CN100410369C - Fi-C31 integrase modified TAT and NLS polypeptide and its use - Google Patents
Fi-C31 integrase modified TAT and NLS polypeptide and its use Download PDFInfo
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- CN100410369C CN100410369C CNB200610028021XA CN200610028021A CN100410369C CN 100410369 C CN100410369 C CN 100410369C CN B200610028021X A CNB200610028021X A CN B200610028021XA CN 200610028021 A CN200610028021 A CN 200610028021A CN 100410369 C CN100410369 C CN 100410369C
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Abstract
PHIC31 integrase protein with TAT and NLS polypeptide modification and its use are disclosed. It consists of PHIC31 integrase protein TAT-PHIC31 and PHIC31 integrase protein TAT-PHIC31-NLS. It's safe and efficient.
Description
Technical field
The invention belongs to field of transgenic technology, be specifically related to a kind of new modified Phi C 31 integrase and the application in transgenosis thereof.
Background technology
Utilize gene transfer technique effectively with the goal gene locus specificity be incorporated in the genome of mammalian cell, thereby avoid foreign gene to insert genome at random, this is the unsolved major issue in gene therapy and the Study on Transgenic Animal.Use retroviral vector in recent years, the clinical treatment of serious severe complex immunity defective disease (SCID-XI) has been obtained success.There is the patient who receives treatment that the leukemia canceration takes place subsequently, mainly be because the gene transfer technique of virus vector causes foreign gene to insert in the cell chromosome at random, cause the result (S.Hacein-Bey-Abina of transgenation, et al.A serious adverse event after successful gene therapy for X-linked severe combinedimmunodeficiency.N.Engl.J.Med.2003,346,1185-1193).In order to increase the security of transgenosis, foreign gene preferably can specific site-directed integration in the cellular genome site of safety.All there are many deficiencies in the method for several genetic manipulations that exist aspect the security of transgenosis approach at present.Virus vector such as adenovirus, adeno-associated virus, retrovirus and lentiviral vectors can cause inserting sudden change by random integration in host genome; Non-virus carrier such as naked DNA carrier also have random integration phenomenon (J.Kimmelman.Recent developments in gene transfer:riskand ethics.B.M.J.2005,330,79-82).Non-virus carrier is considered to relatively safe as the naked DNA plasmid vector, preparation and use simple gene transfer method, but also have potential carrier DNA random integration to cause the possibility of transgenation, and vector gene long-term expression and cause the toxicity of pair cell to cell chromosome; In addition, the transgenosis of plasmid DNA is subjected to the influence of different cell types, has only very low gene transfering efficiency in some cell.
In mammalian cell, streptomycete phage Phi C 31 integrase is proved to be the intergrase that can react with specific sequence in the genomic dna, it can stably be incorporated into foreign gene on genomic those the specific sequence sites of mammalian cell, thereby greatly reduces because foreign gene inserts the transgenation risk that causes at random.The cytogene group-specific site of found Phi C 31 integrase identification has more than 100, bioinformatic analysis to integration site shows that they have security (T.W.Chalberg preferably, et al.Integration specificity of phage phiC31integrase in the human genome.J Mol Biol.2006,357,28-48), point out it as a useful instrument that in mammalian cell, carries out genetic manipulation and gene therapy.Have in the cytologic experiment of report with the Phi C 31 integrase of naked DNA plasmid vector and find, merge the nuclear localization signal (NLS of one section SV40 virus large T antigen, aminoacid sequence is PKKKRKV, be designated as SEQ.NO4), can be good at improving the integration function (S.Andress of Phi C 31 integrase in mammalian cell, et al.Enhanced efficiency through nuclear localization signal fusion on phage Φ C31-integrase:activity comparison with Cre and FLPe recombinase in mammalian cells.NucleicAcids Res.2002,30,2299-2306), thereby, in order to use Phi C 31 integrase better in mammalian cell, it is modified is necessary.
The polypeptide that a kind of utilization has the protein transduction function has newly appearred in recent years, polypeptide (TAT as the proteic 47-57 amino acids composition of HIV-1 virus Tat, aminoacid sequence is YGRKKRRQRRR, be designated as SEQ.NO5), the VP22 albumen of HSV virus etc., form the protein product that merges with goal gene, directly shift the protein transduction technology that enters cell.This protein transduction technology is because shift an instantaneous existence, enter degradable activated protein form in the cell, thereby greatly reduce the unfavorable aspect of other transgenosis approach, the toxicity problem that insertion sudden change that causes as random integration and long-term expression cause, thereby have better security.The protein transduction that existing report proof utilizes TAT to merge, be subjected to the restriction of cell type hardly, can enter all mammalian cells and tissue in the mode of density dependency, even can be by the hemato encephalic barrier (A.Ho of mouse, et al.Synthetic protein transduction domains:enhanced transduction petentialin vitro and in vivo.Cancer Res.2001,61,474-477).So protein transduction is a kind of new novel method that can be used for genetic manipulation and gene therapy in the mammalian cell.
Summary of the invention
The object of the present invention is to provide a kind of through peptide modified Phi C 31 integrase albumen of TAT and NLS and preparation method thereof and the application in cell.
One aspect of the present invention provides a kind of Phi C 31 integrase albumen that has expression vector and utilize the preparation of Ni ion affinity column chromatography, and its aminoacid sequence is SEQ.NO1.
The present invention designs on the other hand and has prepared the Phi C 31 integrase fusion rotein of modifying through TAT, and its aminoacid sequence is SEQ.NO2.
Further aspect of the present invention is design and has prepared through TAT and the common Phi C 31 integrase fusion rotein of modifying of NLS that its aminoacid sequence is SEQ.NO3.
The present invention is with above-mentioned three kinds of albumen and the eukaryotic cell lines 293-PB[EGFP that detects the Phi C 31 integrase function] acting in conjunction respectively, constitute the protein transduction function and the active system of intergrase that detect the Phi C 31 integrase of modifying.
The present invention also provides use cellular immunization histochemical method, the Phi C 31 integrase of modifying is carried out the analytical system of Subcellular Localization.
The albumen that the invention allows for the Phi C 31 integrase that uses TAT and NLS modification is as the new way of using Phi C 31 integrase in mammalian cell, because TAT merges Phi C 31 integrase albumen it can be entered in all mammalian cells of hatching jointly, thereby improved the transfer efficiency of goal gene greatly.The albumen form of this instantaneous existence and the mode of action of its distinctive density dependency have improved the security of its application in mammalian cell.Utilize NLS to merge Phi C 31 integrase albumen and promote more fusion rotein in nucleus, to distribute, improved the function of its site-directed integration in cellular genome.The present invention has improved the deficiency of using other transgenosis approach of Phi C 31 integrase, is the new instrument that is applied to genetic manipulation and field of gene.
The preparation of the fusion rotein that the present invention proposes and the method for application specifically describe as follows:
1. the proteic preparation of Phi C 31 integrase.
Utilize the PCR reaction to increase out the Phi C 31 integrase gene, with pCMV-Φ C31 plasmid is template, in the primer of upstream and downstream, add the recognition sequence of restriction enzyme Nhe I and Xho I respectively, the PCR product is through after these two enzymic digestions, recovery is connected to same process enzyme and cuts on the postdigestive prokaryotic expression carrier pET28b (+), obtains pET28b-Φ C31 recombinant plasmid; After the analysis of recombinant plasmid process dna sequencing is correct, be transformed in e. coli bl21 (DE3) bacterial strain, select 5-8 male colony clone in the LB of kalamycin resistance substratum, with the pre-abduction delivering Phi C 31 integrase of IPTG albumen; Whole bacterial protein SDS-PAGE after the expression, Coomassie brilliant blue R250 dyes, and determines the condition and the positive colony clone of abduction delivering, then at 16-22 ℃, great expression albumen under the 0.2mM IPTG condition;
The proteic bacterium of abduction delivering is through the ultrasonication thalline, after discharging target protein, affinity chromatography column purification through the Ni ion chelating, albumen behind the purifying is 2mg/ml through ultrafiltration and concentration, leave in-70 ℃ the refrigerator, through experiment of the protein immunization marking and protein N terminal order-checking proof, its aminoacid sequence is shown in SEQ.NO1.
2.TAT-the preparation of Φ C31 fusion rotein.
The nucleotide sequence of TAT and Phi C 31 integrase gene are spliced together in the PCR reaction, be cloned on the prokaryotic expression carrier pET28b (+) as above-mentioned 1 said same method then, after order-checking is identified correctly, be transformed in BL21 (DE3) bacterial strain, carry out a large amount of abduction delivering purifying with above-mentioned 1 said same steps as, finally obtain TAT-Φ C31 fusion rotein, its aminoacid sequence is shown in SEQ.NO2.
3.TAT-the preparation of Φ C31-NLS fusion rotein.
The sequence of the nucleotide sequence of NLS and TAT-Φ C31 is spliced together in the PCR reaction, and according to above-mentioned 1 said same method, the clone expresses, and purifying concentrates and prepares TAT-Φ C31-NLS albumen, and its aminoacid sequence is shown in SEQ.NO3.
Three kinds of Φ C31 of all above-mentioned preparations albumen can further confirm (Fig. 1) through the protein immunization marking experiment of the antibody of Φ C31.
4. different Φ C31 albumen respectively with Phi C 31 integrase Function detection clone 293-PB[EGFP] hatch jointly, detect their the protein transduction function and the activity of intergrase.
The detection clone 293-PB[EGFP that derives from the human embryos nephrocyte] cell inoculation is on 12 porocyte culture plates, when cell density reaches 50-70%, in different holes, add respectively through the filtering Φ C31 of 0.2 μ M filter, TAT-Φ C31, TAT-Φ C31-NLS fusion rotein and cell were hatched 3 hours jointly, every histone is done the cell parallel laboratory test in 3 holes, and the cell that stays one group of 3 hole is as negative control.Hatch finish after, remove nutrient solution, clean cell with 1 times PBS solution, adding normal cell culture fluid continues to cultivate 36 hours, fluorescent microscope is observed down then, the cell number of green fluorescence how much represent Φ C31 fusion rotein transduction to enter cell after, the active size of the recombining reaction of the specific sequence on the catalysis cell chromosome.For the activity of Φ C31 fusion rotein in mammalian cell quantitatively is described, we digest each sample cell get off with tryptic solution, the ratio of the cell of green fluorescence in each sample of flow cytometry analysis, thus calculate the recombination efficiency of different Φ C31 fusion roteins and the activity in mammalian cell.
Experimental result illustrates that TAT fusion Φ C31 albumen of the present invention can be in the mode of density dependency, in mammalian cell, show the activity of intergrase through protein transduction, after merging NLS, this function is further strengthened (Fig. 2), show albumen that TAT merges Φ C31 be one effectively, safety, the instrument that can on mammalian cell karyomit(e) level, carry out genetic manipulation.
5. immunohistochemical analysis confirms that TAT-Φ C31 fusion rotein enters the cell that each is hatched, and the TAT-Φ C31 albumen that merges NLS enters nuclear horizontal showed increased.
Mammals African green monkey kidney cell line COS-7 cell inoculation in being covered with 12 porocyte culture plates of Cover Glass, when cell when the wave plate upper density reaches 30-50%, Φ C31-TAT and Φ C31-TAT-NLS albumen join hatch 3 hours jointly in the cell culture medium after, remove substratum, PBS changed normal culture medium culturing cell 24 hours after cleaning, and prepared cell specimen with immunohistochemical method.Fix with 4% Paraformaldehyde 96,0.2%TritonX-100 handles cell, after the horse serum sealing, with Φ C31 specificity antibody response, the distribution of second antibody colour developing Φ C31 fusion rotein in cell of rhodamine mark, use the nuclear staining of DAPI pair cell at last, sample is observed photograph, image Photoshop software processes on the two-photon laser confocal microscope of Zeiss company.The albumen that presentation of results TAT merges Φ C31 enters into each and its cell of hatching altogether, and the TAT-Φ C31 albumen of fusion NLS enters nuclear horizontal showed increased (Fig. 3).
Utilize TAT to merge Φ C31 protein transduction and be applied to DNA reorganization in the mammalian cell.Comprise the preparation of fusion rotein, and in report clone the detection of cytological Effect and the Subcellular Localization in the cell, illustrate that the Phi C 31 integrase through TAT and NLS modification will be applied in the prospect of carrying out fields such as genetic manipulation and gene therapy in the mammalian cell.Consider that there are the potential deficiency in virus vector and plasmid naked DNA carrier carrying the Phi C 31 integrase application facet, this non-virus provided by the invention, non-nucleic acid form, can effectively enter the TAT-Φ C31-NLS fusion rotein through modifying of cell performance function fast, be one effectively, safety, the instrument that can on mammalian cell karyomit(e) level, carry out genetic manipulation.
Description of drawings
Fig. 1, Φ C31 fusion rotein and purifying protein synoptic diagram thereof are modified in design.A is the overview diagram of the Φ C31 fusion rotein of different modifying; B is the SDS-PAGE detection figure of Φ C31 Expression of Fusion Protein and purifying; C is 3 kinds of Φ C31 fusion roteins of the different modifying of preparation; D utilizes the result of the specificity antibody of Φ C31 to the immune marking of 3 kinds of Φ C31 fusion roteins.
Fig. 2,3 kinds of Φ C31 fusion roteins are at clone 293-PB[EGFP] in use and the Function detection synoptic diagram.A is 293-PB[EGFP] the active system schematic of detection intergrase; After B is the TAT-Φ C31-NLS protein transduction cell of different concns, the result under the fluorescent microscope, it is the cell that green fluorescence is arranged that the grey cytochrome that wherein print to show is represented cell; After C was the warm protein transduction cell of 3 kinds of Φ C31 of different concns, flow cytometer detected the result of gfp positive cell.
Fig. 3, in mammalian cell COS-7, the cellular localization situation of the Φ C31 protein transduction that TAT and NLS modify.A is that laser confocal microscope detection TAT-Φ C31 albumen distributes in cell; B is the cellular localization comparison diagram of TAT-Φ C31 and TAT-Φ C31-NLS protein transduction.Wherein print the grey color part that shows and represent the distribution of Φ C31 fusion rotein in cell.
Embodiment
Below in conjunction with specific examples, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, people such as David and for example, condition described in the cell experiment guide (New York:Cold Spring HarborLaboratory Press, 1998).
1.3 plant the structure of Φ C31 fusion rotein, express purifying:
The Expression of Fusion Protein purifying of 3 kinds of Φ C31 adopts essentially identical affinity chromatography method, their different modifications are shown in the sign shown in Fig. 1-A wherein the polypeptide of 6 Histidines mainly is its preparation scheme to be described for Ni is mainly wherein a kind of below affine for example.
With pCMV Φ C31 (pCMVint) is template, the condition of the pcr amplification reaction of Φ C31: primer 1,
5-GCTC
ATGACACAAGGGGTTGTG-3 (being designated as SEQ.NO6), primer 2,5-GAG
GGGTGTC-3 (being designated as SEQ.NO.7).5 ' end of these two sections primers contains NheI and XhoI restriction enzyme site (box indicating) respectively, be respectively the encoding sequence of target gene 5 ' end and 3 ' end, in the reaction volume of 50 μ l, contain 50mmol/LKCl, 10mmol/L Tris-Cl thereafter, (pH8.5), 1.5mmol/L MgCl
2, 200 μ mol/L dNTP, 10pmol primer, the pyrobest archaeal dna polymerase of 1U (TaKaRa company product).Go up by 35 cycles of following conditioned response at DNA thermal cycler (Eppendorf company): 94 ℃, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 2 minutes.Amplified production reclaims the test kit purifying with the quick glue of the dna segment of vast Imtech, with the dna ligation kit of TaKaRa company be connected to expression vector plasmid pET-28b (+) (Novagen company product, Cat.No.69865.3).After respectively amplification PCR products and plasmid pET-28 (+) being carried out double digestion digestion with restriction enzyme NheI and XhoI, reclaim dna fragmentation respectively, and with the T4 ligase enzyme 16 ℃ connect 16 hours after, connect product with transforming big enterobacterial DH5 α, after the flat board of the LB substratum that contains kantlex (final concentration 30 μ g/ml) spends the night, after the positive colony enzyme of screening cut evaluation, and check order.The dna sequence analysis result shows shown in aminoacid sequence after the translation of PCR product representative and the SEQ.NO1. identical.Then with recombinant plasmid transformed e. coli bl21 (DE3) (Novagen company product).In the LB liquid nutrient medium that contains kantlex (final concentration 30 μ g/ml), host bacterium BL21 (pET-28b-Φ C31) is cultured to logarithmic phase at 37 ℃, and adding IPTG is 0.2mM to final concentration, and 18 ℃ are continued to cultivate 30 hours.Centrifugal collection thalline, destroy thalline with the broken bacterium instrument of ultrasonic wave, centrifugal collection supernatant with carrying out affinity chromatography with Histidine polypeptide (6His-Tag) bonded affinity column His-Bind Quick Cartridge (Novagen company product), has obtained the Φ C31 fusion rotein of purifying.Through the SDS-PAGE electrophoresis, obtain a single band (Fig. 1-B) at about 70kDa place.Collect the protein sample of purifying, utilize the ultrafiltration post of Millipore company to concentrate Φ C31 fusion rotein, be stored in-70 ℃ of refrigerators to 2mg/ml.
The fusion rotein of TAT Φ C31 and TAT-Φ C31-NLS is used and prepared to same method respectively.Be template with pCMV-Φ C31 (pCMVint) respectively, the primer of the PCR reaction of TAT-Φ C31 is:
5-GCTC
CATGCCGGCGTTCTTTGCGGCGGTCGCGGCGGCGATGACACAAGGGGTTGTG-3 (being designated as SEQ.NO8) and 5-GAG
GGGTGTC-3/ (being designated as SEQ.NO9).The primer of the PCR reaction of TAT-Φ C31-NLS is: 5-GCTC
CATGCCGGCGTTCTTTGCGGCGGTCGCGGCGGCGATGACACAAGGGGTTGTG-3 (being designated as SEQ.NO10) and 5-AAA
CTCGAGTTACTAAACCTTCCTCTTCTTCTTAGGCGCCGCTACGTCTTCC-3 (being designated as SEQ.NO11).Square frame in the primer sequence is represented restriction enzyme NheI and XhoI site respectively.The pET-28b-TAT-Φ C31 and the pET-28b-TAT-Φ C31-NLS expression plasmid that make up show shown in aminoacid sequence after the translation of gene wherein and SEQ.NO2 and the SEQ.NO3 identical through the dna sequence analysis result.Express purifying and concentrated TAT-Φ C31 and the TAT-Φ C31-NLS fusion rotein of obtaining respectively.
The SDS-PAGE detected result of the fusion rotein of 3 kinds of Φ C31 is shown in Fig. 1-C, and the immune marking result of specificity antibody who utilizes Φ C31 is shown in Fig. 1-D.
2.TAT-Φ C31 fusion rotein transduction Mammals report clone 293-PB[EGFP]:
Human embryonic kidney cell 293-PB[EGFP] mono-clonal report clone prepares by us, and the reaction process under the Phi C 31 integrase effect is seen shown in Fig. 2-A.Inoculating cell in 12 porocyte culture plates of the DMEM cell culture fluid that contains 10% calf serum, 37 ℃, 5%CO
2Cultivate in the cell culture incubator.When cell density reaches 50-70%, use 5uM respectively, 10uM, three kinds of Φ C31 fusion roteins of 15uM add in the cell culture fluid hatched 3 hours altogether with cell, and PBS changes the normal cell nutrient solution after cleaning, and observes after 36 hours.The DNA recombination function of the then qualitative explanation of green fluorescence cell Φ C31 fusion rotein transduction in mammalian cell arranged under fluorescent microscope.Result under the fluorescent microscope, it is that cell that green fluorescence is arranged (is seen Fig. 2-B) that the grey cytochrome that wherein print to show is represented cell.Use trypsin digestion and cell, detect the cell number that green fluorescence is arranged, can detection by quantitative go out the activity of Φ C31 fusion rotein and recombination efficiency and (see Fig. 2-C) through flow cytometer.
3. the location of TAT-Φ C31 protein transduction in mammalian cell detected by cellular immunization histochemical method.
Mammals African green monkey kidney cell line COS-7 cell inoculation in being covered with 12 porocyte culture plates of Cover Glass, when cell when the wave plate upper density reaches 30-50%, Φ C31-TAT and Φ C31-TAT-NLS albumen join hatch 3 hours jointly in the cell culture medium after, remove substratum, PBS changed normal culture medium culturing cell 24 hours after cleaning, and prepared cell specimen with immunohistochemical method.Fix with 4% Paraformaldehyde 96,0.2%TritonX-100 handles cell, after the horse serum sealing, with Φ C31 specificity antibody response, the distribution of second antibody colour developing Φ C31 fusion rotein in cell of rhodamine mark, use the nuclear staining of DAPI pair cell at last, sample is observed photograph, image Photoshop software processes on the two-photon laser confocal microscope of Zeiss company.The grey color part that wherein print to show represent the distribution of Φ C31 fusion rotein in cell, and the albumen of Fig. 3-A presentation of results TAT fusion Φ C31 enters into each and its cell of hatching altogether.The TAT-Φ C31 albumen that immunohistochemical analysis confirm to merge NLS enters nuclear horizontal showed increased (Fig. 3-B).
Sequence table
SEQ.NO1.(ΦC31)
MGSSHHHHHHSSGLVPRGSHMASRRVADMTQGVVTGVDTYAGAYDRQSRERENSSA
ASPATQRSANEDKAADLQREVERDGGRFRFVGHFSFAPGTSAFGTAERPEFERILNECR
AGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRL
DASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGRMVNVVINKL
AHSTTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPT
RGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITL
RPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRG
EESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLAL
LWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAYDGPVGRKHFR
KQQAALTLRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKSWWGRASVDDK
RVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQDGTEDVAA
SEQ.NO2.(TAT-ΦC31)
MGSSHHHHHHSSGLVPRGSHMASRRVADYGRKKRRQRRRMTQGVVTGVDTYAGAYD
RQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSEAPGTSAFGTAER
PEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNV
MDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGR
MVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCK
RMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPTTKIE
GYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCE
CGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRH
AEGDEETLALLWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAY
DGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKS
WWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQD
GTEDVAA
SEQ.NO3.(TAT-ΦC31-NLS)
MGSSHHHHHHSSGLVPRGSHMASRRVADYGRKKRRQRRRMTQGVVTGVDTYAGAYD
RQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSEAPGTSAFGTAER
PEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVTIVSTQEGVFRQGNV
MDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGR
MVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCK
RMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPDGTPTTKIE
GYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKGLSRGQAILSAMDKLYCE
CGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQHEGTCNVSMAALDKFVAERIFNKIRH
AEGDEETLALLWEAARRFGKLTEAPEKSGERANLVAERADALNALEELYEDRAAGAY
DGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKS
WWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQD
GTEDVAAPKKKRKV
SEQ.NO4：
PKKKRKV
SEQ.NO5：
YGRKKRRQRRR
SEQ.NO6：
5′-GCTC
ATGACACAAGGGGTTGTG-3′
SEQ.NO.7：
5′-GAG
GGGTGTC-3′
SEQ.NO8：
5′-GCTC
CATGCCGGCGTTCTTTGCGGCGGTCGCGGCGGCGATGACACAAGGGGTTGTG-3′
SEQ.NO9：
5′-GAG
GGGTGTC-3′
SEQ.NO10：
5′-GCTC
CATGCCGGCGTTCTTTGCGGCGGTCGCGGCGGCGATGACACAAGGGGTTGTG-3′
SEQ.NO11：
5′-AAA
CTCGAGTTACTAAACCTTCCTCTTCTTCTTAGGCGCCGCTACGTCTTCC-3′
Claims (3)
1. one kind through TAT and the peptide modified Phi C 31 integrase albumen of NLS, it is characterized in that aminoacid sequence is SEQ.NO3.
2. the proteic preparation method of Phi C 31 integrase that modifies of TAT as claimed in claim 1 and NLS, it is characterized in that concrete steps are as follows: the sequence of the nucleotide sequence of NLS and TAT-Φ C31 is spliced together in the PCR reaction, with pCMV-Φ C31 plasmid is template, in the primer of upstream and downstream, add the recognition sequence of restriction enzyme Nhe I and XhoI respectively, the PCR product is through after these two enzymic digestions, recovery is connected to same process enzyme and cuts on the postdigestive prokaryotic expression carrier pET28b (+), obtains pET28b-TAT-Φ C31-NLS recombinant plasmid; After the analysis of recombinant plasmid process dna sequencing is correct, be transformed in e. coli bl21 (DE3) bacterial strain, select 5-8 male colony clone in the LB of kalamycin resistance substratum, with the pre-abduction delivering Phi C 31 integrase of IPTG albumen; Whole bacterial protein SDS-PAGE after the expression, Coomassie brilliant blue R250 dyes, and determines the condition and the positive colony clone of abduction delivering, then at 16-22 ℃, great expression albumen under the 0.2mM IPTG condition;
The proteic bacterium of abduction delivering is through the ultrasonication thalline, after discharging target protein, affinity chromatography column purification through the Ni ion chelating, albumen behind the purifying is 2mg/ml through ultrafiltration and concentration, leave in-70 ℃ the refrigerator, through experiment of the protein immunization marking and protein N terminal order-checking proof, its aminoacid sequence is shown in SEQ.NO3.
A Phi C 31 integrase albumen of modifying through TAT and NLS as claimed in claim 1 on mammalian cell karyomit(e) level as the application of genetic manipulation instrument.
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| WO2001049832A2 (en) * | 2000-01-07 | 2001-07-12 | Artemis Pharmaceuticals Gmbh | Transduction of recombinases for inducible gene targeting |
| WO2002038613A2 (en) * | 2000-11-10 | 2002-05-16 | Artemis Pharmaceuticals Gmbh | Modified recombinase |
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| In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Helena M. Thorpe, et al.Proc. Natl. Acad. Sci. USA,Vol.95 No.10. 1998 |
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