CN102533821B - Recombinant lysostaphin gene and expression vector and recombinant cell constructed by recombinant lysostaphin gene - Google Patents

Abstract

The invention discloses a recombinant lysostaphin gene and an expression vector and a recombinant cell constructed by the recombinant lysostaphin gene. The nucleotide sequence of the recombinant lysostaphin gene is shown as SEQ.ID.NO.1, a mammary gland-specific expression vector pBELS containing the recombinant lysostaphin gene and a green fluorescent protein (EGFP) gene is constructed, the mammary gland-specific expression vector pBELS is transfected to the ear fibroblast of a high producing dairy cow by using Fugen HD, the expression condition of labeled genes EGFP is observed by using a fluorescent microscope, the positive transgenic cells are obtained through G418 screening, the PCR (Polymerase Chain Reaction) and the Southern bloting identification are carried out, so that the condition that the recombinant lysostaphin gene is integrated into the genome of the ear fibroblast of the cow is confirmed. A positive transgenic cell in good growing state is taken as a donor cell, and a transgenic embryo is constructed and is implanted into a synchronized receptor cow uterus through embryo transplantation, so that cows with transferred lysostaphin genes are obtained.

Description

A kind of expression vector and reconstitution cell of recombinate dissolving staphylococcal bacteria plain gene and structure thereof

Technical field

The invention belongs to the transgene clone milk cow technical field of Effect of Anti mastitis, relate to a kind of expression vector and reconstitution cell of recombinate dissolving staphylococcal bacteria plain gene and structure thereof.

Background technology

Mammitis of cow (mastitis) has another name called mastadenitis of cow, mainly caused by cause pathogeny imcrobe infection, it is one of principal disease that endangers world's milk cattle cultivating industry, caused huge loss for the milk cattle cultivating industry, there is the annual loss that causes because of mammitis of cow of the document announcement U.S. to reach 2,000,000 dollars of (Sordillo LM, Streicher KL.Mammary gland immunity and mastitis susceptibility.JMammary Gland Biol 2002; 7:135-146).Mammitis of cow is divided into clinic mastitis and Subclinical mastitis.The microbe species that causes mammitis of cow is a lot, and streptococcus aureus is one of topmost pathogenic bacteria of mammitis of cow, and this bacterium can cause acute, chronic and subclinical property (latent mammitis) mastitis, produces for the milk industry and brings massive losses.

for many years, the treatment mammitis of cow is all the method that adopts the spectrum antibiotic therapy, although effectively, but curative ratio less than 50% (Maidhof H, Reinicke B, Blumel P, Berger-Bachi B, Labischinski H.femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistantand methicillin-susceptible Staphylococcus aureus strains.J Bacteriol1991, 173:3507-3513).Moreover the application of Broad spectrum antibiotics has caused the generation of serious Resistant strain, has brought larger difficulty (D.Ferber.Antibiotic resistance-Livestock feed ban preserves drugs ' power.Science 2002 to treatment; 295:27-28).Studies show that: use the special antibiotic of pathogenic bacteria to be expected to reduce the probability that produces resistant strain.Therefore, our target is by the special antibiotic of the mammary gland secretion pathogenic bacteria of transgenic dairy, thus opposing mammitis of cow (D.E.Kerr and O.Wellnitz Mammary expression of new genes to combat mastitis J Anim Sci 2003.81:38-47).

The production of transgenic animal can trace back to for 20 century 70 middle periods, produce transgenic animal (the BOWEN RA that continued more than 20 year with pronuclear microinjection, REED ML, SCHNIEKE A, et al.Transgenic cattle resulting from biopsied embryos:expression of c-ski in a transgenic calf[J] .Biol Reprod, 1994,50:664-8), but the random integration of foreign gene and gamete system transmits the widespread use that the uncertainty of foreign gene has limited this technology.on mouse, embryonic stem cell can replace procaryotic injection (BRANDON EP, IDZERDA RL, MCKNIGHT GS.Targeting the mouse genome:a compendium of knockouts (Part II) [J] .Curr Biol, 1995, 5:758-65), but embryonic stem cell line is not also set up (SHIM H on domestic animal, GUTIERREZ-ADAN A, CHEN LR, et al.Isolation of pluripotent stem cells from cultured porcine primordial germ cells[J] .Biol Reprod, 1997, 57:1089).shown powerful vitality (GOO J and use somatic cell clone technique production transgenic animal, BHUIYANM.M.U., HYUN Y J, et al.An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene[J] .Theriogenology, 2006, 65 (9): 1800-1812.), its outstanding advantage is that the transgenosis step is advanceed to the somatic cell culture stage, directly utilize the fixed somatocyte that is integrated with goal gene to produce transgenic animal for nuclear donor carries out body-cell neucleus transplanting (SCNT).The natality that this technology not only can improve transgenic animal can also reduce its production cost (WHEELER MB, WALTERS EM.Transgenic technology and applications in swine[J] .Theriogeno logy, 2001,56:1345-69).

lysostaphin (Lysostaphin) is by a kind of metalloprotease that imitates staphylococcus (staphylococcus simulans) secretion, catalytic activity with hydrolysis aureus cell wall peptidoglycan pentapeptide bridging, golden Portugal bacterium there is powerful bacteriolysis (Recsei P A, GrussA D, Novick R P.Cloning, sequence, and expression of the lysostaphin gene from S taphylococcus simulans[J] .Proc Natl Acad Sci USA, 1987,84 (5): 1127-1131).a kind of amyloid protein precursor of natural lysostaphin genes encoding, the first expressing protein precursor of the bacterium that produces, be secreted into the outer lysostaphin that becomes maturation by the thiol proteinase effect cuts N a plurality of 13aa tumor-necrosis factor glycoproteinss of end of born of the same parents, mature lysostaphin is than approximately high 4.5 times of (the Thumm G of activity of amyloid protein precursor, Gotz F.Studies on prolysostaphin p rocessing and characterization of the Lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus[J] .MolMicrobiol, 1997, 23 (6): 1251-1265).the external relevant zooscopy that successfully turns the lysostaphin gene has: express the Lysostaphin staphylococcus by transgenic mouse milk and infect, result shows that the economical load by the method enhancing lactation animal health reduction mastitis of genetic engineering is a kind of effective strategy (David E.Kerrl, Karen Plaut, A.John Bramley, etal Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.Nat Biotechnol 2001, 19:66-70).Carried the transgenic cattle birth of Lysostaphin gene in 2005, improve ability (the Wall RJ of the mastitis of milk cow staphylococcus infection, Powell AM, Paape MJ, etal.Genetically enhanced cows resist intramammary Staphylococcus aureus infection.Nat Biotechnol 2005; 23:445-451.).Domestic research to lysostaphin only sees as the antibiotic therapy trauma infection contamination, and the treatment of mammitis of cow and endometritis has obtained good effect.But, with the BLG regulating and controlling sequence instruct the mammary gland expression vector of lysostaphin and transfecting animal cells particularly the somatocyte transgenic cell line of milk cow have no report; Also not with the report of lysostaphin as the goal gene transgenic dairy.

Summary of the invention

The problem that the present invention solves is to provide a kind of cell strain of recombinate dissolving staphylococcal bacteria plain gene mammary gland expression vector and transfection, and the transgenic dairy of producing the anti-Staphylococcus aureus mazoitis for body-cell neucleus transplanting provides donorcells.

The present invention is achieved through the following technical solutions:

A kind of restructuring dissolving staphylococcal bacteria plain gene, its nucleotide sequence is as shown in SEQ.ID.NO.1.

Described restructuring dissolving staphylococcal bacteria plain gene also also connects the BLG5 controlling element as shown in SEQ.ID.NO.2 in the upstream of restructuring dissolving staphylococcal bacteria plain gene, connect the BLG3 controlling element as shown in SEQ.ID.NO.3 in its downstream.

A kind of mammary gland specific expression vector of the lysostaphin of recombinating, comprise the restructuring dissolving staphylococcal bacteria plain gene shown in SEQ.ID.NO.1, at the BLG5 controlling element of its 5 ' end connection as shown in SEQ.ID.NO.2, at the BLG3 controlling element of its 3 ' end connection as SEQ.ID.NO.3, also be connected with the insulator sequence as shown in SEQ.ID.NO.4 in the upstream of BLG5 ' controlling element, be provided with CMV promotor and green fluorescence marker gene in the insulator upstream.

Also be provided with antibiotic-screening gene neo in the mammary gland specific expression vector of described restructuring lysostaphin ^r

The mammary gland specific expression vector of described restructuring lysostaphin, this expression vector is the pBELS expression vector, be that following element is spliced successively: then promotor-marker gene EGFP-insulator sequence-controlling element BLG5-restructuring dissolving staphylococcal bacteria plain gene-controlling element BLG3 is cloned on the pEGFP-C1 carrier.

The reconstitution cell that the mammary gland specific expression vector of described restructuring lysostaphin builds, take holstein cow ear inoblast as host cell, the mammary gland specific expression vector pBELS of linearizing restructuring lysostaphin is transfected in host cell.

Described linearizing mammary gland specific expression vector pBELS cuts with ApaL I enzyme to carry out linearizing.

Described transfection is transfection host cell after the mammary gland specific expression vector pBELS with linearizing restructuring lysostaphin mixes with FuGen HD transfection reagent.

Described host cell is the 1-3 milk the ears of an ox or cow inoblast in generation.

Described reconstitution cell build as nuclear transplantation the transgene clone embryo nuclear donor cell application and turn the production of restructuring lysostaphin genic cow.

Compared with prior art, the present invention has following useful technique effect:

1, because the glycosylation that notes abnormalities in the test with natural lysostaphin gene transfection mammalian cell makes this albumen lose activity.For recovering its activity, restructuring dissolving staphylococcal bacteria plain gene provided by the invention is to have carried out the fixed point glycosylation to suddenly change to guarantee to produce in the mammalian cell at this gene of transfection and have bioactive dissolving staphylococcal bacteria fibroin in natural mature polypeptide coding sequence.Secondly the dissolving staphylococcal bacteria plain gene mature peptide with sudden change is connected to human growth hormone signal peptide back composition restructuring dissolving staphylococcal bacteria plain gene.The dissolving staphylococcal bacteria fibroin of helping suddenly change with the human growth hormone signal peptide passes through mammalian cell membrane and is secreted into outside born of the same parents.Be the activity of checking restructuring dissolving staphylococcal bacteria fibroin, the dissolving staphylococcal bacteria plain gene of recombinating is inserted into and builds carrier for expression of eukaryon pCDNA-lys on pCDNA3.1 (+) carrier.With plasmid pCDNA-lys transfection bovine fibroblasts, obtain to turn the positive cell of restructuring dissolving staphylococcal bacteria plain gene through the G418 screening.With the positive cell cracking, do the dull and stereotyped bacteriostatic test of Bovine Mastitis Caused by Staphylococcus aureus (ATCC25923), result shows that the restructuring lysostaphin has the activity (as shown in Figure 2) of sterilization.

2, the present invention builds restructuring lysostaphin mammary gland specific expression vector pBELS.

The present invention (comprises 5 ' promotor with the control region of the bovine beta-lactoglobulin gene of mammary gland specifically expressing, First Exon partial sequence and 3 ' the 6th exon partial sequence, the 7th exon and polyA sequence) instruct restructuring dissolving staphylococcal bacteria plain gene specific expressed in the mammary tissue of ox, thus built restructuring lysostaphin mammary gland specific expression vector pBELS.With its linearizing and be transfected into bovine ear fibroblast, obtain the transgenic cell line of the expression of witness marking gene EGFP under fluorescence microscopy through the G418 screening.Extract the genome of transgenic cell line, carry out PCR and Southern bloting and identify that confirmation restructuring lysostaphin gene integration is in the genome of bovine ear fibroblast.

3, with the bovine ear fibroblast of integrating restructuring dissolving staphylococcal bacteria plain gene as nuclear donor cell, obtains the transgene clone embryo by SCNT, the milk cow that turns the dissolving staphylococcal bacteria plain gene of recombinating is produced in the uterus that these embryos is moved into synchronized recipient cattle.

4, the transgene clone embryo is moved into 80 recipient cattle uterus, 16 pregnancies are arranged during pregnant inspection in 60 days.5 transgenic dairies have been obtained when growing to birth.

Description of drawings

Fig. 1-1～1-2 is collection of illustrative plates and the restructuring lysostaphin genetic transcription part drawing schematic diagram of mammary gland specific expression vector pBELS;

Fig. 2 is the dull and stereotyped bacteriostatic test result of the active restructuring of eukaryotic expression dissolving staphylococcal bacteria fibroin;

Fig. 3-1～3-2 is the figure as a result that the reporter gene EGFP of the fluorescence microscope positive bovine ear fibroblast that turns the pBELS carrier expresses;

Fig. 4 is from the bovine ear fibroblast genome that turns the pBELS carrier positive, the detected result figure of the agarose gel electrophoresis of pcr amplification restructuring dissolving staphylococcal bacteria plain gene;

Fig. 5 is in the bovine ear fibroblast genome that turns the pBELS carrier positive, and Southernbloting identifies restructuring dissolving staphylococcal bacteria plain gene figure as a result;

Fig. 6-1～6-2 is the fate map that fluorescence microscope comprises pBELS positive cell nuclear transplantation reconstructed embryo;

Fig. 7 is the restructuring dissolving staphylococcal bacteria plain gene agarose gel electrophoresis figure as a result in pcr amplification reconstruct embryo genome;

Fig. 8-1～8-2 is the transgenic cattle ear tissue piece EGFP expression figure of fluorescence microscope birth;

Fig. 9 is that multiplex PCR detects EGFP in 5 transgenic cattle genomes of being born, neo ^rAnd the detected result figure of restructuring dissolving staphylococcal bacteria plain gene agarose gel electrophoresis.

Embodiment

At first the present invention builds the mammary gland specific expression vector pBELS that contains restructuring lysostaphin and green fluorescent protein (EGFP) gene, secondly use Fugen HD with linearizing mammary gland-specific expression vector pBELS transfection milk the ears of an ox or cow inoblast, the expression of fluorescence microscope marker gene EGFP, and obtain positive cell through the G418 screening, carry out PCR and Southern bloting and identify, confirm that goal gene restructuring lysostaphin is incorporated in the genome of bovine ear fibroblast.At last, the recombinate bovine ear fibroblast of dissolving staphylococcal bacteria plain gene of transfection is moved in the ox enucleation oocyte as nuclear donor, obtain the transgenosis reconstituted embryo, the performing PCR of going forward side by side is identified, the transgene clone embryo is moved into 80 recipient cattle uterus, and when obtaining 60 days, pregnant inspection has 16 pregnancies.5 cow heads have been obtained during to birth.Multiplex PCR detects restructuring dissolving staphylococcal bacteria plain gene, EGFP gene and neo ^rThe result of gene shows that 5 oxen of birth are all positive transgenic cattles.

Concrete related reagent is as follows: G418, DMEM substratum and foetal calf serum are all available from U.S. GIBCO (Invitrogen) company, and EDTA and Trypsin are available from U.S. Sigma company, and transfection reagent Fugen HD is the product of Switzerland Roche company.Tissue Culture Plate and culture dish are Denmark Nunclon company product, and plasmid extraction kit and cellular genome are extracted test kit all available from sky root company.TaqDNA polysaccharase and restriction enzyme are available from Takara company.

The present invention is described in further detail below in conjunction with accompanying drawing, and the explanation of the invention is not limited.

1, the structure of mammary gland specific expression vector pBELS

1.1 the clone of goal gene and structure

Dissolving staphylococcal bacteria plain gene mature peptide is done point mutation, and the albumen the 125th of mature peptide, the N point mutation of 232 are Q, and namely protein is that the Asn point mutation is Gln.Be that AAT is mutated into CAG on DNA sequence dna, do respectively point mutation 373～375,694～696 of sequence, with the biological activity of dissolving staphylococcal bacteria fibroin in eukaryotic cell of recovering to express.

The lysostaphin gene order point mutant primer of announcing according to GenBank ID:M15686.With the full gene of synthetic lysostaphin (XAFW021, TaKaRa numbering: CAG593) be template, utilize overlap extension pcr to realize the mature peptide point mutation, primer sequence is as follows:

TB1：5-gctgcaacac atgaacattc agcac-3 25；

TB2：5-gatcttgggc agttgactgt gaaaatgaat taac-3 34；

TB3：gttaattcat tttcacagtc aactgcccaa gatccaatgc c 41；

TB4：tcactttata gttccccaaa gaacacctaa agtattagta gatttctgcc atgttcttac agg 63

TB5：tcactttata gttccccaaa gaacacctaa ag 32；

The PCR reaction system of 50 μ L is:

Buffer (Mg ²⁺Plus): 10 μ L, dNTP Mixture (each 2.5mM): 4 μ L, TB1 (10 μ mol/L): 1 μ L, TB2 (10 μ mol/L): 1 μ L,

HS DNA Polymerase (2.5U/ μ L)): 0.5 μ L, template 1 μ L adds sterilization ultrapure water to 50 μ L;

Reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 64 ℃ of annealing 15s, 72 ℃ are extended 25s, 30 PCR circulations, 72 ℃ are extended 7min again.

At first utilize primer TB1, the front 391bp of TB2 amplification dissolving staphylococcal bacteria plain gene mature peptide sequence, the suddenlyd change codon of 373～375 of sequence of PCR product called after fragment 1, this fragment, the 125th amino acid of the albumen that also just suddenlyd change simultaneously;

Use and then primer TB3, TB4, reaction system and reaction conditions are the same, the rear 384bp of amplification dissolving staphylococcal bacteria plain gene mature peptide sequence.The suddenlyd change codon of 694～696 of sequences of PCR product called after fragment 2, this fragment.The 232nd amino acid of albumen has also just suddenlyd change simultaneously.

Use at last primer TB1 and TB5, take the mixture of segment 1 and fragment 2 equal proportions as template, reaction system is the same again.The PCR reaction conditions is: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 62 ℃ of annealing 15s, 72 ℃ are extended 45s, 30 PCR circulations, 72 ℃ are extended 10min again and finish to add afterwards 0.5 μ L rTaq archaeal dna polymerase, 0.5 μ L TB1 and 0.5 μ L TB5 inside the products of reaction, put in the PCR instrument 72 ℃ of reactions 10 minutes (purpose of this reaction be add at the resulting amplified production of previous step two ends A be convenient to next step and complete the TA clone).After being reclaimed, the PCR product purification that increases is the TA clone, be connected to pMD-18T Vector, transformed competence colibacillus cell E.coli DH5 α, recon is selected in blue hickie screening by α-complementary, cut through BamH I enzyme and identify positive recombinant plasmid and deliver Shanghai biotechnology Services Co., Ltd and check order, result shows that the dissolving staphylococcal bacteria plain gene is consistent with the sequence of announcement, and successfully realize the point mutation of lysostaphin mature peptide in predetermined site, the gained carrier is called recombinant plasmid plys.

Human growth hormone signal peptide length is 78bp, GenBank ID:CS075524.Utilize round pcr the human growth hormone signal peptide to be connected to 5 ' end of the lysostaphin mature peptide of sudden change, obtain restructuring dissolving staphylococcal bacteria plain gene.

This PCR plys plasmid that reacts to recombinate is template, realizes the clone of goal gene (restructuring lysostaphin), and the primer sequence of pcr amplification is as follows:

hGH1：gctctgcctg ccctggctcc aggagggctc cgcggctgca acacatgaac attcagc 57

hGH2：gggtcccgca cctccctcct cctggccttc ggcctgctct gcctgccctg gctccagg 58

hGH3：atggcgaccg ggtcccgcac ctccctcctc ctggc 35

hGH4：tcactttata gttccccaaa gaacacc 27

Take restructuring plys plasmid as template, at first be PCR with hGH1 and hGH4 primer.Reaction system the same (be all to adopt the high-fidelity enzyme of Takara to be PCR, prevent unnecessary sudden change), it is 60 ℃ that reaction conditions just changes annealing temperature, extends time 45s, 30 circulations.With resulting PCR product called after h14;

And then use hGH2 and hGH4 primer, take h14 as template, do the PCR reaction.Reaction system is the same with reaction conditions and previous step, with resulting PCR product called after h24;

Again take h24 as template, do the PCR reaction with hGH3 and hGH4 primer at last, reaction system is constant, and the time of extending in reaction conditions changes 50s into, 30 circulations.Add 0.5 μ L rTaq archaeal dna polymerase inside the product of reaction more at last after extending 10 minutes, 0.5 μ L hGH3 and hGH4, put in the PCR instrument 72 ℃ of reactions 10 minutes (purpose of this reaction be add at the resulting amplified production of previous step two ends A be convenient to next step and complete the TA clone).With resulting PCR product called after h34;

Be exactly to synthesize desired gene by primer like this, the method that PCR puts up a bridge is connected to the human growth hormone signal peptide gene at 5 ' end of restructuring lysostaphin mature peptide gene order.

To be connected on the pMD-18T carrier by doing the TA cloning experimentation through the h34 of DNA test kit purifying.Transformed competence colibacillus cell E.coli DH5 α selects recon by the blue hickie screening of α-complementary, cuts through BamH I enzyme and identifies that positive recombinant plasmid ph34 delivers Shanghai biotechnology Services Co., Ltd and checks order.

Constructed restructuring dissolving staphylococcal bacteria plain gene, its concrete nucleotide sequence is as shown in SEQ.ID.NO.1, wherein 5 ' connect human growth hormone signal peptide sequence (1～78bp), and mature peptide the 125th, 232 bit tables are reached the Asn point mutation for expressing Gln, at 451～453bp, AAT is mutated into CAG, at 772～774bp, AAT is mutated into CAG.

To check order recombinant plasmid ph34 correct as template, use following primer again, the upstream adds Kpn I, and the downstream adds the BamH I purpose restructuring dissolving staphylococcal bacteria plain gene that increases:

LS1：cgg ggtacca tggcgaccgg gtcccgcacc t 31

LS2：cgc ggatcct cactttatag ttccccaaag aacacct 37

With Kpn I and BamH I difference double digestion PCR product and pEGFP-C1, the PCR product that enzyme is cut reclaims the restructuring dissolving staphylococcal bacteria plain gene of 819bp, and the pEGFP-C1 that enzyme is cut reclaims 4.7kb carrier vector fragment, these two fragments are connected with T4DNAase spend the night and transformed competence colibacillus cell E.coliDH5 α, build intermediate carrier and name pLS.

1.2 the activity identification of restructuring lysostaphin in eukaryotic cell.

With Kpn I and BamH I difference double digestion PCR product and pCDNA3.1 (+) plasmid.Reclaim the restructuring lysostaphin gene fragment of 819bp and the carrier segments of 5428bp, these two fragments are connected with T4DNAase spend the night and transformed competence colibacillus cell E.coli DH5 α, build carrier for expression of eukaryon pCDNA-lys.With plasmid pCDNA-lys transfection bovine fibroblasts, obtain to turn the positive cell of restructuring dissolving staphylococcal bacteria plain gene through the G418 screening.About 10 ⁶Positive cell digested and centrifugal, outwell the PBS solution that contains 0.1%TrionX-100 that supernatant adds 25 microlitre precoolings, of short duration ultrasonic degradation positive cell.The centrifuging and taking supernatant is done the expression that western blot identifies the restructuring lysostaphin.The cracking supernatant of 10 microlitres is used for being coated with fresh Bovine Mastitis Caused by Staphylococcus aureus (ATCC25923) agarose plate.37 ℃ of incubated overnight can clearly be seen the sterilization ring on flat board, and specifically as shown in Figure 2, result shows that the restructuring lysostaphin has the activity of sterilization.

1.3 the pcr amplification of regulating and controlling sequence

Utilize the regulating and controlling sequence of cattle beta-casein gene to regulate and control goal gene specific expression in bovine mammary epithelial cell, specifically 5 ' control region at 5 of goal gene ' end connection bovine beta-lactoglobulin comprises the First Exon partial sequence, the regulating and controlling sequence of promotor and upstream thereof, called after BLG5.3 ' the control region that connects bovine beta-lactoglobulin at 3 of goal gene ' end comprises the 6th exon partial sequence, the 7th exon and polyA sequence, called after BLG3.Controlling element shows as the adjusting of goal gene, under rational factor is induced, as the stimulation of prolactin antagonist, foreign gene restructuring lysostaphin is transcribed by the endogenous mode of transcribing of bovine beta-lactoglobulin.

The gene order of the bovine beta-lactoglobulin of announcing according to GenBank ID:x14710, the 5 ' ending regulating sequence of the about 2.8kb before this albumen initiator codon of pcr amplification ATG also adds Bgl II in the upstream, and Sal I restriction enzyme site is added in the downstream.After the terminator codon that increases simultaneously TAG about 1.4kb 3 ' ending regulating sequence and add Xba I in the upstream, Mlu I site is added in the downstream.Primer sequence following (underscore marks restriction enzyme site):

BLG5F：5′-gga agatctcgccttccagtgcaggggatgtg-3′

BLG5R：5′acgc gtcgacggctgcagctggggtcacgctt-3′

BLG3F：5′-tgc tctagagtgagcccctgccggcgcct-3′

BLG3R：5′-cg acgcgtgttcagttcagtcgctcagtcgtgtcc-3′

Take holstein cows blood, extracting test kit extraction genome with a day root poba gene group is template, pcr amplification bovine beta-lactoglobulin gene 5 ' control region gene.Concrete operations are as follows:

50 μ L reaction systems: 10 * LA PCR Buffer II (Mg2+Plus), 5 μ L; DNTP Mixture (each 2.5mM) 8 μ L; Template (0.5 μ g) 2 μ L; Each 2 μ L of primer (10 μ M); TaKaRa LA Taq (5U/ μ L) 0.5 μ L; The ultrapure water 31.5 μ L of sterilization.

Reaction conditions: 94 ℃ 5 minutes; 94 ℃ 30 seconds; 64 ℃ 30 seconds; 72 ℃ 2 minutes 50 seconds; 30 circulations; 72 ℃ 10 minutes; 4 ℃ of preservations.

During the regulating and controlling sequence of pcr amplification 3 ' end, reaction system is constant, gets final product and the extension time of reaction conditions is made as 1 minute and 25 seconds.Be connected on the pMD20-T carrier being the TA clone after resulting PCR product purification, transformed competence colibacillus cell E.coli DH5 α extracts plasmid and send order-checking to obtain correct plasmid, respectively called after pBLG5 and pBLG3.The controlling element BLG5 that increases and the nucleotide sequence of BLG3 are as shown in SEQ.ID.NO.2 and SEQ.ID.NO.3.

1.4 the pcr amplification of 2 * chicken betaglobulin insulator

With business-like expression vector pBC1 as template, pcr amplification 2 * chicken betaglobulin insulator fragment.Use insulator to insert in the carrier that builds the impact that is not subjected near the silencer insertion point genome with protection goal gene and EGFP, make them separately can normal expression.

The upstream and downstream primer adds the BspEI restriction enzyme site, adds TAA terminator codon (underscore) simultaneously after upstream primer BspEI restriction enzyme site, to stop the translation of green fluorescent protein, is unlikely to occur the frameshit phenomenon.Primer sequence is as follows:

BIF：5-agg tccggataagggacagcccccccccaaag-3

BIR：5-agg tccggactcactgactccgttcctggagttgg-3

50 μ L reaction systems: 10 * LA PCR Buffer II (Mg2+Plus), 5 μ L; DNTP Mixture (each 2.5mM) 8 μ L; Template (0.5 μ g) 2 μ L; Each 2 μ L of primer BIF and BIR (10 μ M); TaKaRaLATaq (5U/ μ L) 0.5 μ L; The ultrapure water 31.5 μ L of sterilization.

Reaction conditions: 94 ℃ 5 minutes; 94 ℃ 30 seconds; 64 ℃ 30 seconds; 72 ℃ 2 minutes 25 seconds; 30 circulations; 72 ℃ 10 minutes; 4 ℃ of preservations.

Be the TA clone after the product purification of PCR reclaims and be connected on the pMD20-T carrier vector, transformed competence colibacillus cell E.coli DH5 α extracts plasmid and send order-checking to obtain correct plasmid, respectively called after pBI.

1.5 construction of expression vector

Building final expression vector divided for four steps these fragments were coupled together: the first step is with Xba I and Mlu I double digestion pLS and pBLG3 plasmid simultaneously, reclaims the approximately regulating and controlling sequence of the BLG3 ' end of the linearizing pLS skeleton fragment of 5.5kb and 1.4kb of purifying.These two sections sequences are connected with T4DNAase spend the night and transformed competence colibacillus cell JM110, kantlex screening recon is cut through enzyme and is identified that order-checking obtains correct intermediate carrier plasmid, called after pLSG3.Second step is with restriction enzyme Bgl II and Sal I double digestion pBLG5 and pEGFP-C1 plasmid, reclaims respectively the skeleton fragment of the pEGFP-C1 of the regulating and controlling sequence fragment of BLG5 ' end of purifying 2.8kb and 4.7kb.These two fragments that reclaim are connected with T4DNAase spend the night and transformed competence colibacillus cell E.coli DH5 α, kantlex screening recon is cut through enzyme and is identified that order-checking obtains correct intermediate carrier plasmid, called after pEG5.The 3rd step was to cut pEG5 and pBI plasmid with restriction enzyme BspEI while enzyme, the skeleton fragment of the pEG5 of recovery purifying 7.5kb and 2 * chicken betaglobulin insulator fragment of 2.4kb.These two fragments are connected with T4DNAase spend the night and transformed competence colibacillus cell E.coli DH5 α, kantlex screening recon is cut evaluation through enzyme and is sent order-checking to determine the direction of inserting, and obtains the recon called after pBEG5 that correctly inserts.The 4th step is with restriction enzyme Sal I and Mlu I double digestion pBEG5 and pLSG3 plasmid simultaneously, and the skeleton fragment that reclaims the pBEG5 of purifying 9.9kb is connected fragment (the restructuring lysostaphin connects the regulating and controlling sequence of BLG3 ' end) with 2.2kb.These two fragments that reclaim are connected with T4 DNAase spend the night and transformed competence colibacillus cell E.coli DH5 α, kantlex screening recon is cut through enzyme and is identified that order-checking obtains correct final carrier pBELS.

Referring to Fig. 1-1,1-2, be connected to promotor-marker gene EGFP-insulator sequence-controlling element BLG5-restructuring dissolving staphylococcal bacteria plain gene-controlling element BLG3 based on the gene constructed Expression element of restructuring lysostaphin in constructed expression vector pBELS, concrete enzyme is cut mode of connection as Figure 1-1, in the position in carrier as shown in Fig. 1-2.

The endotoxic plasmid purification test kit that goes with Promega company extracts concentration and the purity of measuring plasmid after plasmid with the nucleic acid-protein determinator, and after measured, the concentration of plasmid is 450ng/ μ l, and OD260/280 is 1.90, illustrates that plasmid is purer, can be used for transfection.

Get 20 μ g pBELS expression vectors, cut 24 hours at 37 ℃ of enzymes with ApaL I (NEB), after cutting fully, adopt acetate ethanol sodium precipitator method purifying linearizing DNA as transfection.

2, pBELS expression vector transfection bovine ear fibroblast and screen nuclear donor cell system

2.1 the cultivation of bovine ear fibroblast

The bovine ear fibroblast (＜3 generation) of getting a pipe holstein cow from liquid nitrogen is thawed in 38 ℃, the centrifugal supernatant of abandoning, add DMEM (Dulbecco ' s Modified Eagle Media) the cell culture fluid re-suspended cell that 1ml contains 10% foetal calf serum, then be inoculated in the culture dish of diameter 60mm, be placed in CO ₂Cultivate under 38.5 ℃ of conditions in incubator.

Reach 90% when converging until bovine ear fibroblast, inhale and abandon nutrient solution, use without Ca ²⁺, Mg ²⁺PBS rinse cell, add pancreatin and EDTA mixture slaking liquid, peptic cell.Observation of cell under inverted microscope until the most cells retraction, when becoming circle, intercellular substance expansion, stops digestion with the DMEM cell culture fluid that contains 10% foetal calf serum, after pipettor piping and druming, centrifugal collection suspends, be inoculated in 60 wares in the ratio of 1: 3, put into CO ₂Cultivate in incubator.

2.2G418 the mensuration of minimum lethal concentration

The present invention as screening of medicaments, has G418 resistant gene neo with G418 on the skeleton due to expression vector pBELS ^r, foreign gene neo ^rExpressed, the bovine ear fibroblast of transfection makes under can survive in the nutrient solution that contains finite concentration G418, and the normal cell of untransfected can be dead under this concentration, and the minimum lethal concentration that therefore needs to determine normal cell G418 is used as screening concentration.

Get the bovine ear fibroblast that grows into 80% degree of converging one 60 wares, be inoculated in 24 orifice plates overnight incubation with trysinization.Add respectively Different concentration of G418, in 1 week of screening, examine under a microscope all death of the cell of institute in porose of G418 concentration 〉=500 μ g/ml.Therefore the minimum lethal concentration of adding of Normocellular G418 that obtains untransfected is 500 μ g/ml.

2.2, pBELS expression vector transfection bovine ear fibroblast

The present invention specifically adopts FuGen HD (Roche) transfection reagent under the condition that has serum to exist, and transfection linearizing pBELS (cutting with ApaL I enzyme) is in bovine ear fibroblast.Carry out transgenosis with FuGen HD transfection reagent and compare with other method, be not subjected to the restriction of DNA size, inorganization specificity and immunogenicity, do not need serum starvation, little to cell injury.Concrete operations are as follows:

Transfection the day before yesterday, with the 2nd generation bovine ear fibroblast with 1 * 10 ⁵Being inoculated in spends the night in 12 orifice plates makes cell reach 70-80% to converge.FuGen HD transfection reagent with 2 μ g (571ng/ μ L) linearizing pBELS expression vector and 8 μ l joins in 88.5 μ l Opti-MEM nutrient solutions respectively, and final volume is 100 μ l.After incubated at room 10min, the transfection composite of 100 μ l is slowly added in a porocyte, after hatching 24h, renew the bright DMEM cell culture fluid that contains 10% serum and continue to cultivate.

2.3, pBELS carrier positive cell screening

Cultivate transfectional cell after 24 hours with the fresh medium that does not contain transfection reagent, the expression of visual report gene EGFP under fluorescent microscope.As Fig. 3-1 (transfection 24 hours), as shown in can see that genetically modified bovine fetal fibroblast sends green fluorescence, illustrate that pBELS has entered cell and positive expression.Trysinization, centrifugal, inoculate the ratio inoculating cell of 90 wares and add 500 μ g/ml G418 screening positive cells in nutrient solution according to a porocyte of 12 orifice plates.With the negative contrast of the bovine fetal fibroblast of untransfected pBELS, its cell culture fluid is added with the G418 of same concentration.Changed liquid once in 3-4 days, and screened the 8th day, the cell of negative control is all dead.The concentration of G418 is reduced to 250 μ g/ml and is continued to cultivate until form island shape monoclonal cells (greatly about the 15th day).The method picking monoclonal cell of trysinization is to 96 porocyte culture plates, with the cell culture fluid enlarged culturing monoclonal cell that does not add G418.As shown in Fig. 3-2, after the positive cell of enlarged culturing, can see that under fluorescent microscope the transgenosis the ears of an ox or cow inoblast of enlarged culturing sends green fluorescence.

The positive cell of the present invention's screening is all the cell of stable transfection pBELS.Linearizing pBELS is incorporated on the genome of cell rather than is free on outside genome, and the position of integration is random.Screen 7 days lasting purposes that reach stable transfection of screening of half-value dose again by G418, show as reporter gene continuous expression in transfected positive cell, therefore the positive cell of screening continues to send green fluorescence and presents the G418 resistance.

2.4, the PCR of pBELS carrier positive cell identifies

Get the positive transgenosis the ears of an ox or cow inoblast of passing to for the 8th generation, extract cell genomic dna, take genomic dna as template, PCR identifies whether restructuring dissolving staphylococcal bacteria plain gene is incorporated in cellular genome, and negative control is the bovine ear fibroblast of untransfected.PCR identifies that the primer sequence is as follows:

P1：atggcgaccg ggtcccgcac ct

P2：tcactttata gttccccaaa gaacacct

The PCR reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 65 ℃ of annealing 30s, 72 ℃ are extended 50s, 30 PCR circulations, 72 ℃ are extended 7min again;

Reclaim the PCR product and carry out 0.8% agarose gel electrophoresis detection, detected result as shown in Figure 4, wherein, swimming lane M is Trans15K DNA Marker, swimming lane 1 is the bovine ear fibroblast of untransfected, the positive genetically modified bovine ear fibroblast of swimming lane 2, and swimming lane 1 is compared with 2, obviously can see the band about a treaty 800bp, and goal gene is 820bp; Swimming lane 1 as negative control is not seen, restructuring lysostaphin gene integration is described in the genome of positive genetically modified bovine ear fibroblast, and the regulating and controlling sequence that is attached thereto also can be incorporated into into together.

2.5, the Southern blotting of positive genetically modified the ears of an ox or cow cell detects

Detect the whether fully integrated genome that advances bovine ear fibroblast of restructuring dissolving staphylococcal bacteria plain gene according to the requirement of the Roche DIG High Prime DNA Labeling and Detection Starter KitII of company (Cat.11585614910).Probe sequence T1 and T2 used are as follows:

T1：tggtcaaata atcggttggt ctg

T2：ccaaacatga ccgtcttgtt tca

Extract the positive cell genomic dna, cut with BamH I enzyme, compare with the pBELS plasmid simultaneously.All experimental implementation are completed according to the experimental program that Roche company provides, detected result is as shown in Figure 5: the 1st road is the band of pBELS positive cell genomic dna hybridization, the 2nd road is the band of the ox fetal cell genomic dna hybridization of untransfected, and the 3rd road is the band of hybridizing after plasmid enzyme restriction.The genomic swimming lane of positive cell can hybridize to band explanation restructuring dissolving staphylococcal bacteria plain gene be entirely integrated into cellular genome in and occur to reset or sudden change, provide foundation for the dissolving staphylococcal bacteria plain gene carries out follow-up qualitative and quantitative analysis.

3, with the genetically modified bovine ear fibroblast of well-grown positive as nuclear donor cell, build the transgene clone embryo.

3.1, the maturation in vitro of ovocyte cultivates

Ovary picks up from ox slaughterhouse, Xi'an, the aseptic holstein cow ovary of taking, transport the laboratory back in 2-3 hour, extract the ovarian follicle of 3～8mm diameter, collect ovocyte ovarian cumulus complex body, select under stereomicroscope and have complete cumulus cell more than three layers, the uniform ovocyte of kytoplasm is used for ripe the cultivation.The maturation culture solution of ovocyte is: TCM199 (Gibaco) adds 10% foetal calf serum, the Urogastron of 10ng/ml, and culture condition is: 38.5 ℃, 5%CO ₂, 95% air atmosphere surrounding, saturated humidity; Remove cumulus cell with 0.2% Unidasa after the ripe 20h of cultivation, with the criterion of first polar body discharging as oocyte maturation, select mature oocyte and be used for the nuclear transplantation test.

3.2 the structure of transgene clone embryo

The present invention adopts body-cell neucleus transplanting (SCNT) technology that donor nuclei is transferred to and removes in nuclear mature oocyte, wherein, the donorcells that is integrated with foreign gene controlled in 10 generations, and this mainly considers to reduce the accumulation of sudden change in culturing process that vitro culture causes.

Nuclear transplantation is specially: micrurgy liquid is for containing 10%FBS, the PBS of 5 μ g/ml cytochalasin Bs, stoning pipe sucking-off first polar body and part kytoplasm on the micrurgy instrument with internal diameter 20 μ m, with 10 μ g/ml Hoechst 33342 dyeing 10min, choose complete non-nucleus egg mother cell under fluorescent microscope; Remove first polar body fully and chromosomal ovocyte is used to nuclear transplantation.

Notes core and the fusion of ovocyte, detailed process is as follows: the transfection restructuring lysostaphin bovine ear fibroblast in 6～10 generations of 0.25% tryptic digestion contact inhibition 3d is as donor cell.With the stoning pipe, donorcells is injected under the ovocyte zona pellucida of stoning success.The caryoplasm complex body merges balance 3min in liquid at electricity, merge with the microelectrode method, use the microelectrode tip arrangement recombinant chou that is connected with the micrurgy instrument, make the film contact surface vertical with the line of two electrodes, push down gently recombinant chou with the microelectrode point, give electricimpulse and carry out the electricity fusion.Fusion voltage is 28V, and time of fusion is 10 μ m.Recombinant chou after fusion is put into the M199 of 10%FBS, 38.5 ℃, 5%CO ₂, full closing under humidity cultivate, and observes the fusion situation after 2h.

3.3 the activation of transgene clone embryo and vitro culture

The reconstituted embryo (cell-ooecium matter recombinant chou) that merges is in containing the M199 of 10%FBS after balance 2h, with containing 5 μ mol/L ionomycin (Ionomycin, available from SIGMA company) mSOFaa nutrient solution (available from SIGMA company) process 5min, then cultivate 5h in the mSOFaa nutrient solution that contains 2mmol/L dimethylaminopurine (6-DMAP), transfer to after washing 3 times in mSOF aa droplet and cultivate, culture density is 5 each reconstituted embryo of μ L, at 38.5 ℃, 5%CO ₂, cultivate under saturated humidity, every 48h half amount is changed liquid 1 time, checks the blastaea developmental state on the 7th～9 day, shown in Fig. 6-1 (transgenic embryos, light field), Fig. 6-2 (transgenic embryos is observed EGFP), the normal development of clone's embryo is to blastaea.

3.4 transgene clone embryo pcr amplification restructuring lysostaphin

The extraction of restructuring embryo genomic dna: the embryo's sample of recombinate is put into the centrifuge tube of 100 μ l sterilizations, adds the 20 μ l distilled waters of sterilizing, and boils after 5min slightly centrifugally, is placed in-20 ℃ and preserves or be directly used in PCR.

Take the genome of the recombinant clone embryo that extracts as template, PCR identifies restructuring dissolving staphylococcal bacteria plain gene, and the PCR reaction: reaction system and process are with step 2.4.With the negative contrast of not genetically modified clone's embryo.Pcr amplification product is through 0.8% agarose gel electrophoresis, obtained the amplified band of size for the 800bp left and right, conform to the expection size, as shown in Figure 7, wherein, swimming lane M is 100bp DNA Ladder, swimming lane 1 is not genetically modified clone's embryo, swimming lane 2 is the recombinant clone embryo, and swimming lane 2 is compared with 1, obviously can see the band about a 820bp.Illustrate that restructuring lysostaphin gene integration is in the embryonic gene group.

3.4 the embryo transfer of transgene clone embryo and gestation are identified

The blastaea of the 7th day moves into after spontaneous estrus the He Sitan recipient cattle intrauterine of the 7th day with Nonoperative method, each recipient cattle is transplanted 2 pieces of blastaeas.Observe it after recipient cattle is transplanted and return the feelings situation, the 60d after transplanting that does not return feelings is done conceived the inspection with B ultrasonic.Check once every January later on, to observe the pregnancy maintenance situation.

Co-transplantation of the present invention turns restructuring lysostaphin gene clone embryo to 80 a recipient cattle intrauterine, and 16 gestation are arranged during pregnant inspection in 60 days.5 transgenic dairies have been obtained when growing to birth.

4. the evaluation of transgenic dairy

Take out the ear-edge tissue of the transgenic dairy of giving birth to, cut the square size of big or small 1mm with eye scissors, tissue block is attached in 60 wares, contain two anti-cell culture fluids until fully adherent rear carefully adding.At 38.5 ℃, 5%CO ₂, full closing under humidity cultivate, about one week the left and right the time inoblast of can moving out around tissue block.Can know and see that tissue block sends green fluorescence as examining under a microscope shown in Fig. 8-1 (EGFP observation), 8-2 (light field).The milk cow that birth is described is complete genetically modified milk cow.Get the blood of transgenic dairy, extract the poba gene group, the foreign gene that changes over to the amplification of the multiple PCR method lysostaphin (765bp) of recombinating, EGFP (458bp) and neo ^r(599bp), primer sequence is as follows:

EGFP1：5-gccgctaccccgaccacatg-3

EGFP2：5-cacgaactccagcaggaccatg-3

NEO1：5-tgctctgatgccgccgtgtt-3

NEO2：5-tcagcaatatcacgggtagccaa-3

Lys1：5-ctgccctggctccaggagggctc-3

Lys2：5-ctttatagttccccaaagaacacctaaagt-3

The PCR reaction system;

Buffer (Mg ²⁺Plus): 10 μ L; DNTP Mixture (each 2.5mM): 4 μ L; Each 0.5 μ L of EGFP1 and EGFP2 (10 μ mol/L); Each 0.7 μ L of NEO1 and NEO2 (10 μ mol/L); Each 1 μ L of Lys1 and Lys2 (10 μ mol/L); HS DNA Polymerase (2.5U/ μ L)): 0.5 μ L; Template 1 μ L adds sterilization ultrapure water to 50 μ L;

Reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 64 ℃ of annealing 15s, 72 ℃ are extended 45s, 30 PCR circulations, 72 ℃ are extended 7min again.

The product of PCR is through 0.8% agarose gel electrophoresis result as shown in Figure 9: can both increase out simultaneously these three genes in the genome of five oxen and not expand out foreign gene in negative control, showing that the ox that gives birth to is all transgenic cattle.

Claims (9)

1. a restructuring dissolving staphylococcal bacteria plain gene, is characterized in that, its nucleotide sequence is as shown in SEQ.ID.NO.1.

2. the mammary gland specific expression vector of the lysostaphin of recombinating, it is characterized in that, comprise the restructuring dissolving staphylococcal bacteria plain gene shown in SEQ.ID.NO.1, the BLG5 controlling element of 5' end connection as shown in SEQ.ID.NO.2 at restructuring dissolving staphylococcal bacteria plain gene, 3' at restructuring dissolving staphylococcal bacteria plain gene holds the BLG3 controlling element that connects as SEQ.ID.NO.3, also be connected with the insulator sequence as shown in SEQ.ID.NO.4 in the upstream of BLG5 controlling element, be provided with CMV promotor and green fluorescence marker gene in the insulator upstream.

3. the mammary gland specific expression vector of restructuring lysostaphin as claimed in claim 2, is characterized in that, also is provided with antibiotic-screening gene neo in the mammary gland specific expression vector of described restructuring lysostaphin ^r

4. the mammary gland specific expression vector of restructuring lysostaphin as claimed in claim 2, it is characterized in that, this expression vector is the pBELS expression vector, be that following element is spliced successively: then insulator sequence-controlling element BLG5-restructuring dissolving staphylococcal bacteria plain gene-controlling element BLG3 is cloned on the pEGFP-C1 carrier.

5. the reconstitution cell that builds based on the mammary gland specific expression vector of restructuring lysostaphin claimed in claim 4, it is characterized in that, take holstein cow ear inoblast as host cell, the mammary gland specific expression vector pBELS of linearizing restructuring lysostaphin is transfected in host cell.

6. reconstitution cell as claimed in claim 5, is characterized in that, the mammary gland specific expression vector pBELS of described linearizing restructuring lysostaphin cuts with ApaL I enzyme to carry out linearizing.

7. reconstitution cell as claimed in claim 5, is characterized in that, described transfection is transfection host cell after the mammary gland specific expression vector pBELS with linearizing restructuring lysostaphin mixes with FuGene HD transfection reagent.

8. reconstitution cell as claimed in claim 5, is characterized in that, described host cell is the 1-3 milk the ears of an ox or cow inoblast in generation.

9. reconstitution cell claimed in claim 5 builds the application of the nuclear donor cell of transgene clone embryo as nuclear transplantation.

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