CN100526462C - Bovine genome pseudo-attP site and its use - Google Patents

Abstract

Method for separating and determining bovine genome pseudo attp site and its usage are disclosed. The process is carried out by separating out tow pseudo attp sites from bovine gene, having 38% and 41% homology with attp site of streptomycete phagicin phiC31 genome, discriminating by streptomycete phagicin phiC31 integrase and integrating foreign gene containing attB site with the site of bovine geneome under mediation of integrase. It has higher integration efficiency.

Description

Bovine genome pseudo-attP site and application thereof

Technical field

The invention belongs to bioengineering field, specifically, be about streptomycete phage phi C31 intergrase discernible false attP site in the cow genome group and be used for application in ox cell integrate foreign genes.

Background technology

Transgenic technology has a wide range of applications at bioengineering field.Except (as cationic-liposome) method of (as electric shock) of using multiple physics and chemistry with in the foreign gene transfered cell genome, people also utilize multiple toolenzyme to realize the transfer of gene.Cre recombinase as coliphage P1 plays a role in yeast, Mammals and vegetable cell, mouse embryo stem cell and mouse effectively, and recombinases such as FLP and β also can efficiently be carried out homologous recombination at specific site.

Recently, streptomycete phage phi C31 intergrase (phiC31 integrase) begins to become the strong instrument in the genetic modification research.But the attB site in this enzyme catalysis bacterial genomes and the homologous recombination between the attP site in the phage genome (Kuhstoss S.and Rao RN.J.Mol.Biol.1991,222 (4): 897-908).Wherein, attB is made up of the sequence that is called BOB ', and attP is made up of POP '.O is a core sequence, is attB and attp institute common.And the sequence of its both sides is B, B ' and P, P ', is called as arm.Phage DNA is a cyclic, is integrated into during reorganization in the bacterial chromosome, becomes linear order.Integrating remark forms two new hybrid att sites after taking place, and the left side is called attL, be made up of BOP ', and the right side is attR, is made up of POB '.

Recombinases such as Cre, FLP and β are not only integrated but also shear at same target site, and therefore, the clean integration rate of their mediations is very low.Different with these enzymes is, streptomycete phage phi C31 intergrase is only carried out integrating remark at its target site, and need additional factor just can finish (Thorpe HM., Smith MC.Proc.Natl.Acad.Sci.U.S.A.1998,95 (10): 5505-10) by the cleavage reaction of its mediation; Therefore under the situation that does not have additional factor to exist, its clean integration rate is higher.This makes φ C31 intergrase become candidate's enzyme that an attractive site-specific is integrated.

The att site that streptomycete phage phi C31 intergrase is discerned is relatively simple for structure, only the have an appointment identical sequence (TTG) of 3bp of attB and attP overlap, and flank is two inverted repeats.Pack in the carrier by attP site that length is different and attB site, investigate the efficient of these two site homologous recombination of intergrase catalysis, finally determine attP and the shortest sequence of attB that intergrase discerns and be respectively 39bp and 34bp, be called core sequence.Wherein, the 39bp sequence of attP site core is CCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGG, the 34bp sequence of attB site core is that GTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCG is (referring to Groth A.C., Olivares E.C., Thyagarajan B.andCalos M.P.Proc.Natl.Acad.Sci.U.S.A.97 (2000): 5995-6000).

Except the attP site, but the also integration in some site in catalysis attB site and some genomes of streptomycete phage phi C31 intergrase, the core sequence in the core sequence in these sites and phage attP site has certain homology, have similar function, therefore be called in " false attP site ".Confirmed that now the intergrase of streptomycete phage phi C31 all can mediate site-specific integration (the Groth AC. of foreign DNA effectively in human cell, mouse cell, rat cell and drosophila cell, Olivares EC., Thyagarajan B., Calos MP.Proc Natl Acad Sci U S A 2000,97 (11): 5995-6000; Thyagarajan B., Olivares EC., Hollis RP., Ginsburg DS., Calos MP.Mol Cell Biol 2001,21 (12): 3926-34; Chalberg, T.W., Genise, H.L, Vollrath, D.and Calos, M.P.Invest.Ophthalmol.Vis.Sci., 2005,46,2140-2146; Groth AC., Fish M., Nusse R.and Calos MP.Genetics 2004,166:1775-1782).These integration mainly occur in some focus sites (or being called preferred sites), that is so-called in the genome " false attP site ".

The integration efficiency of dna fragmentation that the catalysis of phage intergrase contains the attB site in these sites be random integration 5-10 doubly.And, there is experiment to show, in the mouse body, after integrase mediated plasma thromboplastin component (FIX) gene of φ C31 was integrated in the locus specificity mode, its expression level was 20 times of (Olivares EC., Hollis RP. with expression level after the transgenosis of lentiviral vectors mediation, Chalberg TW., Meuse L., Kay MA., Calos MP.Nat Biotechnol.20 (2002): 1124-1128).

From the genome of people, mouse, rat and fruit bat, isolated at present a plurality of false attP site.The separation in these sites proves that further phage phi C31 intergrase catalysis foreign gene in these cells integrates in the locus specificity mode.But in the genome of ox, whether exist the discernible false attP of phage phi C31 intergrase site to yet there are no report.

Summary of the invention

The present inventor is contained the plasmid of attB sequence and foreign gene by structure and is contained the plasmid of streptomycete phage phi C31 integrase gene; two kinds of plasmid carriers are mixed by a certain percentage; transfection ox cell; screening positive clone strain then; extract the positive cell genomic dna; behind digestion with restriction enzyme; recovery also connects again; be that template is carried out inverse PCR then to connect back DNA; at last amplified production is carried out sequencing analysis; by sequence alignment, seek cow genome group sequence and carrier attB sequence fusion sequence, identify two false attP sites that obtained the cow genome group.

Therefore, primary and foremost purpose of the present invention just is to provide the discernible false attP of streptomycete phage phi C31 intergrase site in the cow genome group.

Another object of the present invention is to provide described false attP site to be used for application in ox cell integrate foreign genes.

The present invention has the dna sequence dna shown in SEQ ID NO.1 and 2 from the cow genome component respectively from two false attP sites that obtain, and lays respectively in the cow genome group on No. 19 karyomit(e) and No. 28 karyomit(e), and the core sequence of its 39bp is as follows:

CCCGGGGGAGGCTATGGCTTACCTGGGGGCCGTTTGTTG (corresponding SEQ ID NO.1); With

CCCGAAAGATGCTATAAGTTATCTAAGGACCTAGAAGGG (corresponding SEQ ID NO.2).

The core sequence in they and phage attP site has 41% and 38% homology respectively.Experiment confirm, φ C31 intergrase can be discerned their sequence and mediate the plasmid that contains the attB site and integrate in these two sites, and therefore, these two false attP sites have the function that is similar to phage attP site.

Bovine genome pseudo-attP site of the present invention can be used in ox cell integrate foreign genes, and the step of integrate foreign genes comprises:

A, structure contain the plasmid of streptomycete phage phi C31 integrase gene;

B, structure contain the plasmid vector in goal gene and attB site;

C, step a gained plasmid and step b gained plasmid vector are mixed transfection ox cell by a certain percentage;

D, screening positive clone cell strain.

Empirical tests, the integration efficiency of foreign gene in two false attP sites of the present invention that contains the attB site improved more than 2 times than random integration.

Utilize two false attP sites of the present invention, foreign gene can be integrated in the cow genome group with higher integration rate under the mediation of phage phi C31 intergrase.Because these two false attP sites are not positioned at the inside of known, foreign gene can't destroy the normal function of cell in the integration in these sites, does not influence expression of exogenous gene.Therefore the resulting ox cell that carries foreign gene also can be used as the donorcells of transgenic cattle, has promoted the further application of fixed point integration of foreign gene technology in transgenic cattle is produced.

Description of drawings

Fig. 1 is an inverse PCR product electrophorogram in the MDBK cell: swimming lane 1 is cut the inverse PCR amplified production that the back connects product for MDBK cell DNA enzyme; Swimming lane 2 is blank (water); Swimming lane 3 negative contrasts, promptly the MDBK cell DNA enzyme of untransfected is cut back connection product; Swimming lane 4 is 1Kb DNA Marker.

Fig. 2 is that foreign gene is integrated the product electrophorogram at the bovine genome pseudo-attP site place: swimming lane 1 is 100bp DNAMarker; Swimming lane 2 is MDBK resistant cell DNA after the transfection; Swimming lane 3 is a MDBK resistance monoclonal cell DNA of picking after the transfection; Swimming lane 4 is blank (water); Swimming lane 5 negative contrasts, i.e. the MDBK cell DNA of untransfected; Swimming lane 6 positive contrasts promptly contain the pGEM-T carrier of inverse PCR product.

Embodiment

The present invention is a foreign gene with the green fluorescence protein gene (EGFP) that is connected with the attB site sequence; with ox inoblast or ox renal epithelial cell is host cell; carry out the gene integration under streptomycete phage phi C31 is integrase mediated, isolation identification obtains the discernible false attP site sequence of streptomycete phage phi C31 intergrase in the cow genome group.

The present invention is further illustrated below in conjunction with embodiment and accompanying drawing, it is pointed out that present embodiment only is used to explain the present invention, but not limitation of the scope of the invention.

Embodiment 1, contain the structure of the plasmid pCMVInt of streptomycete phage phi C31 integrase gene

According to document (Groth A.C., Olivares E.C., Thyagarajan B.and Calos M.P..Proc.Natl Acad.Sci.USA 97 (2000): 5995-6000) described method, structure contains the plasmid pCMVInt of streptomycete phage phi C31 integrase gene, make up in two steps, specific as follows:

The first step, the structure of plasmid pTA-Int:

(1) be template with streptomycete phage phi C31 (center, Braunschweig, Germany, DSM No.49156 are gathered in German microorganism and cell culture) genomic dna, with PCR method amplification integrase gene.

Primer sequence is:

5 '-GAGTTCTCTCAGTACTAGTCGTAGGGTCGC-3 '; With

5’-CGCCTAACAGGGGATCCGGTGTCTCGCTAC-3’；

PCR reaction system: 1 * PCR buffered soln (TaKaRa company), 1.5mM Mg ²⁺, each 10 μ M of primer, 200 μ M dNTPs, template DNA 100～200ng, 2.5U LATaq archaeal dna polymerase (TaKaRa company).

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 1min, 62 ℃ of 1min, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min.

(2) reclaim test kit (match Parkson, Beijing gene engineering company limited, UltraPure with gel ^TMPCR product purification test kit) the PCR product of the 1.9Kb of purifying gained, operation steps is undertaken by product description.

(3) get purified product 5 μ l, be connected among the plasmid pCR2.1 (invitrogen company), condition of contact is: purified product 5 μ l, pCR2.1 carrier 1 μ l, T ₄Dna ligase (Promega company) 1 μ l, 5 * T ₄Dna ligase damping fluid (Promega company) 2 μ l, distilled water 1 μ l, total reaction volume 10 μ l.4 ℃ are spent the night.

(4) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with BamH I and SpeI enzyme.The endonuclease reaction system is: BamH I, 0.2 μ l; Spe I, 0.2 μ l; 10 * K (TaKaRa company), 2 μ l; Sterilization distilled water 16.6 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

(5) enzyme is cut product and carry out electrophoretic analysis, deposition condition is: 9 μ l enzymes are cut sample-loading buffer (10 * sample-loading buffer: 1%SDS that product and 1 μ l contain tetrabromophenol sulfonphthalein, 50% glycerine, 0.05% tetrabromophenol sulfonphthalein) mix after, the agarose gel electrophoresis by 1% separates.Electrophoretic buffer is that (pH8.3), electrophoresis is about 1 hour under the voltage of about 90V left and right sides for 0.04M Tris-acetate, 0.001M EDTA, takes out gel, and (0.5 μ g/ml) soaked about 15 minutes in the aqueous solution that contains ethidium bromide for TAE.

Under ultraviolet lamp, observe, with the digital gel imaging system photographic analysis of strategene eagle sight (Strategene company).Enzyme is cut and produced 3904bp, 1872bp, 49bp and 6bp band person is required plasmid pTA-Int.

Second step, the structure of plasmid pCMVInt:

(1) digested plasmid pTA-Int: the first step gained plasmid pTA-Int, 25 μ l; BamH I, 4 μ l; Spe I, 4 μ l; 10 * K, 20 μ l; The sterilization distilled water, 147 μ l; Total reaction volume, 200 μ l.37 ℃ are spent the night.

(2) digested plasmid pCMVSPORT β Gal: plasmid pCMVSPORT β Gal (Invitrogen company), 25 μ l; BamH I, 4 μ l; Spe I, 4 μ l; 10 * K, 20 μ l; The sterilization distilled water, 147 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(3) with test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the band that contains the integrase gene sequence from the 1872bp of pTA-Int cutting-out, and the pCMVSPORT β Gal linearizing fragment of 4358bp, add and connect test kit (TaKaRa company, DNAligation Kit Ver.2.1) connect in, 16 ℃ connect 45 minutes.

(4) will connect product transformed into escherichia coli competent cell, the plasmid of amplification is cut with BamH I and Spe I enzyme.The endonuclease reaction system is: BamH I, 0.2 μ l; Spe I, 0.2 μ l; 10 * K, 2 μ l; Sterilization distilled water 16.6 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 4358bp and 1872bp band person is required plasmid pCMVInt.

Embodiment 2, contain the structure of the plasmid vector in attB site

According to document (Groth A.C., Olivares E.C., Thyagarajan B.and Calos M.P..Proc.Natl Acad.Sci.USA 97 (2000): 5995-6000) described method, make up the plasmid pBCPB+ that contains the attB site, detailed process is as follows:

The first step, the structure of plasmid pTA-attB:

(1) with a pair of primer:

5 '-CAGGTACCGTCGACGATGTAGGTCACGGTC-3 '; With

5’-GTCGACATGCCCGCCGTGACCG-3’

With streptomycete (Streptomyces lividans, the center is gathered in Germany microorganism and cell culture, Braunschweig, Germany, DSM No.46482) genomic dna is a template, with the increase sequence of a segment length 293bp of PCR method, contain the attB site of φ C31 intergrase identification in this sequence.

PCR reaction system: 1 * PCR buffered soln (TaKaRa company), 1.5mM Mg ²⁺, each 10 μ M of primer, 200 μ MdNTPs, template DNA 100～400ng, 2U Taq archaeal dna polymerase (TaKaRa company).

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 45s, 64 ℃ of 45s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 10min.

(2) reclaim test kit (match Parkson, Beijing gene engineering company limited, UltraPure with gel ^TMPCR product purification test kit) the PCR product of purifying gained, operation steps is undertaken by product description.

(3) get purified product 5 μ l, be connected among the pCR2.1 (invitrogen company), condition of contact is: comprise purified product 5 μ l in the 10 μ l reaction systems, pCR2.1 carrier 1 μ l, T ₄Dna ligase (Promega company) 1 μ l, 5 * T ₄Dna ligase damping fluid (Promega company) 2 μ l, water 1 μ l, 4 ℃ are spent the night.

(4) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with the SalI enzyme.The endonuclease reaction system is: Sal I, 0.2 μ l; 10 * H, 2 μ l; Sterilization distilled water 16.8 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 3943bp and 279bp band person is required plasmid pTA-attB.

Second step, the structure of plasmid pTA-attP:

Use a pair of primer:

5 '-CGACTAGTACTGACGGACACACCGAA-3 '; With

5’-GTACTAGTCGCGCTCGCGCGACTGACG-3’，

(center is gathered in German microorganism and cell culture with streptomycete phage phi C31, Braunschweig, Germany, DSM No.49156) genomic dna be template, the increase sequence of a segment length 234bp contains the attP site of φ C31 intergrase identification in this sequence.

PCR reaction system: 1 * PCR buffered soln (TaKaRa company), 1.5mM Mg ²⁺, each 10 μ M of primer, 200 μ MdNTPs, template DNA 100～300ng, 2U Taq archaeal dna polymerase (TaKaRa company).

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 45s, 60 ℃ of 45s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 10min.

(2) reclaim test kit (match Parkson, Beijing gene engineering company limited, UltraPure with gel ^TMPCR product purification test kit) purifying gained PCR product, operation steps is undertaken by product description.

(3) get purified product 5 μ l, be connected among the pCR2.1 (invitrogen company), condition of contact is: comprise purified product 5 μ l in the 10 μ l reaction systems, pCR2.1 carrier 1 μ l, T ₄Dna ligase (Promega company) 1 μ l, 5 * T ₄Dna ligase damping fluid (Promega company) 2 μ l, distilled water 1 μ l, 4 ℃ are spent the night.

(4) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with Spe I enzyme.The endonuclease reaction system is as follows: Spe I, 0.2 μ l; 10 * H, 2 μ l; Sterilization distilled water 16.8 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 3901bp, 224bp and 38bp band person is required plasmid pTA-attP.

The 3rd step, the structure of plasmid pBC β Gal:

(1) digested plasmid pBCSK+:pBCSK+ (Stratagene company), 25 μ l; Pvu I, 4 μ l; Kpn I, 4 μ l; 10 * K, 10 μ l; The sterilization distilled water, 157 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(2) after enzyme is cut, mend the protruding terminus of flat carrier DNA: cut product with dehydrated alcohol precipitation enzyme, 70% ethanol is washed once, treat the DNA drying after, carry out filling-in.In dried protruding terminus DNA, add: sterilization distilled water, 34.5 μ l; 10 * T4DNA polymerase buffer (TaKaRa), 5 μ l; 0.1%BSA, 5 μ l; 2.5mM dNTPs, 3.5 μ l; T4DNA polysaccharase (TaKaRa), 2 μ l; Total reaction volume 50 μ l.Behind 37 ℃ of 5min, thermal agitation, and put into ice at once.

(3) according to the method for (5) in embodiment 1 the first step benefit thing of showing no increases in output is carried out electrophoresis, reclaim test kit (match Parkson, Beijing gene engineering company limited, UltraPure with gel ^TMPCR product purification test kit) flat 3244bp fragment is cut and mended to purifying enzyme.

(4) carrier is from connecting: condition of contact is: comprise the purified product 5 μ l of previous step in the 10 μ l reaction systems, T ₄Dna ligase (Promega company) 1 μ l, 5 * T ₄Dna ligase damping fluid (Promega company) 2 μ l, water 2 μ l, 4 ℃ are spent the night.

(5) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with BssH II enzyme.The endonuclease reaction system is: BssHII, 0.2 μ l; 10 * M, 2 μ l; Sterilization distilled water 16.8 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme cut only produce 3250bp band person for required from connection carrier.

(6) enzyme is cut from connection carrier: go up the step amplification from connection carrier, 25 μ l; Spe I, 4 μ l; HindIII, 4 μ l; 10 * M, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(7) digested plasmid pCMVSPORT β Gal: plasmid pCMVSPORT β Gal (invitrogen company), 25 μ l; Spe I, 4 μ l; HindIII, 4 μ l; 10 * M, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(8) after enzyme is cut, with test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the band that contains the LacZ gene from the 3514bp of pCMVSPORT β Gal cutting-out, and from connection carrier linearizing fragment (3214bp), add in the connection test kit (TaKaRa company, DNA ligation Kit Ver.2.1), 16 ℃ connect 45 minutes.

(9) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with Spe I and HindIII enzyme.The endonuclease reaction system is: Spe I, 0.2 μ l; HindIII, 0.2 μ l; 10 * M, 2 μ l; Sterilization distilled water 16.6 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut and is only produced 3514bp and 3214bp band person is required plasmid pBC β Gal.

The 4th step, the structure of plasmid pBC β Gal-attP:

With Spe I the attP sequence is excised from pTA-attP, connect into the Spe I site of pBC β Gal, make up and form plasmid pBC β Gal-attP.

(1) enzyme is cut pTA-attP: the second step gained plasmid pTA-attP, 25 μ l; Spe I, 4 μ l; 10 * M, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume, 200 μ l.37 ℃ are spent the night.

(2) enzyme is cut pBC β Gal: the 3rd step gained plasmid pBC β Gal, 25 μ l; Spe I, 4 μ l; 10 * M, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(3) the segmental dephosphorylation reaction of plasmid pBC β Gal linearizing: 2 times of volume dehydrated alcohol precipitation pBC β Gal enzymes are cut product, 70% ethanol is washed once, 43 μ l sterilization distilled water dissolving linearized vector, add 5 μ l10 * alkaline phosphatase damping fluid (TaKaRa company), 2 μ l alkaline phosphatases (TaKaRa company), 50 ℃ were reacted 30 minutes.65 ℃ subsequently, 30 minutes deactivation alkaline phosphatases.

(4) after enzyme is cut, with test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the band that contains the attP sequence from the 224bp of plasmid pTA-attP cutting-out, and the plasmid pBC β Gal linearizing fragment (6.7Kb) behind the dephosphorylation, add and connect test kit (TaKaRa company, DNA ligation Kit Ver.2.1) in, 16 ℃ connect 45 minutes.

(5) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with Spe I enzyme.The endonuclease reaction system is: Spe I, 0.2 μ l; 10 * M (TaKaRa company), 2 μ l; Sterilization distilled water 16.8 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 6728bp and 224bp band person is required plasmid pBC β Gal-attP.

The 5th step, the structure of plasmid pBCPB+:

With Sal I attB is excised from pTA-attB, connect into the Sal I site of pBC β Gal-attP, obtain plasmid pBCPB+ as shown in Figure 2.

(1) digested plasmid pTA-attB: the first step gained pTA-attB, 25 μ l; Sal I, 4 μ l; 10 * H, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume, 200 μ l.37 ℃ are spent the night.

(2) digested plasmid pBC β Gal-attP: the 4th step gained pBC β Gal-attP, 25 μ l; Sal I, 4 μ l; 10 * H, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(3) the segmental dephosphorylation reaction of plasmid pBC β Gal-attP linearizing: the enzyme of 2 times of volume dehydrated alcohol precipitation pBC β Ga1-attP is cut product, 70% ethanol is washed once, 43 μ l sterilization distilled water dissolving linearized vector, add 5 μ l10 * alkaline phosphatase damping fluid (TaKaRa company), 2 μ l alkaline phosphatases (TaKaRa company), 50 ℃ were reacted 30 minutes.65 ℃ subsequently, 30 minutes deactivation alkaline phosphatases.

(4) after enzyme is cut, with test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the band that contains the attB sequence from the 279bp of plasmid pTA-attB cutting-out, and the pBC β Gal-attP linearizing fragment (6.9Kb) behind the dephosphorylation, add and connect test kit (TaKaRa company, DNA ligation Kit Ver.2.1) in, 16 ℃ connect 45 minutes.

(5) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with Sal I enzyme.The endonuclease reaction system is: Sal I 0.2 μ l; 10 * H (TaKaRa company), 2 μ l; Sterilization distilled water 16.8 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 6952bp and 279bp band person is required plasmid pBCPB+.

Embodiment 3, contain the structure of the plasmid in goal gene (EGFP) and attB site

In the present embodiment, except that indicating especially, all restriction enzymes are all available from TaKaRa company.

The structure of plasmid pEGFP-N1-attB divided for three steps:

The first step: the structure of plasmid pSL301-attB

(1) digested plasmid pBCPB: embodiment 2 gained pBCPB, 25 μ l; Xho I, 4 μ l; 10 * H, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume, 200 μ l.37 ℃ are spent the night.

(2) digested plasmid pSL301:pSL301 (invitrogen company), 25 μ l; Xho I, 4 μ l; 10 * H, 20 μ l; The sterilization distilled water, 151 μ l; Total reaction volume 200 μ l.37 ℃ are spent the night.

(3) the segmental dephosphorylation reaction of plasmid pSL301 linearizing: the enzyme of 2 times of volume dehydrated alcohol precipitation pSL301 is cut product, 70% ethanol is washed once, 43 μ l sterilization distilled water dissolving linearized vector, add 5 μ l10 * alkaline phosphatase damping fluid (TaKaRa company), 2 μ l alkaline phosphatases (TaKaRa company), 50 ℃ were reacted 30 minutes.65 ℃ subsequently, 30 minutes deactivation alkaline phosphatases.

(4) after enzyme is cut, with test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the band that contains the attB sequence from the 313bp of plasmid pBCPB cutting-out, and the pSL301 linearizing fragment (3.2Kb) behind the dephosphorylation, add and connect test kit (TaKaRa company, DNA ligation Kit Ver.2.1), 16 ℃ connect 45 minutes.

(5) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with the HindIII enzyme.The endonuclease reaction system is as follows: HindIII, 0.2 μ l; 10 * M, 2 μ l; Sterilization distilled water 16.8 μ l, plasmid 1 μ l; Cumulative volume, 20 μ l.37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 3238bp and 338bp band person is required plasmid pSL301-attB.

Second step: the structure of plasmid pcDNA3.1-zeo (+)-attB

(1) digested plasmid pcDNA3.1-zeo (+): pcDNA3.1-zeo (+) (invitrogen company), 20 μ l; HindIII, 1.5 μ l; 10 * M, 15 μ l; The sterilization distilled water, 113.5 μ l; Total reaction volume, 150 μ l.37 ℃ are spent the night.

(2) digested plasmid pSL301-attB:pSL301-attB, 20 μ l; HindIII, 1.5 μ l; 10 * M, 15 μ l; The sterilization distilled water, 113.5 μ l; Total reaction volume; 150 μ l.37 ℃ are spent the night.

(3) the segmental dephosphorylation reaction of plasmid pcDNA3.1-zeo (+) linearizing: the enzyme of 2 times of volume dehydrated alcohol precipitation pcDNA3.1-zeo (+) is cut product, 70% ethanol is washed once, 43 μ l sterilization distilled water dissolving linearized vector, add 5 μ l10 * alkaline phosphatase damping fluid (TaKaRa company), 2 μ l alkaline phosphatases (TaKaRa company), 50 ℃ were reacted 30 minutes.65 ℃ subsequently, 30 minutes deactivation alkaline phosphatases.

(4) after enzyme is cut, with test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the band that contains the attB sequence from the 338bp of pSL301-attB cutting-out, and pcDNA3.1-zeo (+) the linearizing fragment (5Kb) behind the dephosphorylation, add and connect test kit (TaKaRa company, DNA ligation KitVer.2.1) in, 16 ℃ connect 45 minutes.

(5) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with Pme I enzyme.The endonuclease reaction system is: Pme I (NEB company), 0.2 μ l; 10 * NEB4,2 μ l; 100 * BSA, 0.2 μ l; The sterilization distilled water, 16.6 μ l; Plasmid, 1 μ l; Cumulative volume, 20 μ l.37 ℃, 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 4913bp and 440bp band person is required plasmid pcDNA3.1-zeo (+)-attB.

The 3rd step: the structure of pEGFP-Nl-attB

(1) digested plasmid pcDNA3.1-zeo (+)-attB:pcDNA3.1-zeo (+)-attB, 20 μ l; AflII, 3.5 μ l; 10 * M, 15 μ l; 10 * BSA, 15 μ l; The sterilization distilled water, 96.5 μ l; Total reaction volume, 150 μ l.37 ℃ are spent the night.

(2) digested plasmid pEGFP-N1:pEGFP-N1 (invitrogen company), 20 μ l; Afl II, 3.5 μ l; 10 * M, 15 μ l; 10 * BSA, 15 μ l; The sterilization distilled water, 96.5 μ l; Total reaction volume, 150 μ l.37 ℃ are spent the night.

(3) the segmental dephosphorylation reaction of plasmid pEGFP-N1 linearizing: the enzyme of 2 times of volume dehydrated alcohol precipitation pEGFP-N1 is cut product, 70% ethanol is washed once, 43 μ l sterilization distilled water dissolving linearized vector, add 5 μ l, 10 * alkaline phosphatase damping fluid (TaKaRa company), 2 μ l alkaline phosphatases (TaKaRa company), 50 ℃ were reacted 30 minutes.65 ℃ subsequently, 30 minutes deactivation alkaline phosphatases.

(4) after enzyme is cut, reclaim test kit (match Parkson, Beijing gene engineering company limited, UltraPure with gel ^TMPCR product purification test kit) recovery contains the band of attB sequence from the 338bp of pcDNA3.1-zeo (+)-attB cutting-out, and the p EGFP-N1 linearizing fragment (4.8Kb) behind the dephosphorylation, and operation steps is undertaken by product description.

Add in the connection test kit (TaKaRa DNA ligation Kit Ver.2.1), 16 ℃ of connections are spent the night.

(5) will connect product transformed into escherichia coli competent cell to increase, the plasmid of amplification is cut with Xho I enzyme.The endonuclease reaction system is as follows: Xho I, 0.2 μ l; 10 * H, 2 μ l; 10 * BSA, 2 μ l; The sterilization distilled water, 14.8 μ l; Plasmid, 1 μ l; Cumulative volume, 20 μ l; 37 ℃ of enzymes were cut 2.5 hours.

According to the method for (5) in embodiment 1 the first step enzyme is cut product and carry out electrophoretic analysis.Enzyme is cut generation 4010bp and 1061bp band person is required plasmid pEGFP-N1-attB.

Embodiment 4, be integrated with the structure of the ox renal epithelial cell of goal gene

Test used ox renal epithelial cell strain (MDBK cell), available from Chinese Academy of Sciences's Shanghai cell bank.

Cell is cultivated in the RPMI-1640 (Gibco BRL company) of the Streptomycin sulphate (Gibco BRL company) of penicillin that contains 10% calf serum (Hyclone company) and 50U/ml and 50mg/ml nutrient solution, carries out the integration of foreign gene, comprises the steps:

(a) transfection: with embodiment 1 gained plasmid pCMVInt and embodiment 3 gained plasmid pEGFP-N1-attB is the mixed (1600ng:40ng) of 40:1 with the mass ratio, according to the process specifications transfection MDBK cell of liposome Lipofectamine 2000 test kits (invitrogen company).

(b) screening: transfection second day, passage, added screening of medicaments G418 (Promega company) in back second day in going down to posterity, making its concentration is 300 μ g/ml, changed nutrient solution (containing penicillin, the 50mg/ml Streptomycin sulphate of 10% calf serum, 50U/ml, the RPMI-1640 nutrient solution of 300 μ g/mlG418) in per 3 days once, and the death condition of observation of cell.Screened 14 days, all dead to negative cells, the positive colony in the transfectional cell grows gradually.

(c) amplification of G418 resistant cell: after treating that positive colony in the transfectional cell covers with gradually, it is gone down to posterity, increase according to required cell concentration.Keep screening with the nutrient solution that contains G418 (containing penicillin, the 50mg/ml Streptomycin sulphate of 10% calf serum, 50U/ml, the RPMI-1640 nutrient solution of 150 μ g/mlG418) during amplification.

Embodiment 5, bovine genome pseudo-attP site the clone

(1) extraction of MDBK cell genomic dna: get embodiment 4 gained G418 resistant cells, with the centrifugal force of 800g centrifugal 10 minutes, with cell pyrolysis liquid (100 μ g/ml Proteinase Ks, 10mM Tris-HCl, 15mM NaCl, 10mM EDTA, 0.4%SDS) resuspended, 37 ℃ are spent the night.

Respectively with Tris balance phenol, Tris balance phenol: chloroform: primary isoamyl alcohol (25:24:1), chloroform: each extracting of primary isoamyl alcohol (24:1) once, dehydrated alcohol precipitation, 70% washing with alcohol once is dissolved in TE (pH8.0) after the drying.

(2) preparation of inverse PCR template: get 10 μ g MDBK cell genomic dnas, cut with BamH I and 100U Bgl II (TaKaRa company) enzyme of 100U.The endonuclease reaction system is: BamH I, 10 μ l; Bgl II, 10 μ l; 10 * K (TaKaRa company), 20 μ l; Sterilization distilled water 150 μ l, MDBK cell genomic dna 10 μ l; Cumulative volume, 200 μ l.37 ℃ are spent the night.

Ethanol precipitation reclaims enzyme and cuts product, carries out from connecting after the TE dissolving.The ligation system: (the T4 dna ligase of New England Biolabs company attaches damping fluid to 200 μ l 1 * connection damping fluid, and 1 * connection damping fluid composition is: 50mMTris-HCl (pH7.5), 10mM MgCl ₂, 10mM dithiothreitol (DTT), 1mM ATP, 25 μ g/ml bovine serum albumins), 1000U T4 dna ligase (New England Biolabs company).16 ℃ are spent the night.

The connection product promptly can be used as template DNA and is used for inverse PCR after the phenol extracting.

(3) inverse PCR: design a pair of inverse PCR primer in the attB of plasmid vector sequence both sides:

The upstream primer sequence is: 5 '-TGGATTGCACGCAGGTTCTC-3 ';

The downstream primer sequence is: 5 '-CAACACTCAACC CTATCTCG-3 '.

PCR reaction system: 1 * PCR buffered soln, 1.5mM Mg ²⁺, each 10 μ M of primer, 200 μ M dNTPs, template DNA 200ng～500ng, 2.5U LA Taq archaeal dna polymerase (TaKaRa company).

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 1min, 59 ℃ of 1min, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min.

(4) carry out electrophoretic analysis according to the method for (5) in embodiment 1 the first step, electrophoresis result as shown in Figure 1.The result of Fig. 1 shows: inverse PCR (swimming lane 1) amplification that is connected product after the MDBK resistant cell DNA enzyme of transfection pCMVInt and pEGFP-N1-attB is cut obtains the band of a treaty 4.6Kb; this band contains the known carrier sequence of the 3.1Kb that has an appointment; all the other are the cow genome group unknown nucleotide sequence at vector integration position, promptly false attP site sequence.

And in swimming lane 2 blanks (water) and swimming lane 3 negative controls (the MDBK cell DNA enzyme of untransfected is connected product after cutting), all do not have any band amplification.

Embodiment 6, false attP site integrates the mensuration and the analysis of flanking sequence

Reclaim test kit (match Parkson, Beijing gene engineering company limited, UltraPure with gel ^TMPCR product purification test kit) purifying embodiment 5 gained PCR products, operation steps is undertaken by product description.

Get purified product 5 μ l, be connected to pGEM-T carrier (Promega company), condition of contact is: purified product 5 μ l, pGEM-T carrier 1 μ l, T ₄Dna ligase (Promega company) 1 μ l, 5 * T ₄Dna ligase damping fluid (Promega company) 2 μ l, water 1 μ l, total reaction volume 10 μ l.4 ℃ are spent the night.

To connect product transformed into escherichia coli competent cell increasing, and the plasmid of amplification be carried out electrophoretic analysis according to the method for (5) in embodiment 1 the first step.

Choosing the clone who contains big fragment plasmid after the electrophoresis detection checks order.

With BLASTN systematic search (http://www.ncbi.nlm.nih.gov/genome/seq/BtaBlast.html); with sequencing result and cow genome group sequence alignment; the fusion sequence that contains cow genome group and carrier attB sequence is the attR sequence of integration site, and cow genome group sequence herein is the left arm (left arm) in false attP site.

Embodiment 7, false attP site right arm (right arm) amplification

Sequence according to embodiment 6 resulting false attP site left arms, design a upstream primer:

5’-CCGAACCTGCCCTCAT-3’，

Again according to primer of false attP site downstream sequence design of announcing among the GENEBANK:

5’-TGTAGTCCACCAGTCTCCTCA-3’，

Increase.

PCR reaction system: 1 * PCR buffered soln, 1.5mM Mg ²⁺, each 10 μ M of primer, 200 μ M dNTPs, template DNA 200ng～500ng, 2U Taq archaeal dna polymerase (TaKaRa company).

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 45s, 60 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min.

With test kit (match Parkson, Beijing gene engineering company limited, UltraPure ^TMPCR product purification test kit) reclaims the PCR product.

The PCR product is 990bp, is connected with the pGEM-T carrier, and order-checking.Order-checking obtains the partial sequence of both sides, false attP site.

Resulting sequence in the present embodiment and embodiment 6 resulting sequences are spliced, obtained the sequence SEQ ID NO.1 in a false attP site in the cow genome group.

This vacation attP site is positioned on No. 19 karyomit(e), and 1929bp is long altogether, and wherein the 39bp sequence of core is 1542-1580bp, promptly CCCGGGGGA GGC TATGGC TTACC TGGGGG CC GTTT GTT GWherein have the base of underscore identical with the base of phage attP site core 39bp, there is 41% homology in this site sequence and phage attP core site.

Embodiment 8, the detection integrated at the bovine genome pseudo-attP site place of foreign gene:

1, pcr amplification:, design a pair of primer according to " false attP site " in the cow genome group and the attB sequence of carrier:

The upstream primer sequence is: 5 '-CGCTGCTTCGCTTTCTG-3 ';

The downstream primer sequence is: 5 '-ACCGTCGAGAACCCGCTGAC-3 '.

PCR reaction system: 1 * PCR buffered soln, 1.5mM Mg ²⁺, each 10 μ M of primer, 200 μ M dNTPs, template DNA (sequence after the attB sequence of " false attP site " and carrier is integrated) 200ng～500ng, 2U Taq archaeal dna polymerase (Takara company).

PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 45s, 56 ℃ of 45s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 10min.

Above-mentioned a pair of primer lays respectively at the left arm in false attP site and the right arm in attB site, when integrating generation, the left arm in false attP site and the right arm in attB site promptly are connected to form attR, therefore can detect by PCR, and, can learn the size that back PCR product takes place to integrate according to the primer position.According to Theoretical Calculation, PCR product size should be 436bp.

2. carry out electrophoretic analysis according to the method for (5) in embodiment 1 the first step, the result as shown in Figure 2, the electrophoresis result of Fig. 2 shows: it be 436bp that back PCR product size takes place to integrate, big or small consistent with theory.

Therefore in these two cell masses, foreign gene is in the integrase mediated false attP site that all is integrated in down in the strain of ox renal epithelial cell.

Embodiment 9, integrase mediated exogenous origin gene integrator efficient investigates

Press two groups of MDBK cells of embodiment 4 described method transfections.First group is: pCMVInt and pEGFP-N1-attB; Second group is: pcDNA3.1-zeo (plasmid of intergrase is not expressed by invitrogen company) and pEGFP-N1-attB.

Last plasmid is 40:1 with the mass ratio of back one plasmid in two groups of transfections.

When transfection was gone down to posterity in second day, get half cell detection transfection efficiency, second half passage carries out cell counting before going down to posterity, write down the inoculating cell number of each transfection.By the fluorescent microscope microscopy, the cell of counting expressing green fluorescent protein in a certain area of visual field accounts for the per-cent of this visual field total cellular score, is transfection efficiency.Count three area of visual field, average.

After drug screening in 14 days, count the resistant cell clone number in each transfection, calculate the integration efficiency of each transfection.Integration efficiency=resistant cell clone number/(transfection efficiency * inoculating cell number).The integration rate of first group of transfection is that the integration rate of 5.47 ± 0.62, second group of transfection is 2.57 ± 0.50.Above integration rate is the mean value of three experiments.

The result shows that under the situation that intergrase exists, integration rate has improved 2.1 times.

Embodiment 10, be integrated with the fibroblastic structure of ox of goal gene and the detection in false attP site

Similar with embodiment 4 to 9 described methods, be host cell with the ox inoblast, construct the ox inoblast that is integrated with goal gene, by detecting, found another false attP site.

Second false attP site sequence is shown in SEQ ID NO.2, and 3496bp is long altogether, and wherein the 39bp sequence of core is 1713-1751 bp's CCCG AAAGAT GC TATAAG TTATC TAAGGA CCTAGAA GGGWherein there is the base of underscore identical, 38% homology arranged with phage attP core site with the base of phage attP site core 39bp.Therefore, its this site also is the false attP site in the cow genome group.

This site is positioned on No. 28 karyomit(e).

The present invention has found two false attP site sequences in the cow genome group; these two sites can be discerned by streptomycete phage phi C31 intergrase; therefore can make the foreign gene that contains the attB site under the mediation of intergrase, be incorporated into this site in the cow genome group, thereby obtain carrying the ox cell of foreign gene with higher integration rate.

Because these two false attP sites are not positioned at the inside of known, foreign gene can't destroy the normal function of cell in the integration in these sites, does not influence expression of exogenous gene.Therefore the gained positive cell can be used for the production of transgenic cattle, and can not influence the growth of transgenosis blastaea.

Sequence table

＜110〉The Children's Hospital Attached to Shanghai Jiaotong Univ.

＜120〉bovine genome pseudo-attP site and application thereof

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<160>2

<170>PatentIn?version?3.3

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<213>Bos?taurus

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<221>gene

<222>(1)..(1929)

<400>1

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<211>3496

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Claims (7)

1, the dna sequence dna of being discerned by phage phi C31 intergrase in a kind of isolating cow genome group is characterized in that the 39bp sequence of its core is:

CCCGGGGGAGGCTATGGCTTACCTGGGGGCCGTTTGTTG。

2, the application of the dna sequence dna of being discerned by phage phi C31 intergrase in the described cow genome group of claim 1 is characterized in that, is used in ox cell integrate foreign genes.

3, application as claimed in claim 2 is characterized in that, described in the ox cell step of integrate foreign genes comprise:

A, structure contain the plasmid of streptomycete phage phi C31 integrase gene;

B, structure contain the plasmid vector in goal gene and attB site;

C, step a gained plasmid and step b gained plasmid vector are mixed back transfection ox cell by a certain percentage;

D, screening positive clone cell strain.

4, application as claimed in claim 3 is characterized in that, the plasmid that contains streptomycete phage phi C31 integrase gene among the described step c is 40:1 with the plasmid blended mass ratio that contains goal gene and attB site.

5, application as claimed in claim 3 is characterized in that, cell transfecting adopts the liposome transfection method among the described step c.

As claim 2 or 3 described application, it is characterized in that 6, described ox cell is ox renal epithelial cell or ox inoblast.

7, the separation method of the dna sequence dna of being discerned by phage phi C31 intergrase in the described cow genome group of claim 1 is characterized in that, may further comprise the steps:

A, structure contain the plasmid of attB sequence and foreign gene;

B, structure contain the plasmid of streptomycete phage phi C31 integrase gene;

C, two kinds of plasmid carriers that step a and b are obtained mix transfection ox cell by a certain percentage;

D, screening positive clone strain;

E, extract the positive cell genomic dna, behind digestion with restriction enzyme, reclaim and connect again;

F, be that template is carried out inverse PCR to connect back DNA, the amplified production sequencing analysis by sequence alignment, is sought cow genome group sequence and carrier attB sequence fusion sequence, identifies to obtain in the cow genome group by the dna sequence dna of phage phi C31 intergrase identification.

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