CN101265483A - Mammary gland specificity expression vector and construction method thereof - Google Patents
Mammary gland specificity expression vector and construction method thereof Download PDFInfo
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Abstract
The invention discloses a mammary gland-specific expression vector and a construction method thereof. The construction method of the mammary gland-specific expression vector provided by the method comprises the following steps: (1) a DNA fragments that are M0 are constructed; the M0 is T1 to T6 that are homologous arms in sequence from upstream position to downstream position; (2) the M0 is inserted into the vector, and a continuous three-time gene capturing vector is obtained; (3) T5 and T6 capture a 3<1> regulatory region of lactoprotein loca through homologous recombinations for three times; T3 and T4 capture an integral exogenous proteinic genome sequence; T1 and T2 capture a 5<1> regulatory region of the lactoprotein loca. The method gets a lactoprotein-objective protein heterozygous loca, an upstream expression regulation sequence and a downstream expression regulation sequence of the lactoprotein are contained in the heterozygous loca, and the exogenous proteinic genome sequence is contained in the middle part. The mammary gland-specific expression vector and the construction method thereof inject new energies into the individual expression system of animal mammary glands, and greatly promote the further application of a mammary gland bioreactor expression system.
Description
Technical field
The present invention relates to mammary gland specific expression vector and construction process thereof.
Background technology
Protein expression system commonly used at present mainly contains bacterium, yeast, mammalian cell, animal bioreactor etc., and above-mentioned some protein expression system respectively has its relative merits and the scope of application, and the protein of different qualities need be selected corresponding expression system.
Animal mammary gland bioreactor (mammary gland bioreactor) belongs to an important branch in the animal bioreactor, claim animal individual mammary gland expression system again, it belongs to the category of transgenic animal, its core content is by various transgenic technologys, foreign gene with the driving of mammary tissue specificity promoter, in the organizationally efficient expression of animal's mammary gland, production purpose product in milk.How to make exogenous gene high-efficient, stable, specific expressed be a main difficult problem of preparation galactophore biological reactor, the major obstacle of existence comprises: 1) expression amount is low or do not express, and does not have business development and is worth; 2) to cause each transgenic animal be that expression level difference is big to genetically modified site effect, needs a large amount of screening operations, causes workload and cost significantly to rise; 3) ectopic expression problem, i.e. position leakage expression foreign protein beyond the mammary gland causes animal physiological to go to bits and is difficult to obtain produce system.
The key that overcomes an above difficult problem is the structure of expression vector.Most cDNA with target gene adds the upstream and downstream regulating and controlling sequence construction of expression vector that milk protein gene is limited in the early stage research, also there is minority to use the genome sequence or the minigene of target gene, finite capacity in view of cloning vector, such carrier generally all is no more than 30Kb, in order to resist the influence of site effect, also can in expression vector, add some controlling elements sometimes as nuclear matrix land (MAR) sequence of lysozyme of chicken, locuscontrol region (LCR) sequence of betaglobulin etc.Use so little expression vector to obtain a collection of animal mammary gland bioreactor, but most expression amount can't reach the requirement of business development, does not possess actual application value.A large amount of evidences, little expression vector can't satisfy the requirement of preparation galactophore biological reactor, although a small amount of successful story is arranged, little expression vector does not obviously have versatility, can't obtain the specific expression pattern of efficient stable reliably.
In view of the problem that occurs in the galactophore biological reactor preparation, external a lot of correlative study group has been strengthened the research work to some important milk protein gene expression regulations, accumulate from domestic and international secular correlative study, seek out good expression effect, need satisfy following key element: 1) enough perfect expression regulation sequence will be arranged, except promotor, also need enhanser, hormone response district (hormone response element; HRE), locuscontrol region (LCR), scaffolding/nuclear matrix land (S/MAR), insulator a series of controlling elements such as (insulator); In addition, estimating to also have a lot, we do not find that as yet the important factor of its function participates in expression regulation; 2) preferably can use the genome sequence rather than the cDNA of target gene, genome sequence more helps efficiently expressing, and this has obtained to generally acknowledge; 3) expression vector will be among the good karyomit(e) environment, avoids the influence of site effect, and perhaps expression vector has the ability of antagonism site effect.
The locus that latest developments are come out (gene locus) expression vector is for the above opportunity that requires to provide is provided.Locus is meant one section zone that certain gene occupies on karyomit(e), be about tens of hundreds of Kb of arriving and do not wait, and in the both sides of locus elements such as insulator is often arranged, and is as the border of same little kingdom, separated this locus and karyomit(e) environment on every side.Use whole locus to carry out transgene expression and have following advantage: 1) have natural perfect expression regulation sequence and genome sequence in the locus, use whole locus to carry out the endogenous expression level that transgene expression generally can both meet or exceed this gene; 2) transcribing of locus is relatively independent, is not subjected to the influence of karyomit(e) environment on every side, therefore uses whole locus to carry out the expression pattern that transgene expression generally can both obtain to insert the non-dependence in site, copy number dependence.
Because locus is about tens of hundreds of Kb of arriving and does not wait, common cloning vector can't hold, and generally all uses carriers such as BAC, YAC to clone.A series of milk-protein BAC locus, expression effect as mouse WAP, milk cow α s1-casein, people's opalescin, goat beta-lactoglobulin etc. has all obtained checking in mouse mammary gland, all obtained the expression pattern that non-dependence in site and copy number rely on, and expression level all near or surpassed the expression level of endogenous gene, expression amount can reach the level of every liter of tens of grams of milk.Yet sequence is expressed if the regulating and controlling sequence that only uses upstream and downstream to count Kb adds the milk protein gene group, and expression level often has only the 1/10-1/15 of BAC locus expression level, and is difficult to obtain to insert the expression pattern of the non-dependence in site.These results show that milk-protein BAC locus has the ability of surprising ability to express and good antagonism site effect, are the valuable sources of the individual expression system of exploitation mammary gland.But up to this point, result of study also only is confined to milk-protein BAC locus and expresses, and people more interested be how to utilize the regulating and controlling sequence in the milk-protein BAC locus to instruct expression of exogenous gene, particularly express some and have important medical value and the very low protein factor of endogenous expression level, only in this way, could use the milk protein gene seat veritably, make animal mammary gland bioreactor become general and easily and effectively an expression system.Want to reach this purpose, the key of problem is how the regulating and controlling sequence in encoding sequence of waiting to express target gene and the milk protein gene seat is reasonably fitted together, and the research of this respect now also is in the starting stage.In order to make the cre recombinase in the mouse mammary tissue, express specifically, people are inserted into cre recombinase cDNA sequence under the promotor in the milk-protein BAC locus and express, and found that the cre recombinase can efficiently express in mouse mammary gland specifically.
Summary of the invention
The invention provides a kind of mammary gland specific expression vector and construction process thereof.
The construction process of mammary gland specific expression vector provided by the invention comprises the steps:
1) constructed dna fragment M
0M
0Be followed successively by homology arm T to the downstream from the upstream
1, homology arm T
2, homology arm T
3, homology arm T
4, homology arm T
5, homology arm T
6The length of described 6 homology arms is 400-600bp; Homology arm T
5With homology arm T
6Can be by obtaining milk protein gene seat 3 ' control region with milk-protein BAC homologous recombination; Homology arm T
3With homology arm T
4Can be by obtaining complete exogenous object protein genome sequence with exogenous object protein BAC homologous recombination; Homology arm T
1With homology arm T
2Can be by obtaining milk protein gene seat 5 ' control region with milk-protein BAC homologous recombination;
2) with described dna fragmentation M
0Insert in the carrier, obtain continuous three genes and arrest carrier;
3) by carrying out twice homologous recombination, carry out homologous recombination one time with described exogenous object protein BAC, with step 2 with described milk-protein BAC) continuous three genes of obtaining arrest the dna fragmentation M in the carrier
0Replace with dna fragmentation M
1, obtain the mammary gland specific expression vector of described exogenous object protein; M
1Be followed successively by described milk protein gene seat 5 ' control region, complete described exogenous object protein genome sequence, described milk protein gene seat 3 ' control region to the downstream from the upstream.
Described milk protein gene seat 5 ' control region is the sequence of the upstream from start codon of the described milk-protein open reading frame in the described milk protein gene seat; Described milk protein gene seat 3 ' control region is the sequence in the terminator codon downstream of the described milk-protein open reading frame in the described milk protein gene seat; Guarantee that so the described milk-protein open reading frame (by initiator codon to terminator codon) in the described milk protein gene seat accurately is replaced into described exogenous object protein genome sequence.
Described milk protein gene seat can be the milk protein gene seat of all mammiferous all kinds, as the milk protein gene seat of ox, sheep, horse, pig, rabbit, people or mouse.
Described milk protein gene seat specifically can be sheep beta-casein gene seat, cattle beta-casein gene seat, mouse WAP locus, rabbit WAP locus, milk cow α s1-casein gene seat, milk cow α s2-casein gene seat, human milk albumin gene seat or goat beta-lactoglobulin locus, bovine beta-lactoglobulin locus.
Described exogenous object protein can be any foreign protein, as human serum albumin.
Following two kinds of dna fragmentations also belong to protection scope of the present invention.
A kind of dna fragmentation is followed successively by homology arm T to the downstream from the upstream
1, homology arm T
2, homology arm T
3, homology arm T
4, homology arm T
5, homology arm T
6The length of described 6 homology arms is 400-600bp; Homology arm T
5With homology arm T
6Can be by obtaining milk protein gene seat 3 ' control region with milk-protein BAC homologous recombination; Homology arm T
3With homology arm T
4Can be by obtaining complete exogenous object protein genome sequence with exogenous object protein BAC homologous recombination; Homology arm T
1With homology arm T
2Can be by obtaining milk protein gene seat 5 ' control region with milk-protein BAC homologous recombination.
A kind of dna fragmentation is followed successively by milk protein gene seat 5 ' control region, complete exogenous object protein genome sequence, milk protein gene seat 3 ' control region to the downstream from the upstream.
In described two kinds of dna fragmentations, described milk protein gene seat can be the milk protein gene seat of all mammiferous all kinds, as the milk protein gene seat of ox, sheep, horse, pig, rabbit, people or mouse.
Described milk protein gene seat specifically can be sheep beta-casein gene seat, cattle beta-casein gene seat, mouse WAP locus, rabbit WAP locus, milk cow α s1-casein gene seat, milk cow α s2-casein gene seat, human milk albumin gene seat or goat beta-lactoglobulin locus, bovine beta-lactoglobulin locus.
Described exogenous object protein can be any foreign protein, as human serum albumin.
The recombinant vectors, expression cassette, transgenic cell line and the reorganization bacterium that carry described dna fragmentation all belong to protection scope of the present invention.
Described method, described dna fragmentation and described recombinant vectors, expression cassette, transgenic cell line or reorganization bacterium all can be applicable to the preparation of galactophore biological reactor.
The present invention utilizes the BAC homologous recombination technique, milk protein gene seat 5 ' control region, complete exogenous object protein genome sequence (from the initiator codon to the terminator codon), three gene fragments of milk protein gene seat 3 ' control region fit together in order, realized that the genome sequence of milk protein gene seat inside accurately is replaced into the genome sequence of exogenous object protein from the initiator codon to the terminator codon, thereby form the heterozygous genes seat of a milk-protein-target protein, include milk-protein upstream and downstream expression regulation sequence in this heterozygous genes seat, the centre but is the genome sequence of exogenous object protein.The benefit of this scheme is both to have obtained perfect milk-protein expression regulation sequence, the genome sequence rather than the cDNA sequence of exogenous object protein have been used again, the genome sequence that uses exogenous object protein is than using the cDNA sequence more to help efficiently expressing, with respect to using the cDNA sequence, also more meet the natural structural state of eukaryotic gene.The present invention will inject new vitality for the individual expression system of animal's mammary gland, be hopeful to set up a kind of general galactophore biological reactor expression pattern, effectively promote promoting the use of galactophore biological reactor expression system.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 arrests carrier for continuous three genes of sheep beta-casein-human serum albumin
Fig. 2 arrests carrier for continuous three genes of cattle beta-casein-human serum albumin
Fig. 3 is that sheep beta-casein-human serum albumin gene group replacement vector makes up synoptic diagram
Fig. 4 is that cattle beta-casein-human serum albumin gene group replacement vector makes up synoptic diagram
Fig. 5 is that sheep beta-casein-human serum albumin gene group replacement vector transgenic mice southern-blot identifies figure
Fig. 6 is that cattle beta-casein-human serum albumin gene group replacement vector transgenic mice southern-blot identifies figure
Fig. 7 is sheep beta-casein-human serum albumin gene group replacement vector transgenic mice milk SDS-PAGE figure
Fig. 8 is cattle beta-casein-human serum albumin gene group replacement vector transgenic mice milk SDS-PAGE figure
Fig. 9 is the western-blot evaluation figure of the human serum albumin of expressing in sheep beta-casein-human serum albumin gene group replacement vector transgenic mice milk
Figure 10 is the western-blot evaluation figure of the human serum albumin of expressing in cattle beta-casein-human serum albumin gene group replacement vector transgenic mice milk
Embodiment
The present invention can utilize the mammary gland specific expression vector of any exogenous object protein of any milk protein gene seat construction expression.Be example with the mammary gland specific expression vector that utilizes sheep beta-casein gene seat, cattle beta-casein gene seat to make up human serum albumin respectively below, illustrate technical scheme of the present invention.
The reagent of restriction endonuclease, molecular biology aspect is purchased the company in Promega; Other reagent are available from Sigma-Aldrich company; The BAC clone is available from the state-run children's hospital of U.S. BACPAC center; The pKD46 plasmid is available from invitrogen company.The pBR322 plasmid is available from New England Biolab company; The pGEM-T carrier is available from promega company.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
One, the homology arm of sheep beta-casein-human serum albumin clone is the homology arm of 400bp-600bp with the structure three of arresting carrier for continuous three times to (totally 6) length, wherein the first pair of homology arm lays respectively near the terminator codon of 3 ' end of sheep beta-casein gene seat and sheep beta-casein gene group sequence, is used to arrest sheep beta-casein gene seat 3 ' control region; Second pair of homology arm lays respectively near the initiator codon and terminator codon of human serum albumin gene genome sequence, is used to arrest the genome sequence of complete human serum albumin gene; The 3rd pair of homology arm lays respectively near the initiator codon of 5 ' end of sheep beta-casein gene seat and sheep beta-casein gene group sequence, is used to arrest sheep beta-casein gene seat 5 ' control region; Utilize the natural restriction enzyme site that exists in sheep beta-casein gene seat and the human serum albumin gene seat to realize the seamless link of 6 homology arms, make up continuous three genes of acquisition and arrest carrier.
1, the acquisition of homology arm 3f1 and 3f2
(catalog number (Cat.No.): CH243-261D1) being template, is primer with P1 and P2, uses the PCR method clone to obtain sheep beta-casein homology arm 3f1, and sheep beta-casein homology arm 3f1 is positioned at sheep beta-casein gene 3 ' far-end with sheep beta-casein BAC; With sheep beta-casein BAC is template, is primer with P3 and P4, uses the PCR method clone to obtain sheep beta-casein homology arm 3f2, and sheep beta-casein homology arm 3f2 is positioned at sheep beta-casein gene terminator codon downstream nearside; This a pair of homology arm is used to arrest the 3 ' flanking region of the about 8Kb of sheep beta-casein gene jointly.
P1 (upstream primer): 5 '-gtcgacGTTTTTCCTGTGGTCATGTATGGATG-3 ';
P2 (downstream primer): 5 '-gcggccgcCGGAGTTCACTCAGACTCATGTCCATC-3 ';
P3 (upstream primer): 5 '-ccatggGAGGATTTCAATGTGAATGCCCCCTC-3 ';
P4 (downstream primer): 5 '-gtcgacAAATTTTGAGATAAAACAATTG-3 '.
In the above-mentioned primer, small letter is held concurrently and tiltedly write part is restriction enzyme site.
Respectively sheep beta-casein homology arm 3f1 is connected with pGEM-T with 3f2, carries out sequencing analysis, analytical results shows, the inner not sudden change of homology arm.The sequence 1 that the sequence of sheep beta-casein homology arm 3f1 is seen sequence table is the SalI restriction enzyme site from the 1st to 6 deoxyribonucleotide of 5 ' end, and the 357th to 364 deoxyribonucleotide is the NotI restriction enzyme site.The sequence 2 that the sequence of sheep beta-casein homology arm 3f2 is seen sequence table is the NcoI restriction enzyme site from 5 ' the 1st to 6 deoxyribonucleotide of holding; The the 451st to 456 deoxyribonucleotide from 5 ' end is the SalI restriction enzyme site.
2, the acquisition of homology arm g1 and g2
(catalog number (Cat.No.): RP11-580P21) being template, is primer with P5 and P6, uses the PCR method clone to obtain human serum albumin homology arm g1, and human serum albumin homology arm g1 is positioned at the terminator codon upstream proximal with human serum albumin BAC; With human serum albumin BAC is template, is primer with P7 and P8, uses the PCR method clone to obtain human serum albumin homology arm g2, and human serum albumin homology arm g2 is positioned at initiator codon downstream nearside; This a pair of homology arm is used to arrest the human serum albumin gene group fragment from initiator codon to the about 16Kb of terminator codon total length jointly.
P5 (upstream primer): 5 '-gttaacAGCACTTTGTTTTTATCTCCTGCTC-3 ';
P6 (downstream primer): 5 '-ccatggTTATAAGCCTAAGGCAGCTTGACTTG-3 ';
P7 (upstream primer): 5 '-ctcgagATGAAGTGGGTAACCTTTATTTCCCTTC-3 ';
P8 (downstream primer): 5 '-gttaacAACAGCAACCAAGAAGACAGAC-3 '.
In the above-mentioned primer, small letter is held concurrently and tiltedly write part is restriction enzyme site.
Respectively human serum albumin homology arm g1 is connected with pGEM-T with g2, carries out sequencing analysis, analytical results shows, the inner not sudden change of homology arm.The sequence of human serum albumin homology arm g1 is seen the sequence 3 of sequence table, is the HpaI restriction enzyme site from the 1st to 6 deoxyribonucleotide of 5 ' end, and the 513rd to 518 deoxyribonucleotide is the NcoI restriction enzyme site.The sequence of human serum albumin homology arm g2 is seen the sequence 4 of sequence table, is the XhoI restriction enzyme site from 5 ' the 1st to 6 deoxyribonucleotide of holding; The the 514th to 519 deoxyribonucleotide from 5 ' end is the HpaI restriction enzyme site.
3, the acquisition of homology arm 5f1 and 5f2
With sheep beta-casein BAC (catalog number (Cat.No.): CH243-261D1) be template, with P9 and P10 is primer, use the PCR method clone to obtain sheep beta-casein homology arm 5f1, sheep beta-casein homology arm 5f1 is positioned at sheep beta-casein gene upstream from start codon nearside; With sheep beta-casein BAC is template, is primer with P11 and P12, uses the PCR method clone to obtain sheep beta-casein homology arm 5f2, and sheep beta-casein homology arm 5f2 is positioned at sheep beta-casein gene 5 ' far-end; This a pair of homology arm is used to arrest the 5 ' flanking region of the about 13Kb of sheep beta-casein gene jointly.
P9 (last two trip primers): 5 '-agtactTAAGTGAATC AACCACTTAA TTTTTC-3 ';
P10 (downstream primer): 5 '-ctcgagGGCTCTCGATTCCTGTGAATGGGAAG-3 ';
P11 (upstream primer): 5 '-gcggccgcACTCTGGAAC AATTTCTTTT TTG-3 ';
P12 (downstream primer): 5 '-agtactGCAAAACTCACCTCTCTGAATTGC-3 '.
In the above-mentioned primer, small letter is held concurrently and tiltedly write part is restriction enzyme site.
Respectively sheep beta-casein homology arm 5f1 is connected with pGEM-T with 5f2, carries out sequencing analysis, analytical results shows, the inner not sudden change of homology arm.The sequence 5 that the sequence of sheep beta-casein homology arm 5f1 is seen sequence table is the ScaI restriction enzyme site from the 1st to 6 deoxyribonucleotide of 5 ' end, and the 460th to 465 deoxyribonucleotide is the XhoI restriction enzyme site.The sequence 6 that the sequence of sheep beta-casein homology arm 5f2 is seen sequence table is the NotI restriction enzyme site from 5 ' the 1st to 8 deoxyribonucleotide of holding; The the 459th to 464 deoxyribonucleotide from 5 ' end is the ScaI restriction enzyme site.
4, arrest the structure of carrier three times
1) transformation of pBR322 carrier
Synthetic two oligonucleotide small segment N1 and N2; N1 and N2 fragment annealing back form double-stranded, form the sticking end of PstI and PvuI respectively at two ends; PBR322 cuts the back with PstI with the PvuI enzyme and is connected with above-mentioned annealed double chain oligonucleotide small segment, obtains to have the pBR322 plasmid of NotI restriction enzyme site, has destroyed the ammonia benzyl resistant gene of pBR322 simultaneously, has only retained tetracyclin resistance.
N1 and N2 sequence are as follows, and the line part is the NotI site:
N?1：5’-CG
GCGGCCGCCTGCA-3’；
N2：5’-TAGC
GCGGCCGCG-3’。
2) 6 homology arms are linked together in order, specific as follows:
Sheep beta-casein gene homology arm 3f1 and sheep beta-casein gene homology arm 3f2 rely on the SalI restriction enzyme site to link together, sheep beta-casein gene homology arm 3f2 and human serum albumin gene homology arm g1 rely on the NcoI restriction enzyme site to link together, human serum albumin gene homology arm g1 and human serum albumin gene homology arm g2 rely on the HpaI restriction enzyme site to link together, human serum albumin gene homology arm g2 and sheep beta-casein gene homology arm 5f1 rely on the XhoI restriction enzyme site to link together, and sheep beta-casein gene homology arm 5f1 and sheep beta-casein gene homology arm 5f2 rely on the ScaI restriction enzyme site to link together.
3) at the 3 ' end of sheep beta-casein gene homology arm 3f1 and the 5 ' end of sheep beta-casein gene homology arm 5f2 a NotI restriction enzyme site is arranged respectively, use the NotI enzyme to cut 6 homology arms that connect together in order, and transfer is cloned on the improved pBR322 carrier of step 1).
The recombinant vectors that obtains is carried out the enzyme evaluation of cutting and check order, prove that having obtained to connect right-on continuous three genes arrests carrier pBRSV
0, continuous three genes are arrested carrier pBRSV
0Structure see Fig. 1.
Two, the structure of mammary gland specific expression vector
1, the preparation of competent cell
1) the DH10 β bacterium that will contain sheep beta-casein gene BAC is respectively made chemoreception attitude cell with the DH10 β bacterium that contains human serum albumin BAC;
2) will encode Red recombination system pKD46 plasmid respectively heat shock be converted in above-mentioned two kinds of chemoreception attitude cells;
3) with step 2) cell 30 ℃ of substratum on the flat board that contains Ampicillin Trihydrate and paraxin of obtaining spend the night;
4) choosing mono-clonal is forwarded in the liquid LB substratum 30 ℃ to continue to be cultured to OD600 is 0.20～0.25;
5) add the L-arabinose of 1mol/L then, make final concentration be about 6mmol/L, continue to cultivate 45min to 1h, make OD600 reach 0.40～0.50;
6) centrifugal collection bacterium, it is inferior to give a baby a bath on the third day after its birth with 10% sterile glycerol, suspends with 10% sterile glycerol at last, and obtaining final concentration is 1 * 10
10The electric shock competent cell that contains pKD46 and sheep beta-casein gene BAC of cells/ μ L, final concentration is 1 * 10
10The electric shock competent cell that contains pKD46 and human serum albumin gene BAC of cells/ μ L.
2, gene is arrested
The flow process that continuous three genes are arrested is seen Fig. 3.Among Fig. 3, T6: sheep beta-casein gene homology arm 3f1; T5: sheep beta-casein gene homology arm 3f2; T4: human serum albumin gene homology arm g1; T3: human serum albumin gene homology arm g2; T2: sheep beta-casein gene homology arm 5f1; T1: sheep beta-casein gene homology arm 5f2.
1) gene is arrested for the first time
Continuous three genes that 1. will build are arrested carrier pBRSV
0Cut with restriction enzyme SalI enzyme, open the connection between sheep beta-casein gene homology arm 3f1 and the homology arm 3f2, discharge two homology arms, reclaim behind the dephosphorization, constitute linear gene and arrest carrier, concentration is about 50ng/uL;
2. get linearizing that 1. the 2uL step obtain and arrest carrier and add 50uL and contain in the competent escherichia coli cell of pKD46 and sheep beta-casein gene BAC, shock by electricity, shock parameters is 2.5kv, 5ms; Add 500uL SOC nutrient solution after the electric shock immediately and suspend, 37 ℃, 220r/min, recovery 1.5h coats on the LB flat board that contains tsiklomitsin (50ug/mL) then, hatches 24h for 37 ℃.
Bacterial clone extracting plasmid to tetracyclin resistance is identified, finds that the plasmid that obtains is obviously big than arresting carrier, illustrates to arrest to have obtained first gene fragment; Carry out enzyme and cut the evaluation of identifying and check order, prove and successfully arrest the sheep beta-casein gene 3 ' control region that has obtained 8Kb.
What obtained that the clone has sheep beta-casein gene seat a 3 ' control region arrests carrier pBRSV
1
2) gene is arrested for the second time
1. from arresting carrier pBRSV
1Set out, cut with restriction enzyme HpaI enzyme, open the connection between human serum albumin gene homology arm g1 and the homology arm g2, discharge two homology arms, reclaim behind the dephosphorization, constitute linear gene and arrest carrier, concentration is about 50ng/uL;
2. get linearizing that 1. the 2uL step obtain and arrest carrier and add 50uL and contain in the competent escherichia coli cell of pKD46 and human serum albumin gene BAC, shock by electricity, shock parameters is 2.5kv, 5ms; Add 500uL SOC nutrient solution after the electric shock immediately and suspend, 37 ℃, 220r/min, recovery 1.5h coats on the LB flat board that contains tsiklomitsin (50ug/mL) then, hatches 24h for 37 ℃.
Bacterial clone extracting plasmid to tetracyclin resistance is identified, finds that the plasmid that obtains is obviously big than the plasmid of arresting acquisition for the first time, illustrates to arrest to have obtained second gene fragment; Carry out enzyme and cut the evaluation of identifying and check order, prove successfully to arrest to have obtained the human serum albumin gene group sequence complete from initiator codon to terminator codon 16Kb.
What obtained so far that the clone has sheep beta-casein gene seat 3 ' control region and a complete human serum albumin gene genome sequence arrests carrier pBRSV
2
3) gene is arrested for the third time
1. from arresting carrier pBRSV
2Set out, cut with restriction enzyme ScaI enzyme, open the connection between sheep beta-casein gene homology arm 5f2 and the 5f1, discharge two homology arms, reclaim behind the dephosphorization, constitute linear gene and arrest carrier, concentration is about 50ng/uL;
2. get linearizing that 1. the 2uL step obtain and arrest carrier and add 50uL and contain in the competent escherichia coli cell of pKD46 and sheep beta-casein gene BAC, shock by electricity, shock parameters is 2.5kv, 5ms; Add 500uL SOC nutrient solution after the electric shock immediately and suspend, 37 ℃, 220r/min, recovery 1.5h coats on the LB flat board that contains tsiklomitsin (50ug/mL) then, hatches 24h for 37 ℃.
Bacterial clone extracting plasmid to tetracyclin resistance is identified, finds that the plasmid that obtains is obviously big than the plasmid of arresting acquisition for the second time, illustrates to arrest to have obtained the 3rd gene fragment; Carry out enzyme and cut the evaluation of identifying and check order, prove and successfully arrest the sheep beta-casein 5 ' control region that has obtained 13Kb.
So far obtained to clone the sheep-casein-human serum albumin gene group displaced type expression vector pBRS of the sheep beta-casein 5 ' control region that the 3 ' control region of sheep beta-casein gene 8Kb, the complete human serum albumin gene group sequence from initiator codon to terminator codon 16Kb and 13Kb are arranged, successfully realized the genome sequence in the sheep beta-casein gene seat accurately is replaced into the genome sequence of human serum albumin gene from the initiator codon to the terminator codon.
Sheep beta-casein-human serum albumin gene group displaced type expression vector pBRS is carried out the HindIII enzyme and cuts evaluation, 240bp, 3281bp, 4412bp, 5429bp, 6409bp, 7712bp, 9246bp totally 6 fragments appear in the result, with estimate identical, it is correct to show that this sheep beta-casein-human serum albumin gene group displaced type expression vector pBRS makes up.
Three, the preparation of transgenic mice and evaluation
1, the preparation of transgenic mice
Expression vector pBRS is cut with the NotI enzyme, reclaim target gene fragment, microinjection is in the protokaryon of C57 mouse (Military Medical Science Institute's Experimental Animal Center) zygote, mouse fertilized egg through injection is gone in the C57 replace-conceive mouse by common oviduct transplantation, treat the newborn mouse childbirth, promptly obtain transgenic founder (Founder) mouse.
Use the southern-blot method and transgenic founder (Founder) mouse is identified probe sequence is seen the sequence 11 (645bp) of sequence table.Obtain 9 male transgenosis Founder mouse (3 female mouse altogether; 6 male mouse).Southern-blot qualification result such as Fig. 5 of 9 male transgenosis Founder mouse.Among Fig. 5,1-9 is 9 male transgenosis Founder mouse; 10 is common C57 mouse; 11 is the pBRS carrier.
Male transgenosis Founder mouse gone down to posterity set up corresponding transgenic mice system.The mutual mating of 9 positive transgenosis Founder mouse, the filial generation transgenic mice that obtains are F1 generation, and continuous passage is until F6 generation.
2, the evaluation of transgenic mice
Carry out mating from F6 for optional 2 female mices and male C57 mouse the transgenic mice and produce son.Transgenic mice produced son back 12 days, isolated mothers and sons 3 hours, and to the pitocin of female mouse injection 0.2U, anesthesia after 20 minutes is squeezed and got the about 80ul of milk.
Simultaneously son is produced in common C57 mouse male and female mating, female mice produced son back 12 days, isolated mothers and sons 3 hours, and to the pitocin of female mouse injection 0.2U, anesthesia after 20 minutes is squeezed and got the about 80ul of milk, as negative control.
1) mensuration of human serum protein's expression amount in the transgenic mice milk
Frozen standby in-80 ℃ of refrigerators with pressing the every pipe packing of 10ul behind the milk centrifugal degreasing, take out the back and dilute 20 times with PBS, get 10ul and add that the sample damping fluid mixes, boiled 5 minutes, place on ice, the glue of use 12% carries out SDS-PAGE to be analyzed, and uses the content of the human serum albumin of gel sweep measuring expression.
The results are shown in Figure 7.Among Fig. 7,1: common negative mouse milk sample; 2: transgenic mice milk sample; M: low molecular weight protein (LMWP) standard.As seen tangible human serum albumin band, molecular weight is about 66Kda, and expression amount is about 5g/L milk.
2) evaluation of human serum albumin in the transgenic mice milk
Get 10ul after mouse milk uses PBS to dilute 10,000 times and carry out SDS-PAGE, use the western-blot method to identify whether expressed products is human serum albumin really, the antibody that uses is the mouse anti human serum albumin monoclonal antibody of HRP mark.
The results are shown in Figure 9.Among Fig. 9,1,2 is that two transgenic mices are the milk sample, and 3 is common negative mouse milk sample.
Proof express human serum albumin really.
One, the homology arm of cattle beta-casein-human serum albumin clone is the homology arm of 400bp-600bp with the structure three of arresting carrier for continuous three times to (totally 6) length, wherein the first pair of homology arm lays respectively near the terminator codon of 3 ' end of cattle beta-casein gene seat and cattle beta-casein gene group sequence, is used to arrest cattle beta-casein gene seat 3 ' control region; Second pair of homology arm lays respectively near the initiator codon and terminator codon of human serum albumin gene genome sequence, is used to arrest the genome sequence of complete human serum albumin gene; The 3rd pair of homology arm lays respectively near the initiator codon of 5 ' end of cattle beta-casein gene seat and cattle beta-casein gene group sequence, is used to arrest cattle beta-casein gene seat 5 ' control region; Utilize the natural restriction enzyme site that exists in cattle beta-casein gene seat and the human serum albumin gene seat to realize the seamless link of 6 homology arms, make up continuous three genes of acquisition and arrest carrier.
1, the acquisition of homology arm 3f1 and 3f2
(catalog number (Cat.No.): RP42-127E3) being template, is primer with P13 and P14, uses the PCR method clone to obtain cattle beta-casein homology arm 3f1, and cattle beta-casein homology arm 3f1 is positioned at cattle beta-casein gene 3 ' far-end with cattle beta-casein BAC; With cattle beta-casein BAC is template, is primer with P15 and P16, uses the PCR method clone to obtain cattle beta-casein homology arm 3f2, and cattle beta-casein homology arm 3f2 is positioned at cattle beta-casein gene terminator codon downstream nearside; This a pair of homology arm is used to arrest the 3 ' flanking region of the about 11Kb of cattle beta-casein gene jointly.
P13 (upstream primer): 5 '-catatgAGTGAAAGTG AAAATGAAGT TGCTC-3 ';
P14 (downstream primer): 5 '-gcggccgcGAGATGGTGAGGGACAGGGAGGTC-3 ';
P15 (upstream primer): 5 '-ccatggGTCTAAATTT ACTAACTGTG CTG-3 ';
P16 (downstream primer): 5 '-catatgATAATTGTGATAAGGAGAACTTC-3 '.
In the above-mentioned primer, small letter is held concurrently and tiltedly write part is restriction enzyme site.
Respectively cattle beta-casein homology arm 3f1 is connected with pGEM-T with 3f2, carries out sequencing analysis, analytical results shows, the inner not sudden change of homology arm.The sequence of cattle beta-casein homology arm 3f1 is seen the sequence 7 of sequence table, is the NdeI restriction enzyme site from the 1st to 6 deoxyribonucleotide of 5 ' end, and the 416th to 423 deoxyribonucleotide is the NotI restriction enzyme site.The sequence of cattle beta-casein homology arm 3f2 is seen the sequence 8 of sequence table, is the NcoI restriction enzyme site from 5 ' the 1st to 6 deoxyribonucleotide of holding; The the 441st to 446 deoxyribonucleotide from 5 ' end is the NdeI restriction enzyme site.
2, the acquisition of homology arm g1 and g2
(catalog number (Cat.No.): RP11-580P21) being template, is primer with P5 and P6, uses the PCR method clone to obtain human serum albumin homology arm g1, and human serum albumin homology arm g1 is positioned at the terminator codon upstream proximal with human serum albumin BAC; With human serum albumin BAC is template, is primer with P7 and P8, uses the PCR method clone to obtain human serum albumin homology arm g2, and human serum albumin homology arm g2 is positioned at initiator codon downstream nearside; This a pair of homology arm is used to arrest the human serum albumin gene group fragment from initiator codon to the about 16Kb of terminator codon total length jointly.
P5 (upstream primer): 5 '-gttaacAGCACTTTGTTTTTATC TCCTGCTC-3 ';
P6 (downstream primer): 5 '-ccatggTTATAAGCCTAAGGCAGCTTGACTTG-3 ';
P7 (upstream primer): 5 '-ctcgagATGAAGTGGGTAACCTTTATTTCCCTTC-3 ';
P8 (downstream primer): 5 '-gttaacAACAGCAACCAAGAAGACAGAC-3 '.
In the above-mentioned primer, small letter is held concurrently and tiltedly write part is restriction enzyme site.
Respectively human serum albumin homology arm g1 is connected with pGEM-T with g2, carries out sequencing analysis, analytical results shows, the inner not sudden change of homology arm.The sequence of human serum albumin homology arm g1 is seen the sequence 3 of sequence table, is the HpaI restriction enzyme site from the 1st to 6 deoxyribonucleotide of 5 ' end, and the 513rd to 518 deoxyribonucleotide is the NcoI restriction enzyme site.The sequence of human serum albumin homology arm g2 is seen the sequence 4 of sequence table, is the XhoI restriction enzyme site from 5 ' the 1st to 6 deoxyribonucleotide of holding; The the 514th to 519 deoxyribonucleotide from 5 ' end is the HpaI restriction enzyme site.
3, the acquisition of homology arm 5f1 and 5f2
With cattle beta-casein BAC (catalog number (Cat.No.): RP42-127E3) be template, with P17 and P18 is primer, use the PCR method clone to obtain cattle beta-casein homology arm 5f1, cattle beta-casein homology arm 5f1 is positioned at cattle beta-casein gene upstream from start codon nearside; With cattle beta-casein BAC is template, is primer with P19 and P20, uses the PCR method clone to obtain cattle beta-casein homology arm 5f2, and cattle beta-casein homology arm 5f2 is positioned at cattle beta-casein gene 5 ' far-end; This a pair of homology arm is used to arrest the 5 ' flanking region of the about 15Kb of cattle beta-casein gene jointly.
P17 (upstream primer): 5 '-agtactCTAAGACATATCTGGCAATAAAAATTA-3 ';
P18 (downstream primer): 5 '-ctcgagGGCTCTCAATTCCTGGGAATG-3 ';
P19 (upstream primer): 5 '-gcggccgcTCAAGAAGAT CCCCTGGAGA AGG-3 ';
P20 (downstream primer): 5 '-agtactATGGTGACCAGAACGCCTTGTGG-3 '.
In the above-mentioned primer, small letter is held concurrently and tiltedly write part is restriction enzyme site.
Respectively cattle beta-casein homology arm 5f1 is connected with pGEM-T with 5f2, carries out sequencing analysis, analytical results shows, the inner not sudden change of homology arm.The sequence of cattle beta-casein homology arm 5f1 is seen the sequence 9 of sequence table, is the ScaI restriction enzyme site from the 1st to 6 deoxyribonucleotide of 5 ' end, and the 505th to 510 deoxyribonucleotide is the XhoI restriction enzyme site.The sequence of cattle beta-casein homology arm 5f2 is seen the sequence 10 of sequence table, is the NotI restriction enzyme site from 5 ' the 1st to 8 deoxyribonucleotide of holding; The the 459th to 464 deoxyribonucleotide from 5 ' end is the ScaI restriction enzyme site.
4, arrest the structure of carrier three times
1) transformation of pBR322 carrier
With the step 1 of embodiment 14 1).
2) 6 homology arms are linked together in order, specific as follows:
Cattle beta-casein gene homology arm 3f1 and cattle beta-casein gene homology arm 3f2 rely on the NdeI restriction enzyme site to link together, cattle beta-casein gene homology arm 3f2 and human serum albumin gene homology arm g1 rely on the NcoI restriction enzyme site to link together, human serum albumin gene homology arm g1 and human serum albumin gene homology arm g2 rely on the HpaI restriction enzyme site to link together, human serum albumin gene homology arm g2 and cattle beta-casein gene homology arm 5f1 rely on the XhoI restriction enzyme site to link together, and cattle beta-casein gene homology arm 5f1 and cattle beta-casein gene homology arm 5f2 rely on the ScaI restriction enzyme site to link together.
3) at the 3 ' end of cattle beta-casein gene homology arm 3f1 and the 5 ' end of cattle beta-casein gene homology arm 5f2 a NotI restriction enzyme site is arranged respectively, use the NotI enzyme to cut 6 homology arms that connect together in order, and transfer is cloned on the improved pBR322 carrier of step 1).
The recombinant vectors that obtains is carried out the enzyme evaluation of cutting and check order, prove that having obtained to connect right-on continuous three genes arrests carrier pBRCV
0, continuous three genes are arrested carrier pBRCV
0Structure see Fig. 2.
Two, the structure of mammary gland specific expression vector
1, the preparation of competent cell
1) the DH10 β bacterium that will contain cattle beta-casein gene BAC is respectively made chemoreception attitude cell with the DH10 β bacterium that contains human serum albumin BAC;
2) will encode Red recombination system pKD46 plasmid respectively heat shock be converted in above-mentioned two kinds of chemoreception attitude cells;
3) with step 2) cell 30 ℃ of substratum on the flat board that contains Ampicillin Trihydrate and paraxin of obtaining spend the night;
4) choosing mono-clonal is forwarded in the liquid LB substratum 30 ℃ to continue to be cultured to OD600 is 0.20～0.25;
5) add the L-arabinose of 1mol/L then, make final concentration be about 6mmol/L, continue to cultivate 45min to 1h, make OD600 reach 0.40～0.50;
6) centrifugal collection bacterium, it is inferior to give a baby a bath on the third day after its birth with 10% sterile glycerol, suspends with 10% sterile glycerol at last, and obtaining final concentration is 1 * 10
10The electric shock competent cell that contains pKD46 and cattle beta-casein gene BAC of cells/ μ L, final concentration is 1 * 10
10The electric shock competent cell that contains pKD46 and human serum albumin gene BAC of cells/ μ L.
2, gene is arrested
The flow process that continuous three genes are arrested is seen Fig. 4.Among Fig. 4, T6: cattle beta-casein gene homology arm 3f1; T5: cattle beta-casein gene homology arm 3f2; T4: human serum albumin gene homology arm g1; T3: human serum albumin gene homology arm g2; T2: cattle beta-casein gene homology arm 5f1; T1: cattle beta-casein gene homology arm 5f2.
1) gene is arrested for the first time
Continuous three genes that 1. will build are arrested carrier pBRSV
0Cut with restriction enzyme NdeI enzyme, open the connection between cattle beta-casein gene homology arm 3f1 and the homology arm 3f2, discharge two homology arms, reclaim behind the dephosphorization, constitute linear gene and arrest carrier, concentration is about 50ng/uL;
2. get linearizing that 1. the 2uL step obtain and arrest carrier and add 50uL and contain in the competent escherichia coli cell of pKD46 and cattle beta-casein gene BAC, shock by electricity, shock parameters is 2.5kv, 5ms; Add 500uL SOC nutrient solution after the electric shock immediately and suspend, 37 ℃, 220r/min, recovery 1.5h coats on the LB flat board that contains tsiklomitsin (50ug/mL) then, hatches 24h for 37 ℃.
Bacterial clone extracting plasmid to tetracyclin resistance is identified, finds that the plasmid that obtains is obviously big than arresting carrier, illustrates to arrest to have obtained first gene fragment; Carry out enzyme and cut the evaluation of identifying and check order, prove and successfully arrest the cattle beta-casein gene 3 ' control region that has obtained 11Kb.
What obtained that the clone has cattle beta-casein gene seat a 3 ' control region arrests carrier pBRCV
1
2) gene is arrested for the second time
1. from arresting carrier pBRCV
1Set out, cut with restriction enzyme HpaI enzyme, open the connection between human serum albumin gene homology arm g1 and the homology arm g2, discharge two homology arms, reclaim behind the dephosphorization, constitute linear gene and arrest carrier, concentration is about 50ng/uL;
2. get linearizing that 1. the 2uL step obtain and arrest carrier and add 50uL and contain in the competent escherichia coli cell of pKD46 and human serum albumin gene BAC, shock by electricity, shock parameters is 2.5kv, 5ms; Add 500uL SOC nutrient solution after the electric shock immediately and suspend, 37 ℃, 220r/min, recovery 1.5h coats on the LB flat board that contains tsiklomitsin (50ug/mL) then, hatches 24h for 37 ℃.
Bacterial clone extracting plasmid to tetracyclin resistance is identified, finds that the plasmid that obtains is obviously big than the plasmid of arresting acquisition for the first time, illustrates to arrest to have obtained second gene fragment; Carry out enzyme and cut the evaluation of identifying and check order, prove successfully to arrest to have obtained the human serum albumin gene group sequence complete from initiator codon to terminator codon 16Kb.
What obtained so far that the clone has cattle beta-casein gene seat 3 ' control region and a complete human serum albumin gene genome sequence arrests carrier pBRCV
2
3) gene is arrested for the third time
1. from arresting carrier pBRCV
2Set out, cut with restriction enzyme ScaI enzyme, open the connection between cattle beta-casein gene homology arm 5f2 and the 5f1, discharge two homology arms, reclaim behind the dephosphorization, constitute linear gene and arrest carrier, concentration is about 50ng/uL;
2. get linearizing that 1. the 2uL step obtain and arrest carrier and add 50uL and contain in the competent escherichia coli cell of pKD46 and cattle beta-casein gene BAC, shock by electricity, shock parameters is 2.5kv, 5ms; Add 500uL SOC nutrient solution after the electric shock immediately and suspend, 37 ℃, 220r/min, recovery 1.5h coats on the LB flat board that contains tsiklomitsin (50ug/mL) then, hatches 24h for 37 ℃.
Bacterial clone extracting plasmid to tetracyclin resistance is identified, finds that the plasmid that obtains is obviously big than the plasmid of arresting acquisition for the second time, illustrates to arrest to have obtained the 3rd gene fragment; Carry out enzyme and cut the evaluation of identifying and check order, prove and successfully arrest the cattle beta-casein 5 ' control region that has obtained 15Kb.
So far obtained to clone the cattle beta-casein-human serum albumin gene group displaced type expression vector pBRC of the cattle beta-casein 5 ' control region that the 3 ' control region of cattle beta-casein gene 11Kb, the complete human serum albumin gene group sequence from initiator codon to terminator codon 16Kb and 15Kb are arranged, successfully realized the genome sequence in the cattle beta-casein gene seat accurately is replaced into the genome sequence of human serum albumin gene from the initiator codon to the terminator codon.
Cattle beta-casein-human serum albumin gene group displaced type expression vector pBRC is carried out the BglI enzyme and cuts evaluation.10 bands appear in qualification result, be respectively: 663bp, 803bp, 2194bp, 2221bp, 3080bp, 3385bp, 3647bp, 5350bp, 9250bp, 11865bp, it is correct to show that cattle beta-casein-human serum albumin gene group displaced type expression vector pBRC makes up.
Three, the preparation of transgenic mice and evaluation
1, the preparation of transgenic mice
Expression vector pBRC is cut with the NotI enzyme, reclaim target gene fragment, microinjection is in the protokaryon of C57 mouse (available from Military Medical Science Institute's Experimental Animal Center) zygote, mouse fertilized egg through injection is gone in the C57 replace-conceive mouse by common oviduct transplantation, treat the newborn mouse childbirth, promptly obtain transgenic founder (Founder) mouse.
Use the southern-blot method and transgenic founder (Founder) mouse is identified probe sequence is seen the sequence 11 (645bp) of sequence table.Obtain 7 male transgenosis Founder mouse altogether.Southern-blot qualification result such as Fig. 6 of 7 male transgenosis Founder mouse.Among Fig. 6,1 is the pBRC carrier; 2 is common C57 mouse; 3-9 is 7 male transgenic mices.
Male transgenosis Founder mouse gone down to posterity set up corresponding transgenic mice system.The mutual mating of 7 positive transgenosis Founder mouse, the filial generation transgenic mice that obtains are F1 generation, and continuous passage is until F6 generation.
2, the evaluation of transgenic mice
Carry out mating from F6 for optional 3 female mices and male C57 mouse the transgenic mice and produce son.Transgenic mice produced son back 12 days, isolated mothers and sons 3 hours, and to the pitocin of female mouse injection 0.2U, anesthesia after 20 minutes is squeezed and got the about 80ul of milk.
Simultaneously son is produced in the mating of C57 mouse male and female, female mice produced son back 12 days, isolated mothers and sons 3 hours, and to the pitocin of female mouse injection 0.2U, anesthesia after 20 minutes is squeezed and got the about 80ul of milk, as negative control.
1) mensuration of human serum protein's expression amount in the transgenic mice milk
Frozen standby in-80 ℃ of refrigerators with pressing the every pipe packing of 10ul behind the milk centrifugal degreasing, take out the back and dilute 20 times with PBS, get 10ul and add that the sample damping fluid mixes, boiled 5 minutes, place on ice, the glue of use 12% carries out SDS-PAGE to be analyzed, and uses the content of the human serum albumin of gel sweep measuring expression.
The results are shown in Figure 8.Among Fig. 8,1: transgenic mice milk sample; 2: common negative mouse milk sample; M: low molecular weight protein (LMWP) standard.As seen tangible human serum albumin band, molecular weight is about 66Kda, and expression amount is about 8g/L milk.
2) evaluation of human serum albumin in the transgenic mice milk
Get 10ul after mouse milk uses PBS to dilute 10,000 times and carry out SDS-PAGE, use the western-blot method to identify whether expressed products is human serum albumin really, the antibody that uses is the mouse anti human serum albumin monoclonal antibody of HRP mark.
The results are shown in Figure 10.Among Figure 10,1,2,3 is that three transgenic mices are the milk sample, and 4 is common negative mouse milk sample.
Proof express human serum albumin really.
Sequence table
＜110〉Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A
＜120〉mammary gland specific expression vector and construction process thereof
<130>CGGNARY81247
<160>11
<210>1
<211>364
<212>DNA
＜213〉artificial sequence
<400>1
gtcgacgttt?ttcctgtggt?catgtatgga?tgtgagagtt?ggactgtgaa?gaaggctgag 60
caccgaagaa?ttgatgcttt?tgaactgtgg?tgttggagaa?gactcttgag?agtcccttgg 120
actgcaagga?gatccaacca?gtccattctg?aaggagatca?gccctgggat?ttctttggaa 180
gtaatgatgc?taaagctgaa?actccaatac?tttggccacc?tcatgtgaag?agttgactca 240
ttggaaaaga?ctctgatggt?gggagggatt?gggggcaaga?ggagaagggg?acgacagagg 300
atgagatggc?tgaatggcat?cactgactcg?atggacatga?gtctgagtga?actccggcgg 360
ccgc 364
<210>2
<211>456
<212>DNA
＜213〉artificial sequence
<400>2
ccatgggagg?atttcaatgt?gaatgccccc?tcctcacttt?tggtaagctt?taggagatta 60
gaggcagact?gatcattttt?atagttaata?tcttttacat?ttaattttcc?tggataagac 120
ccaatagtag?caatttctat?cagtatacca?gcgtaaagat?tagttttaaa?tttattttca 180
gtgattgact?gttatttact?gacctgaaat?tatgtatctg?ttatatttca?aataatgcaa 240
aactgtatat?atatggtgtt?gacagatttg?attggttttc?tttcaattgc?ctatatcctt 300
attattgatt?gtaatcattt?atagaaaaaa?ctgaaaataa?tttcttatac?ttttatgtaa 360
acctgttaga?gcttatttta?aagatcaact?gcattcacat?ttctaatcta?gtcattatga 420
gcttcaattg?ttttatctca?cttaaaattt?gtcgac 456
<210>3
<211>518
<212>DNA
＜213〉artificial sequence
<400>3
gttaacagca?ctttgttttt?atctcctgct?ctattgtgcc?atactgttaa?atgtttataa 60
tgcctgttct?gtttccaaat?ttgtgatgct?tatgaatatt?aataggaata?tttgtaaggc 120
ctgaaatatt?ttgatcatga?aatcaaaaca?ttaatttatt?taaacattta?cttgaaatgt 180
ggtggtttgt?gatttagttg?attttatagg?ctagtgggag?aatttacatt?caaatgtcta 240
aatcacttaa?aattgccctt?tatggcctga?cagtaacttt?tttttattca?tttggggaca 300
actatgtccg?tgagcttccg?tccagagatt?atagtagtaa?attgtaatta?aaggatatga 360
tgcacgtgaa?atcactttgc?aatcatcaat?agcttcataa?atgttaattt?tgtatcctaa 420
tagtaatgct?aatattttcc?taacatctgt?catgtctttg?tgttcagggt?aaaaaacttg 480
ttgctgcaag?tcaagctgcc?ttaggcttat?aaccatgg 518
<210>4
<211>519
<212>DNA
＜213〉artificial sequence
<400>4
ctcgagatga?agtgggtaac?ctttatttcc?cttctttttc?tctttagctc?ggcttattcc 60
aggggtgtgt?ttcgtcgaga?tgcacgtaag?aaatccattt?ttctattgtt?caacttttat 120
tctattttcc?cagtaaaata?aagttttagt?aaactctgca?tctttaaaga?attattttgg 180
catttatttc?taaaatggca?tagtattttg?tatttgtgaa?gtcttacaag?gttatcttat 240
taataaaatt?caaacatcct?aggtaaaaaa?aaaaaaaggt?cagaattgtt?tagtgactgt 300
aattttcttt?tgcgcactaa?ggaaagtgca?aagtaactta?gagtgactga?aacttcacag 360
aatagggttg?aagattgaat?tcataactat?cccaaagacc?tatccattgc?actatgcttt 420
atttaaaaac?cacaaaacct?gtgctgttga?tctcataaat?agaacttgta?tttatattta 480
ttttcatttt?agtctgtctt?cttggttgct?gttgttaac 519
<210>5
<211>465
<212>DNA
＜213〉artificial sequence
<400>5
agtacttaag?tgaatcaacc?acttaatttt?tctgtaaata?tctgtaactt?ctcttctgtc 60
tttccaaaaa?cactcgtaag?tactgtgaat?gagatgaaaa?agagtgaagt?aggatatagg 120
atgttagcag?aaaacatctg?aatggctggc?agtgaaacat?taacttgaaa?tgtaagatta 180
atgagtaata?gtaaatttta?actttggcca?tatgataaaa?tgtttattaa?tatttttcta 240
aaatacaggg?ctttttgttt?ttgccatgag?gtttgcagga?tcttggttcc?ctgaccaggg 300
atcaaacctg?cgctcccctg?gaagcatgga?gtcttggaca?tttgtattat?acactatctt 360
tggttccttg?taaagggaag?taattttact?taaataagaa?aatagattga?gaagtaatac 420
actgtttcct?catcttccca?ttcacaggaa?tcgagagccc?tcgag 465
<210>6
<211>464
<212>DNA
＜213〉artificial sequence
<400>6
gcggccgcac?tctggaacaa?tttctttttt?gctgaaagtg?agacaaatgt?atcactgtga 60
attggagaat?aaaattgaaa?attaaggttt?agatttcctc?aaatcaaatg?ttttttatgt 120
ggatatgttc?aagtagctca?aaagttattt?acattcatat?attagttatg?atgatagggt 180
ctgatataaa?gtttattttc?tatttccttc?ttactttaaa?aaaattgtct?acttctatta 240
ttttataaaa?aaaataatct?gttactgtga?tataaataac?caatttgttt?ttgaaagatc 300
catgacaact?tgttggatta?atagaagtca?tcttatttct?tgaagagtat?ctcagtaact 360
agacataaag?aatatcaaag?tgaaagttac?cactgctttc?acagaaggag?aaacaaattt 420
agaggaagat?gagagcaatt?cagagaggtg?agttttgcag?tact 464
<210>7
<211>423
<212>DNA
＜213〉artificial sequence
<400>7
catatgagtg?aaagtgaaaa?tgaagttgct?cagtcatgtc?cactcttcgc?aaacccatgg 60
acggtagcct?accaggctct?gccgtccatg?ggattttcca?ggcaagaata?ctgaagtggg 120
ctgccatttc?cttctccagg?ggatcttccc?aacccaggga?tcaaacccgg?gtctcctgca 180
ttgcagacag?acgctttacc?atctgaacca?cccaggaagg?ccatttacat?agagtgcatg 240
taaatgcata?atccataatt?gcttccaaac?aaatttcagt?aaaccagcaa?tctaagtaga 300
agttgaagct?atagacattt?gtgtgtgtgt?gtgtgtttca?gtcaccaagt?tatgtctgac 360
tcttagcgac?ctcatggact?gcagcacacc?agacctccct?gtccctcacc?atctcgcggc 420
cgc 423
<210>8
<211>446
<212>DNA
＜213〉artificial sequence
<400>8
ccatgggtct?aaatttacta?actgtgctgt?ttaacttctg?atgtttgtat?gatattcgag 60
taattaagag?tcctataaaa?aaatgaataa?tgaatggttc?caaaataagc?atagctgaga 120
ttaatgattg?tcagcattag?ttataaatag?aataagctgg?agaaccttca?cctcccctcc 180
accaccagat?ctcaatgtct?aggcttaccc?gtggagattc?tgatgtaatt?gttctttcta 240
tgtagaagaa?acttattggg?aagaaataat?ataatggact?atgatttaat?tggtctgttg 300
agaccaatta?aattagatga?aggcgattaa?ggtacaataa?agccagaatt?gaatttgata 360
atctcatttg?gctaagaata?acaaacctaa?gaaggtttgc?tattttctac?aattttgaag 420
ttctccttat?gcacaattat?catatg 446
<210>9
<211>510
<212>DNA
＜213〉artificial sequence
<400>9
agtactctaa?gacatatctg?gcaataaaaa?ttaataaata?aatattttta?ataagtaaat 60
caatcactta?atttttctgt?aagtatctgt?aacttctctt?ctgtctttcc?aaaaaacact 120
cataagtact?gtgaataaga?tgaaaagagt?gaaataagat?ataggctgtt?agctgaaaac 180
atctggatgg?ctggcagtga?aacattaact?tgaaatgtaa?gattaatgag?taatagtaaa 240
ttttaacctt?ggccgtatga?taaaatgtct?attaatattt?ttctaaaata?cagggctttt 300
tgtttttgcc?atgaggtttg?caggatcttg?gttccctgat?gagggatcaa?acctgggctc 360
ccctggaagc?acggagtctt?agatatttgt?attatacact?atctttggtt?tcttttaaag 420
ggaagtaatt?ctacttaaat?aagaaaatag?attgacaagt?aatacactat?ttcctcatct 480
tcccattccc?aggaattgag?agccctcgag 510
<210>10
<211>464
<212>DNA
＜213〉artificial sequence
<400>10
gcggccgctc?aagaagatcc?cctggagaag?gaaatggcaa?ctactccagt?attcttgact 60
gaaaaatccc?acggacagag?aaacttccgg?ctacaagcca?atgagtcaca?aagagttgga 120
catgatcgaa?ttataaagca?caattagtat?actatgtgta?aaataaataa?ccaataagaa 180
tctactgtat?aacacaggga?agtctactcc?gtgctctgtg?atgttctaac?tgggagagaa 240
gtccaaaaaa?gagtatatgt?ggattattca?ctttgttgta?aagcagaaac?tgacacagca 300
gtgtaaaaaa?gctatactgc?cccaccccct?agaaaaaaga?attattgtaa?tacctatttt 360
gtccatggga?aaattaaagt?ttagaaaaga?ttaggaaaag?ttcaaggttg?tgaaaccgtc 420
aggaatagcc?tacaaccaca?aggcgttctg?gtcaccatag?tact 464
<210>11
<211>645
<212>DNA
＜213〉artificial sequence
<400>11
gctagctttc?ataagcagaa?ggaagtaatg?tgtgtgtgtg?catgtttgtg?tgcatgtgtg 60
tgtgcatgca?cgtgtgtgta?tgtgtgatat?tggcagtcaa?ggccccgagg?atgataattt 120
tttttttttt?tttgagacgg?agtctcgctt?tgttgtccag?gctggagtgc?agtggtgcca 180
tctcggctca?ctgcaacctc?cgcctcccag?gttcaagcca?ttctcctgcc?tcagcctccc 240
aagtagctgg?gactacaggt?gcatgccacc?atgcctggct?aattttttgt?atttttagta 300
gaaaattttc?agcttcacct?cttttgaatt?tctgctctcc?tgcctgttct?ttagctatcc 360
gtggtcctga?accagttatg?tgtgttgcat?gagaaaacgc?cagtaagtga?cagagtcacc 420
aaatgctgca?cagaatcctt?ggtgaacagg?cgaccatgct?tttcagctct?ggaagtcgat 480
gaaacatacg?ttcccaaaga?gtttaatgct?gaaacattca?ccttccatgc?agatatatgc 540
acactttctg?agaaggagag?acaaatcaag?aaacaaacgt?gaggagtatt?tcattactgc 600
atgtgtttgt?agtcttgata?gcaagaactg?tcaattcaag?ctagc 645
Claims (10)
1, the construction process of mammary gland specific expression vector comprises the steps:
1) constructed dna fragment M
0M
0Be followed successively by homology arm T to the downstream from the upstream
1, homology arm T
2, homology arm T
3, homology arm T
4, homology arm T
5, homology arm T
6The length of described 6 homology arms is 400-600bp; Homology arm T
5With homology arm T
6Can be by obtaining milk protein gene seat 3 ' control region with milk-protein BAC homologous recombination; Homology arm T
3With homology arm T
4Can be by obtaining complete exogenous object protein genome sequence with exogenous object protein BAC homologous recombination; Homology arm T
1With homology arm T
2Can be by obtaining milk protein gene seat 5 ' control region with milk-protein BAC homologous recombination;
2) with described dna fragmentation M
0Insert in the carrier, obtain continuous three genes and arrest carrier;
3) by carrying out twice homologous recombination, carry out homologous recombination one time with described exogenous object protein BAC, with step 2 with described milk-protein BAC) continuous three genes of obtaining arrest the dna fragmentation M in the carrier
0Replace with dna fragmentation M
1, obtain the mammary gland specific expression vector of described exogenous object protein; M
1Be followed successively by described milk protein gene seat 3 ' control region, complete described exogenous object protein genome sequence, described milk protein gene seat 5 ' control region to the downstream from the upstream.
2, the method for claim 1 is characterized in that: the milk protein gene seat that described milk protein gene seat is ox, sheep, pig, horse, rabbit, people or mouse.
3, method as claimed in claim 2 is characterized in that: described milk protein gene seat is sheep beta-casein gene seat, cattle beta-casein gene seat, mouse WAP locus, rabbit WAP locus, milk cow α s1-casein gene seat, milk cow α s2-casein gene seat, human milk albumin gene seat or goat beta-lactoglobulin locus or bovine beta-lactoglobulin locus.
4, as arbitrary described method in the claim 1 to 3, it is characterized in that: described exogenous object protein is a human serum albumin.
5, a kind of dna fragmentation is followed successively by homology arm T to the downstream from the upstream
1, homology arm T
2, homology arm T
3, homology arm T
4, homology arm T
5, homology arm T
6The length of described 6 homology arms is 400-600bp; Homology arm T
5With homology arm T
6Can be by obtaining milk protein gene seat 3 ' control region with milk-protein BAC homologous recombination; Homology arm T
3With homology arm T
4Can be by obtaining complete exogenous object protein genome sequence with exogenous object protein BAC homologous recombination; Homology arm T
1With homology arm T
2Can be by obtaining milk protein gene seat 5 ' control region with milk-protein BAC homologous recombination.
6, a kind of dna fragmentation is followed successively by milk protein gene seat 5 ' control region, complete exogenous object protein genome sequence, milk protein gene seat 3 ' control region to the downstream from the upstream.
7, as claim 5 or 6 described dna fragmentations, it is characterized in that: the milk protein gene seat that described milk protein gene seat is ox, sheep, pig, horse, rabbit, people or mouse; Be preferably sheep beta-casein gene seat, cattle beta-casein gene seat, mouse WAP locus, rabbit WAP locus, milk cow α s1-casein gene seat, milk cow α s2-casein gene seat, human milk albumin gene seat or goat beta-lactoglobulin locus or bovine beta-lactoglobulin locus.
8, as arbitrary described dna fragmentation in the claim 5 to 7, it is characterized in that: described exogenous object protein is a human serum albumin.
9, carry recombinant vectors, expression cassette, transgenic cell line and the reorganization bacterium of arbitrary described dna fragmentation in the claim 5 to 8.
10, arbitrary described dna fragmentation or the described recombinant vectors of claim 9, expression cassette, transgenic cell line, the application of reorganization bacterium in the preparation galactophore biological reactor in arbitrary described method, the claim 5 to 8 in the claim 1 to 4.
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| CN102492722A (en) * | 2011-12-06 | 2012-06-13 | 广西大学 | Mammary gland-specific expression buffalo prolactin (PRL) gene vector, construction method and application thereof |
| CN102653773A (en) * | 2012-04-26 | 2012-09-05 | 天津农学院 | Bovine mammary gland specific expression vector pBC-alphas1 and preparation method |
| CN103131722A (en) * | 2011-11-25 | 2013-06-05 | 华中农业大学 | Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof |
| CN103409462A (en) * | 2013-06-28 | 2013-11-27 | 曹更生 | Construction method of efficient specific expression vector of mammary gland |
| CN105779496A (en) * | 2016-03-18 | 2016-07-20 | 青岛农业大学 | Method of utilizing goat mammary gland bioreactor to prepare Canine distemper fusion protein gene recombinant vaccine |
| CN115838733A (en) * | 2022-11-04 | 2023-03-24 | 成都碧傲竞生物科技有限公司 | Specific site on rabbit alpha-S1 casein gene seat and application thereof |
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| CN1493693A (en) * | 2002-10-31 | 2004-05-05 | 中国人民解放军军事医学科学院生物工 | Mammal cell expression system and its application |
| CN1659959A (en) * | 2004-02-24 | 2005-08-31 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing and producing tissue type activator of plasminogen from mutant of transgene goat by nuclear transplantation |
| CN1706954A (en) * | 2004-06-10 | 2005-12-14 | 中国人民解放军军事医学科学院生物工程研究所 | Construction process of pig serum albumin locus targeting vector for producing foreign protein |
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| CN103131722A (en) * | 2011-11-25 | 2013-06-05 | 华中农业大学 | Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof |
| CN102492722A (en) * | 2011-12-06 | 2012-06-13 | 广西大学 | Mammary gland-specific expression buffalo prolactin (PRL) gene vector, construction method and application thereof |
| CN102492722B (en) * | 2011-12-06 | 2014-09-10 | 广西大学 | Mammary gland-specific expression buffalo prolactin (PRL) gene vector, construction method and application thereof |
| CN102653773A (en) * | 2012-04-26 | 2012-09-05 | 天津农学院 | Bovine mammary gland specific expression vector pBC-alphas1 and preparation method |
| CN102653773B (en) * | 2012-04-26 | 2015-01-14 | 天津农学院 | Bovine mammary gland specific expression vector pBC-alphas1 and preparation method |
| CN103409462A (en) * | 2013-06-28 | 2013-11-27 | 曹更生 | Construction method of efficient specific expression vector of mammary gland |
| CN103409462B (en) * | 2013-06-28 | 2015-08-26 | 曹更生 | A kind of construction process of efficient specific expression vector of mammary gland |
| CN105779496A (en) * | 2016-03-18 | 2016-07-20 | 青岛农业大学 | Method of utilizing goat mammary gland bioreactor to prepare Canine distemper fusion protein gene recombinant vaccine |
| CN115838733A (en) * | 2022-11-04 | 2023-03-24 | 成都碧傲竞生物科技有限公司 | Specific site on rabbit alpha-S1 casein gene seat and application thereof |
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| Publication number | Publication date |
|---|---|
| CN101265483B (en) | 2010-09-29 |
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