utilize IRES that lysostaphin (lysostaphin) is connected with Polysaccharides, peptide complexes endolysin (Peptidoglycan endolysin B30), can make two kinds of anti-mammitis of cow genes connect and all obtain and express, concrete steps are following:

1, the structure of expression vector pBC1+adapter

The pBC1 carrier only contains the single clone's restriction enzyme site of XhoI; The carrying out of cloning for ease; Inserted the joint that contains Apa1, Age1, Xho1, Sac1 restriction enzyme site at the Xho1 of pBC1 cloning site, simultaneously the sequence at designed joint two ends is to have removed two Xho1 restriction enzyme sites that script can occur after sticky end that TCGAT makes it to cut out with the 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, and the reverse sequence of the forward sequence of joint and joint is annealed to be connected, and the back joint obtains annealing; Specific as follows: in the reaction system that annealing connects, forward sequence and reverse sequence final concentration all are 100nM, add 10 * annealing buffer (100mM Tris pH 7.5; 1M NaCl; 10mM EDTA), supply remaining volume with DEPC water, 65 ℃ of water-bath incubations are after 10 minutes; Room temperature (25 ℃) cooling 1 to 2 hour, the back joint obtains annealing.

Joint after the above-mentioned annealing that obtains is connected with the pBC1 carrier of cutting through same enzyme after the XhoI enzyme is cut, obtains pBC1+adapter, be the XhoI site that annealing back joint is inserted into.

2, the structure of recombinant vectors

1) acquisition of pBC1+adapter+seq2

(sequence 1 is from 5 ' terminal 1-799 position Nucleotide in the sequence table, and amino acid is the sequence 2 in the sequence table with the lysostaphin gene after modifying.) insert between the Apa1 and Xho1 restriction enzyme site of pBC1+adapter, obtain carrier pBC1+adapter+seq2, specific as follows:

(sequence 1 is from 5 ' terminal 1-799 in the sequence table for synthetic lysostaphin gene; And the upper reaches are carried Apa1 recognition sequence downstream and are had the Xho1 recognition sequence; Called after seq2; Cut seq2 with Apa1 and Xho1 enzyme, the enzyme that obtains is cut product and is cut the pBC1+adapter that obtains and be connected through same enzyme, obtains pBC1+adapter+seq2.

2) acquisition of pIRES-seq3

Polysaccharides, peptide complexes endolysin (Peptidoglycan endolysin B30) is inserted Nco1 and the Sac1 enzyme of pT2KXIG-IRES-EGFP and cut between restriction enzyme site, obtain carrier pIRES-seq3, specific as follows:

(sequence 1 is from 5 ' terminal 1470-2065 in the sequence table for synthetic Polysaccharides, peptide complexes endolysin (Peptidoglycan endolysin B30); And the upper reaches are carried Nco1 recognition sequence downstream and are had the Sac1 recognition sequence; Called after seq3; Cut seq3 with Nco1 and Sac1 enzyme, the enzyme that obtains is cut product and is cut the pT2KXIG-IRES-EGFP that obtains through same enzyme and (is documented in Genes Dev.1998Jul 1; 12 (13): 2048-60.Niwa H, Burdon T, Chambers I, among the Smith A, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.) connect, obtain recombinant plasmid pIRES-seq3, through order-checking, this plasmid is with the downstream of sequence in the sequence table 1 IRES in 5 ' terminal 1470-2065 position Nucleotide insertion pT2KXIG-IRES-EGFP, constitutes 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 cut through same enzyme above-mentioned 1) pBC1+adapter+seq2 that obtains is connected, and obtains connecting product; Above-mentioned connection product is changed in the e.colistraindh5; Obtain transformant, extract the plasmid of transformant and send to order-checking, the result for this plasmid for the carrier that obtains between the Sac1 of the 1 insertion pBC1+adapter of the sequence in the sequence table and Apa1; With 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 about 2 μ L of the plasmid pBC1+adapter+seq2+IRES-seq3 that obtains by embodiment 1; Restriction enzyme A atII that adds and NarI, restriction enzyme reaction damping fluid; Add water and be settled to 40 μ L; Flick the tube wall mixing, put 37 ℃ of thermostat container 1h, get 3 μ L reaction solutions, 1% agarose gel electrophoresis observation enzyme then and cut the result.

Reaction system such as table 1 that the transgene carrier enzyme is cut:

The reaction system that table 1 transgene carrier enzyme is cut

The result of DNA agarose gel electrophoresis is as shown in Figure 1,1:AatII and NarI endonuclease bamhi; M:1000bp DNAladder, the endonuclease bamhi of recovery 18.5kb obtains the microinjection dna fragmentation, and through order-checking, this microinjection dna fragmentation fragment comprises the sequence 1 in the 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 the recovery is measured concentration, and being diluted to concentration is 2ng/ μ L, and 10 μ L/ manage packing, and-80 ℃ of preservations are subsequent use.With the centrifugal 5min of injection dna fragmentation 12000rpm, get middle portion solution and be used for injection before the injection.

Two, the acquisition of anti-mastitis transgene mouse model

To carry out microinjection by the microinjection dna fragmentation that embodiment 1 obtains and go in the 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 available from Test Animal Centre, Academy of Military Medical Sciences, P.L.A, and credit number: SCXK-(army) 2007-004 below is also referred to as wild-type mice.All experimental rats are all isolated bag raising and breeding soft the 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 use 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 pregnancy urine extract (hCG) (Ningbo three lives Pharma Inc.); M2 (sigma, M7167); M16 (sigma, M4287); Unidasa (sigma, H3506) and Yellow Protopet 2A (sigma, M8410).

3) key instrument

Constant temperature incubator (Tianjin Tai Site Instr 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 the pin appearance (David Kopf Instruments, USA); Forge pin appearance (Japanese Nari shi ge MF-400), card grinding appearance (Japanese Nari shigeEG-400); Bechtop (three the scientific instrument ltds in Shanghai); Glass capillary (U.S. Harvard Apparatus); Various instruments (Beijing medicine limited-liability company); Syringe (the good Bioisystech Co., Ltd of friend of Beijing section); Various petridish (U.S. Costar).

4) hold the preparation of ovum pin and microinjection pin

(1) holds the preparation of ovum pin

The Glass tubing of cut-off footpath 1mm is drawing on the pin appearance, and setting and drawing the pin parameter is heating power 15; Pulling force 25 is broken into the flat lancet that diameter is about 80 μ m with forging the pin appearance after drawing again, with the card grinding appearance fracture is polished back roasting pin on the broken needle appearance again; Internal diameter is roasting to 20-30 μ m, preserve subsequent use.

(2) microinjection pin preparation

The Glass tubing of cut-off footpath 1mm, at heating power 17, pulling force 35 pulls into needle point and directly preserves after less than the entry needle of 1 μ m subsequent use.

(3) capillary glass pipette preparation

Select the glass tubule of external diameter 1.5mm, length 10cm for use; The right-hand man pinches the Glass tubing two ends respectively, the middle part is placed to rotate on the flame be heated to rubescent deliquescing, and both hands draw to two ends gently; In the middle of Glass tubing, row dry with emery wheel; Break off with the fingers and thumb and be broken into 2 tubules, it the most carefully locates about 130 to 160 μ m, and port slips over flame gently makes its heating become smooth.Prepare disposable transfusion device, prolong on the transfusion device and cut short tubing respectively about the about 20cm in filter front and back end, its thinner end connects the thick fracture of Glass tubing, and the other end then is a mouthful suction end.

2, the preparation of the male mouse of ligation

Prepare the ICR male mice in 6 ages in week, 2% vetanarcol are pressed the dosage anesthetized mice of 45mg/kg body weight, after the anesthesia with its Baoding of lying on the back; For a short time cut arrow with the elbow operation and remove abdomen mouse hair, 75% alcohol disinfecting operative site, the outside of belly between cross sections two hind legs; The integumentary musculature otch is big (about 1cm) too, finds fat pad to pull out gently with blunt nosed tweezers, and testis, epididymis and vas deferens can be pulled out thereupon; Peel off the vas deferens surrounding tissue with tweezers, pick up the vas deferens that has dissociated with tweezers and form semicircular ring, with burning the semicircle vas deferens of the rapid pinch off of red ophthalmology pincet; Send whole tissue back to intraperitoneal with tweezers, same method pinch off opposite side vas deferens.After all organized renewing original positions, suture muscles, skin are put into mouse cage with mouse behind the sterilization otch, get final product rehabilitation after 2 weeks.Afternoon or evening mate the heat mouse of male mice numbering back with 8 ages in week, test bolt morning next day, and positive mouse is put to death towards ovum, and whether microscopic examination has zygote, if no 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 ages in week; About 2 every mouse peritoneal PMSG Injection that afternoon (10IU), 48h pneumoretroperitoneum injection hCG (10IU), the male ICR mouse with health mates at 1: 1 then; Examine bolt morning next day, positive person 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 about 8 ages in week rutting sedson Healthy female ICR mouse and the successful male ICR mouse of ligation mate at 2: 1, examine bolt morning next day, positive person chooses the acceptor mouse as the same day.

5, reclaim the embryo towards ovum

The female ICR mouse of injection hCG 28h inspection bolt male takes off the cervical vertebra method and puts to death mouse, lies on the back in the operation plate; 75% alcohol disinfecting skin is cut an osculum with eye scissors with skin in the belly mid-way, and both hands pinch skin and gently draw along the mouse cephlad-caudal; Tear skin, open the abdominal cavity, can see uterine tube and ovary that fat pad is other with disinfectant scissors tweezers; Free uterine tube; It is cut put into the M2 that fills 37 ℃ of preheatings and cultivate and drip, under dissecting microscope, tear ampulla of uterine tube, can see that cumulus cell group spills (or to draw M2 insertion fimbriae tubae portions and wash whole uterine tube with joining No. 4 blunt nosed syringe needle 1mL syringes; Liquid is flowed out by another opening of pipe); Can obtain zygote after the Unidasa that adds 500IU/mL is handled about 0.5min, will discharge second polar body with glass pipette immediately and have that two protokaryons, even, the no abnormal spilting of an egg of kytoplasm, cell are full, zona pellucida clearly high-quality be fertilized in another M2 petridish of embryo's immigration, wash 2-3 time with M2; The M16 that changes MO covering and 37 ℃ of preheatings over to puts into 37 ℃ 5%CO in cultivating and dripping ₂Incubator is cultivated.

6, microinjection

On slide glass, do the M2 drop of an about 2.5mm of diameter, drop covers with Yellow Protopet 2A, does the DNA drop of the about 2ng/ μ of a concentration L again and covers with vaporization prevention with Yellow Protopet 2A; The unicellular pronuclear-stage embryos of the high-quality of select is changed in the M2 drop by M16, and slide glass moves on on the microscopical Stage microscope, will hold ovum pin and entry needle and be fixed on the motion arm; Adjust angle direction, inhale and blow liquid affirmation entry needle opening, and with glass needle marshalling zygote; Begin microinjection then, hold an ovum with fixed tube, pressure is not too big; With its position of syringe adjustment, make the protokaryon of zygote high-visible, and make that needle point and protokaryon are clear to be positioned at same focal plane syringe is thrust protokaryon; The microinjection dna fragmentation that obtains of injection embodiment 1 is when seeing the protokaryon quick withdraw of the needle in back that obviously swells.Push zygote to below with holding the ovum pin, hold a new zygote once more and carry out same injection operation, so repeat to finish up to whole zygote injections.Change zygote over to M16 that MO covers 37 ℃ of preheatings after the injection and cultivate and drip, in 37 ℃ 5% CO2gas incubator, cultivate about 60min.Recover to cultivate the back inversion and examine under a microscope embryo and record.Be judged as the survival embryo to have complete ooecium plasma membrane, normal ovum week crack, can be used for embryo transfer; The kytoplasm diffusion, what all cracks of ovum disappeared is dead embryo then, 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, and mouse is lain on the back in the operation plate; Cut off center line both sides, the two hind leg back side, back mouse hair, 75% alcohol disinfecting is on the last rib level of dorsomeson; Vertically cut 1cm left and right sides osculum, mouse is moved on to find fatty body to press from both sides out with blunt nosed tweezers under the stereoscopic microscope, ovary, uterine tube etc. are organized also and are drawn out thereupon; Clamp the fatty body of the ovary of ining succession with Small clamp, fixing.Suck air filled cavity and substratum with the interval 3mm of capillary glass grafts elder generation, suck 12 left and right sides embryos, grafts is placed on safe place at front end.Tear cyst membrane and find uterine tube hydraucone and ampulla, insert grafts by hydraucone, be blown into the embryo, the air filled cavity that examines up to the inside gets into the portion of expanding.Extract grafts out, then tissues such as ovary are reset into intraperitoneal, suture muscles and skin with 75% alcohol disinfecting operation mouthful, are incubated observation and treat it and revive under 37 ℃ of environment.

Use the microinjection dna fragmentation that obtains by embodiment 1 to carry out microinjection and go in the mouse male pronucleus, injected 320 pieces of zygotes altogether, wherein survive 205 pieces, be transplanted to 10 acceptor mouse, 5 pregnancies, 29 F0 that have been born altogether are for transgenic mice.

Three, the evaluation of transgenic mice and going down to posterity

1, the evaluation of transgenic mice

The pEASY-T1 carrier is from full formula King Company.

Extracting the genomic dna of birth 2 all F0 for the about 0.5cm of tail point of transgenic mice, carry out pcr amplification with two pairs of PCR screenings with Auele Specific Primer respectively, is contrast with the wild-type mice.Primer sequence is as shown in table 2 below:

Table 2Seq2, Seq3 and contrast primer sequence

Table 3PCR amplification reaction system

The PCR reaction conditions is: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, and 72 ℃ are extended 15min, 35 circulations, 4 ℃ of preservations.

The result is as shown in Figure 2, for F0 for transgenic mice PCR qualification result, Fig. 2 A is for using the result of seq2 primer amplification; Fig. 2 B is for using the result of seq3 primer amplification; Fig. 2 C is for using 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 (the 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, the Seq2 primer is to obtaining 437bp size amplified band; And the Seq3 primer to the positive F0 that obtains 409bp size amplified band for transgenic mice; Showing has 3 positive F0 of mouse for transgenic mice, and positive rate is 10.3% (3/29), and numbering is respectively F32; F35, F38.

Reclaim positive F0 for the Seq2 primer of mouse to the 437bp size pcr amplification product that amplification obtains, be connected with the 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 the PCR product and has in the sequence table sequence 1 from 5 ' terminal 75-511 position Nucleotide; It is the lysostaphin gene; Further prove numbering and be respectively F32, F35, the positive F0 of F38 is for mouse.

Explain that the numbering that obtains 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 leaves breast, the male and female sub-cage rearing for transgenic mice (former generation is F0 generation) about 3 weeks; Wait to grow to about 8 weeks; Mate to go down to posterity to protect with normal ICR mouse respectively and plant, obtain F1, be used for subsequent experimental for transgenic mice (is that F1 is for transgenic mice like 32#).Again F1 is mated to go down to posterity to protect for transgenic mice and normal ICR mouse and plant, obtain F2 for mouse.Wherein F38 4 weeks during ages because unknown cause is dead.F32 and F35 are in good condition, treat that it grew to for 6 ages in week after, mate with the male mouse of wild-type ICR.

The result sees table 4 and table 5, is F2 generation with the male F1 mouse of 35-9# (the positive F0 of numbering F35 is for the first filial generation of transgenic mice) and the mouse of wild-type ICR female mice mating gained, obtains F2 altogether for 32 of mouse.Wherein, female 12, male 20.The F2 that the founder35# mouse is obtained obtains F3 for 35 of mouse altogether for mouse and wild-type mice mating.Up to the present, two transgenic lines (32# and 35#) that obtained can be passaged to F4 generation.The heredity stably of two transgenic lines is described.

The F1 of founder32# mouse and founder35# mouse is following for the mouse detected result:

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 the transgenic mice

1, destination gene expression in the milk

It is that F1 uses breast appearance damping fluid (breast appearance damping fluid: 125mM/L NaCl, 25mM/L Tris pH7.4,5mM/L KCl, 2mM/L PMSF for the milk of transgenic mice and wild-type mice that PCR is detected 32#.) 1: 4 the dilution after, at 4 ℃ of centrifugal 15min of refrigerated centrifuge 12000rpm, remove the fat on upper strata gently, it is subsequent use in another centrifuge tube-20 ℃ preservation to get the middle layer whey liquid, obtains whey liquid.

Whey liquid is carried out the SDS-PAGE electrophoresis, and the result is as shown in Figure 3, and 1: wild-type mice milk, applied sample amount are 2.5 μ L; 2:32# be F1 for mouse milk, applied sample amount is 2.5 μ L; 3: wild-type mice milk, applied sample amount are 5 μ L; 4:32# be F1 for mouse milk, applied sample amount is 5 μ L; 5: wild-type mice milk, applied sample amount are 10 μ L; 6:32# be F1 for mouse milk, applied sample amount is 10 μ L; 7: wild-type mice milk, applied sample amount are 15 μ L; 8:32# be F1 for mouse milk, applied sample amount is 15 μ L; M: albumen marker; Can find out; With respect to wild-type mice; A tangible protein band in transgenic mice milk, occurred, size is about 20KD (shown in the black arrow), with transgene protein lysostaphin that inserts and endolysin size consistent (size also is about 20KD).

2, goal gene expression in mouse tissue

To be F1 be put in respectively in the homogenizers of different numberings for the liver of mouse, each 200 μ g of mammary gland clip male 32#, extracts RNA with Triozl, and reverse transcription obtains cDNA; With seq3 primer PCR amplification, to as negative control, the result is as shown in Figure 4 with the Gapdh primer; RT-PCR detects transgenic transcription product, 1: wild-type mice mammary tissue, 2: the wild-type mice spleen tissue; 3:32# is that F1 organizes for mouse spleen, 4:32# be F1 for the mouse mammary tissue, 5:32# is that F1 is for the mouse mammary tissue; As can be seen from the figure, be the fragment that F1 has 409bp in for the mouse mammary tissue at 32#, be in the mouse at 32#; Transgenic is expressed in mammary tissue specifically, 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 in Institute of Microorganism, Academia Sinica respectively; 1 bacterium numbering is 1.2386; 2 bacterium numbering is 1.2419) be inoculated in the 2ml substratum, 220 rev/mins, 37 ℃ of shaking table shaking culture are to logarithmic phase.

2) prepare to contain agar concentration and be respectively 1.5% and 0.7% substratum, autoclaving.

3) in the 100mm Micro-Organism Culture Dish, pour the 1.5% nutrient agar 10ml that is cooled to about 55 ℃ into, room temperature (25 ℃) cooling back is subsequent use.

4) the logarithmic phase bacterium is added in the substratum that contains 0.7% agar that is cooled to 55 ℃ in 1: 200 ratio; After mixing; Pour containing in the 1.5% nutrient agar ware of solidifying into, obtain two petridish of A and B, wherein; A is streptococcus aureus CGMCC1.2386, and B is streptococcus aureus CGMCC1.2419; Pour 7ml liquid into according to each 100mm petridish, naturally cooling after 30 minutes in Bechtop, setting-out on each petridish.

5) to be F1 drop on the substratum that petridish solidifies for the milk 15 μ L of mouse the 32# of extracting degreasing.

6) add liquid nutrient medium with volume as negative control, concentration is that the lysostaphin (lysostaphin) of 100 μ g/mL is as positive control.

7) petridish that drips milk is placed on to leave standstill in the super clean bench and observes after changing 37 ℃ of incubator incubated overnight over to after 30 minutes, takes pictures.

The result is as shown in Figure 5, and Spot on lawn analyzes mouse milk,, the streptococcus aureus among the A is CGMCC1.2386, the streptococcus aureus among the B is CGMCC1.2419; 1 is wild-type mice milk among A and the B figure; 2 and 3 to be 32# be that F1 is for 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 F1,32# produces tangible inhibition zone for mouse; Albumen bacterinertness in the transgenic mice milk is described apparently higher than wild-type mice, explaining in the transgenic mice milk specific expressedly to have lysostaphin (Lysostaphin), and it has antibacterial.

Above description of test, the present invention has obtained to have the transgenic mice of anti-Mammitis of cattle gene, thereby can check antibiotic protein gene and the transgenic regulation element expression regulation situation in mammary gland rapidly, for the somatocyte transgenic cattle provides basis and foundation; Can system detect the various unit price antibacterial proteins of transgenic mice expression and the disease-resistant efficient that the multivalence antimicrobial protein resists tiring of artificial inoculation mastitis pathogenic bacterium and whole animal simultaneously.

Claims (10)

1 A and anti-mastitis compositions related proteins, including hormone and peptidoglycan lysostaphin endolysin; said endolysin peptidoglycan from Streptococcus agalactiae phage B30, the amino acid sequence of Sequence Table the sequence 2; wherein the amino acid sequence of lysostaphin prime the sequence in the Sequence 3.

2. the gene of coding claim 1 described protein composition.

3. gene according to claim 2 is characterized in that: said gene comprises the encoding gene of said lysostaphin and the encoding gene of said peptide glycan endolysin;

Said gene specifically is made up of with the internal ribosome entry site that is connected the two said lysostaphin encoding gene, said peptide glycan endolysin encoding gene.

4. according to claim 2 or 3 described genes, it is characterized in that: said gene is connected to form by the encoding gene of the encoding gene of said lysostaphin, said internal ribosome entry site and said peptide glycan endolysin successively; The nucleotides sequence of said gene is classified the sequence 1 in the sequence table as.

5. the recombinant vectors, reorganization bacterium, transgenic cell or the expression cassette that contain arbitrary said gene among the claim 2-4.

6. recombinant vector according to claim 5 is characterized in that: said recombinant vector obtains expressing the recombinant vector of lysostaphin and peptide glycan endolysin for arbitrary said gene among the claim 2-4 is inserted in the pBC1+adapter carrier.

7. arbitrary said gene, the described recombinant vectors of claim 5, reorganization bacterium, transgenic cell or the application of expression cassette in making up the animal model relevant among the described protein composition of claim 1, the claim 2-4 with anti-mastitis;

Or in the described protein composition of claim 1, claim 2-4 arbitrary said gene, the described recombinant vectors of claim 5, reorganization bacterium, transgenic cell or expression cassette in the application of the anti-mastitis product of screening animal;

Said animal is specially mouse or ox.

8. the method for the animal model that a structure is relevant with anti-mastitis for arbitrary said gene among the claim 2-4 is imported in the animal, obtains transgenic animal, promptly obtains the animal model relevant with anti-mastitis.

9. method according to claim 8 is characterized in that:

The method that said gene imports animal comprises the steps:

1) claim 5 or 6 described recombinant vectorss are cut with AatII and NarI enzyme, reclaim the fragment of 18.5kb;

2) fragment of the 18.5kb that step 1) is obtained imports in the animal, obtains transgenic animal, promptly obtains the animal model relevant with anti-mastitis;

Said animal is specially mouse or ox.

10. protein B, its aminoacid sequence is the sequence 3 in the sequence table.

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