CN102851262A - Fusion protein capable of targeting activation of RANK signal pathway and recombinant plasmid and application thereof - Google Patents
Fusion protein capable of targeting activation of RANK signal pathway and recombinant plasmid and application thereof Download PDFInfo
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Abstract
The invention discloses fusion protein capable of targeting activation of a RANK signal pathway, a recombinant plasmid and an application thereof. The fusion protein has an amino acid sequence as shown in SEQ ID NO. 1, and is obtained by substituting a segment of a gyrase B sequence for a C terminal of a mouse RANK; the nucleotide sequence coding the fusion protein is shown in SEQ ID NO. 2. After the fusion protein is introduced into a cell, continuous stimulation of a micromolecular medicament of coumermycin is accepted, and a RANK-dependent signal pathway is targetedly activated; and thus side effect caused by disease treatment though ligand-activated RANK signal pathway using acceptors is prevented. A recombinant vector contains the nucleotide sequence shown in SEQ ID NO. 2. The recombinant vector has very important value for RANK function research and RANK-caused disease treatment research.
Description
Technical field
The present invention relates to a kind of structure of fusion rotein, but particularly a kind of fusion rotein of targeted activation RANK signal path and recombinant plasmid and application.
Background technology
The Nuclear factor kappa B receptor activation factor (receptor activator of nuclear factor-κ B, RANK) signal path has the multiple physiological function.This signal path is absolutely necessary for tooth formation, lymphoglandula formation, osteoclast formation; Thereby the synergy of RANK, CD40 signal path can instruct the Central immunotolerance of the formation control body of thymic medulla microstructure; RANK signal path regulation heating maincenter; The RANK signal path can promote survival and the activity of dendritic cell; The RANK signal path is absolutely necessary for the formation of mammary epithelial cell; The RANK signal path plays an important role for the generation of vascular endothelial cell and the formation of blood vessel; The RANK signal path also has close related etc. with the generation of kinds cancer and development.These result of study indications RANK signal path is extremely important signal path in the above-mentioned disease treatment, and therefore, RANK is the important target molecules of these diseases for the treatment of.This signal path unusually usually cause various diseases, prompting RANK signal path is an extremely important signal path in these disease treatments.First-selection utilizes the respective ligand (RANK ligand, RANKL) of RANK to activate the RANK signal path when generally speaking, utilizing the RANK signal path that above-mentioned disease is treated.Yet, utilize the RANK part to activate the RANK signal path and usually can cause many side effects, such as serious osteoporosis, lethality hypercalcinemia, systemic inflammatory response etc.
Therefore, need to transform RANK, make its function that had both kept RANK, overcome again the defective that it causes side effect.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome the shortcoming and deficiency of prior art, but a kind of fusion rotein of targeted activation RANK signal path is provided.
Another object of the present invention is to provide the recombinant vectors of the nucleotide sequence that contains the above-mentioned fusion rotein of encoding.
A further object of the present invention is to provide the application of above-mentioned recombinant vectors.
Purpose of the present invention is achieved through the following technical solutions: but a kind of fusion rotein of targeted activation RANK signal path, and its aminoacid sequence is as follows:
MYPYDVPDYAMSNSYDSSSIKVLKGLDAVRKRPGMYIGDTDDGTGLHHMVFEVVDNAIDEALAGHCKEIIVTIHADNSVSVQDDGRGIPTGIHPEEGVSAAEVIMTVLHAGGKFDDNSYKVSGGLHGVGVSVVNALSQKLELVIQREGKIHRQIYEHGVPQAPLAVTGETEKTGTMVRFWPSLETFTNVTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGKEDHFHYEGP?SLIVLLLFISVVVVAAIIFGVYYRKGGKALTANLWNWVNDACSSLSGNKESSGDRCAGSHSATSSQQEVCEGILLMTREEKMVPEDGAGVCGPVCAAGGPWAEVRDSRTFTLVSEVETQGDLSRKIPTEDEYTDRPSQPSTGSLLLIQQGSKSIPPFQEPLEVGENDSLSQCFTGTESTVDSEGCDFTEPPSRTDSMPVSPEKHLTKEIEGDSCLPWVVSSNSTDGYTGSGNTPGEDHEPFPGSLKCGPLPQCAYSMGFPSEAAASMAEAGVRPQDRADERGASGSGSSPSDQPPASGNVTGNSNSTFISSGQVMNFKGDIIVVYVSQTSQEGPGSAEPESEPVGRPVQEETLAHRDSFAGTAPRFPDVCATGAGLQEQGAPRQKDGTSRPVQEQGGAQTSLHTQGSGQCAE；
But the nucleotide sequence of the fusion rotein of the described targeted activation RANK signal path of encoding is as follows:
ATGTACCCATACGATGTTCCAGATTACGCTATGTCGAACTCTTATGACTCCTCCAGTATCAAAGTCCTGAAAGGGCTGGATGCGGTGCGTAAGCGCCCGGGTATGTATATCGGCGACACGGATGACGGCACCGGTCTGCACCACATGGTATTCGAGGTGGTAGATAACGCTATCGACGAAGCGCTCGCGGGTCACTGTAAAGAAATTATCGTCACCATTCACGCCGATAACTCTGTCTCTGTACAGGATGACGGGCGCGGCATTCCGACCGGTATTCACCCGGAAGAGGGCGTATCGGCGGCGGAAGTGATCATGACCGTTCTGCACGCAGGCGGTAAATTTGACGATAACTCCTATAAAGTGTCCGGCGGTCTGCACGGCGTTGGTGTTTCGGTAGTAAACGCCCTGTCGCAAAAACTGGAGCTGGTTATCCAGCGCGAGGGTAAAATTCACCGTCAGATCTACGAACACGGTGTACCGCAGGCCCCGCTGGCGGTTACCGGCGAGACTGAAAAAACCGGCACCATGGTGCGTTTCTGGCCCAGCCTCGAAACCTTCACCAATGTGACCGAGTTCGAATATGAAATTCTGGCGAAACGTCTGCGTGAGTTGTCGTTCCTCAACTCCGGCGTTTCCATTCGTCTGCGCGACAAGCGCGACGGCAAAGAAGACCACTTCCACTATGAAGGCCCCAGTCTCATCGTTCTGCTCCTCTTCATCTCTGTGGTAGTAGTGGCTGCCATCATCTTCGGCGTTTACTACAGGAAGGGAGGGAAAGCGCTGACAGCTAATTTGTGGAATTGGGTCAATGATGCTTGCAGTAGTCTAAGTGGAAATAAGGAGTCCTCAGGGGACCGTTGTGCTGGTTCCCACTCGGCAACCTCCAGTCAGCAAGAAGTGTGTGAAGGTATCTTACTAATGACTCGGGAGGAGAAGATGGTTCCAGAAGACGGTGCTGGAGTCTGTGGGCCTGTGTGTGCGGCAGGTGGGCCCTGGGCAGAAGTCAGAGATTCTAGGACGTTCACACTGGTCAGCGAGGTTGAGACGCAAGGAGACCTCTCGAGGAAGATTCCCACAGAGGATGAGTACACGGACCGGCCCTCGCAGCCTTCGACTGGTTCACTGCTCCTAATCCAGCAGGGAAGCAAATCTATACCCCCATTCCAGGAGCCCCTGGAAGTGGGGGAGAACGACAGTTTAAGCCAGTGTTTCACCGGGACTGAAAGCACGGTGGATTCTGAGGGCTGTGACTTCACTGAGCCTCCGAGCAGAACTGACTCTATGCCCGTGTCCCCTGAAAAGCACCTGACAAAAGAAATAGAAGGTGACAGTTGCCTCCCCTGGGTGGTCAGCTCCAACTCAACAGATGGCTACACAGGCAGTGGGAACACTCCTGGGGAGGACCATGAACCCTTTCCAGGGTCCCTGAAATGTGGACCATTGCCCCAGTGTGCCTACAGCATGGGCTTTCCCAGTGAAGCAGCAGCCAGCATGGCAGAGGCGGGAGTACGGCCCCAGGACAGGGCTGATGAGAGGGGAGCCTCAGGGTCCGGGAGCTCCCCCAGTGACCAGCCACCTGCCTCTGGGAACGTGACTGGAAACAGTAACTCCACGTTCATCTCTAGCGGGCAGGTGATGAACTTCAAGGGTGACATCATCGTGGTGTATGTCAGCCAGACCTCGCAGGAGGGCCCGGGTTCCGCAGAGCCCGAGTCGGAGCCCGTGGGCCGCCCTGTGCAGGAGGAGACGCTGGCACACAGAGACTCCTTTGCGGGCACCGCGCCGCGCTTCCCCGACGTCTGTGCCACCGGGGCTGGGCTGCAGGAGCAGGGGGCACCCCGGCAGAAGGACGGGACATCGCGGCCGGTGCAGGAGCAGGGTGGGGCGCAGACTTCACTCCATACCCAGGGGTCCGGACAATGTGCAGAATGA；
A kind of recombinant vectors contains the gene of above-mentioned nucleotide sequence;
Gene clone to the lentiviral vectors that described recombinant vectors is preferably above-mentioned nucleotide sequence obtains;
Described lentiviral vectors is preferably CSII-EF-RfA-IRES2-Venus;
Described recombinant vectors is preferably the CSII-EF-RfA-IRES2-Venus-HG-RANK recombinant plasmid, and it prepares by the following method:
(1) take mycobacterium tuberculosis dna as template, take FP and RP as primer, the PCR reaction obtains the HA-GyraseB gene;
FP:CGGGATCCACCATGTACCCATACGATGTTCCAGATTACGCTATGTCGAACTCTTATGACTCCTCC
RP：CGGAATTCTTAGGTACCGCCTTCATAGTGGAAGTGGTCTTC；
(2) by EcoR I enzyme and the BamH I enzyme HA-Gyrase B gene that obtains of double digestion pENTR-2B carrier and step (1) respectively, enzyme is cut rear purifying, obtains the pENTR-2B carrier of double digestion and the HA-Gyrase B gene of double digestion; The HA-Gyrase B gene of again the pENTR-2B carrier of double digestion being connected with double digestion connects, and obtains recombinant vectors pENTR-2B-HA-Gyrase B; With Kpn I enzyme single endonuclease digestion recombinant vectors pENTR-2B-HA-Gyrase B, dephosphorylation obtains the pENTR-2B-HA-Gyrase B behind the single endonuclease digestion;
(3) take mouse thymus cDNA as template, FP2 and RP2 are primer, and PCR reaction amplification obtains RANK cross-film zone and bag inner compartment gene; With Kpn I enzyme single endonuclease digestion RANK cross-film zone and bag inner compartment gene, obtain RANK cross-film zone and bag inner compartment gene behind the single endonuclease digestion;
FP2：CGGGTACCCCCAGTCTCATCGTTCTGCTC；
RP2：CCGGGTACCTCATTCTGCACATTGTCCGGAC；
(4) the pENTR-2B-HA-Gyrase B behind the single endonuclease digestion that step (2) is obtained is connected 3 with step) RANK cross-film zone behind the single endonuclease digestion that obtains and be connected the connection of inner compartment gene, obtain recombinant vectors pENTR-2B-HG-RANK;
The HG-RANK double chain DNA fragment that (5) will be positioned on the recombinant vectors pENTR-2B-HG-RANK by the Gateway technology is transferred on the lentiviral vectors CSII-EF-RfA-IRES2-Venus, obtains the CSII-EF-RfA-IRES2-Venus-HG-RANK recombinant plasmid;
The condition optimization of the PCR reaction described in step (1) and (2) is: 94 ℃ of 2min; 94 ℃ of 15sec, 55 ℃ of 15sec, 68 ℃ of 30sec, 30 circulations; 72 ℃ of 10min;
The reaction system of the described Gateway technology of step (5) is preferably:
Reaction conditions is preferably: 25 ℃ of lower reactions 6 hours, add 1 μ l Proteinase K (concentration is 2 μ g/ μ l), and 37 ℃ were reacted 10 minutes;
The application of described recombinant vectors in the RANK functional study.
The present invention has following advantage and effect with respect to prior art:
(1) fusion rotein provided by the invention is behind cell inner expression, can accept the lasting stimulation of small-molecule drug Notomycin, the signal path that targeted activation RANK is interdependent, thus can avoid utilizing the part of acceptor to activate the side effect that causes when the RANK signal path carries out disease treatment.
(2) the present invention has made up a recombined small-mouse RANK plasmid, contain above-mentioned fusion rotein, carrier framework is the slow virus plasmid, utilize slow-virus transfection, both be suitable for the interior research of body and also be suitable in vitro study, both can be used for somatoblast and also can be used for Unseparated Cell, and can be used for of short duration research and also can be used for long-term observation; Transfection efficiency can be determined by the green fluorescence of cell.Therefore, recombined small-mouse RANK gene slow virus plasmid all has extremely important value for the disease treatment research that functional study and the RANK of RANK causes.Recombined small-mouse RANK slow virus plasmid has very large commercial value.
Description of drawings
Fig. 1 is the expression of results figure of western blotting technology for detection HG-RANK fusion rotein in the NIH3T3 cell.
Fig. 2 is the Expression of phosphorylated figure as a result of the interdependent I κ B α of target stimulates in the Notomycin different time of 0.05 μ M concentration RANK specificity; α-tubulin is as confidential reference items.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) preparation of recombinant vectors pENTR-2B-Gyrase B-HA
1. obtain Gyrase B gene by the PCR reaction, primer (be 5 '-3 ', the large genome company of China is synthetic) is as follows:
FP:CGGGATCCACCATGTACCCATACGATGTTCCAGATTACGCTATGTCGAACTCTTATGACTCCTCC
RP：CGGAATTCTTAGGTACCGCCTTCATAGTGGAAGTGGTCTTC。
Cooperate the PCR reaction solution according to following system:
Carry out the PCR reaction according to follow procedure: 94 ℃ of 2min of initial sex change; 94 ℃ of 15sec of sex change, 55 ℃ of 15sec of renaturation, 68 ℃ of 30sec of extension, 30 circulations; 72 ℃ of 10min.
2. in the PCR product that 1. step obtains, add the freezing dehydrated alcohol of 2.5 times of volumes (adding simultaneously 0.1 times, the 3M sodium-acetate of pH5.6) precipitation, centrifugation, this throw out cooperates by following reaction system:
The PCR throw out
37 ℃ of processing of above reaction system 2 hours, electrophoresis reclaims HA-Gyrase B gene fragment.
Cooperate the endonuclease reaction system according to following system:
37 ℃ of processing of above reaction system 2 hours, electrophoresis reclaims the purpose fragment.
PENTR-2B carrier after enzyme cut is connected with HA-Gyrase B gene fragment after enzyme is cut, obtains recombinant vectors pENTR-2B-HA-Gyrase B(hereinafter to be referred as pENTR-2B-HG).
Linked system is as follows: double chain DNA fragment 100ng, pENTR-2B carrier 20ng, DNA Ligation Kit Ver.2.0(are available from TAKARA company) 5 μ l, distilled water is mended to 10 μ l.
16 ℃ connect 12 hours, then transform competent escherichia coli cell DH5 α (available from TAKARA company) with 1 μ l connecting fluid, are incubated at the LB solid medium according to the molecular cloning formulated, and the kantlex that wherein contains 50 μ g/ml is selected.The clone who grows out is positive colony, the picking positive colony is incubated in the LB liquid nutrient medium that contains kantlex according to the molecular cloning formulated, extracting, obtain recombinant vectors, by order-checking, determine to obtain containing the recombinant plasmid pENTR-2B-HG of HA sequence (introducing by primers F P) and Gyrase B gene order (as follows).
Gyrase B gene order (660bp):
ATGTCGAACTCTTATGACTCCTCCAGTATCAAAGTCCTGAAAGGGCTGGATGCGGTGCGTAAGCGCCCGGGTATGTATATCGGCGACACGGATGACGGCACCGGTCTGCACCACATGGTATTCGAGGTGGTAGATAACGCTATCGACGAAGCGCTCGCGGGTCACTGTAAAGAAATTATCGTCACCATTCACGCCGATAACTCTGTCTCTGTACAGGATGACGGGCGCGGCATTCCGACCGGTATTCACCCGGAAGAGGGCGTATCGGCGGCGGAAGTGATCATGACCGTTCTGCACGCAGGCGGTAAATTTGACGATAACTCCTATAAAGTGTCCGGCGGTCTGCACGGCGTTGGTGTTTCGGTAGTAAACGCCCTGTCGCAAAAACTGGAGCTGGTTATCCAGCGCGAGGGTAAAATTCACCGTCAGATCTACGAACACGGTGTACCGCAGGCCCCGCTGGCGGTTACCGGCGAGACTGAAAAAACCGGCACCATGGTGCGTTTCTGGCCCAGCCTCGAAACCTTCACCAATGTGACCGAGTTCGAATATGAAATTCTGGCGAAACGTCTGCGTGAGTTGTCGTTCCTCAACTCCGGCGTTTCCATTCGTCTGCGCGACAAGCGCGACGGCAAAGAAGACCACTTCCACTATGAAGGC。
(2) preparation of recombinant vectors pENTR-2B-HG-RANK cross-film zone and bag inner compartment
1. obtain RANK cross-film zone and bag inner compartment gene by the PCR reaction, primer (be 5 '-3 ', the large genome company of China is synthetic) is as follows:
FP2：CGGGTACCCCCAGTCTCATCGTTCTGCTC；
RP2：CCGGGTACCTCATTCTGCACATTGTCCGGAC。
Cooperate the PCR reaction solution according to following system:
Carry out the PCR reaction according to follow procedure: 94 ℃ of 2min of initial sex change; 94 ℃ of 15sec of sex change, 55 ℃ of 15sec of renaturation, 68 ℃ of 30sec of extension, 35 circulations; 72 ℃ of 10min.
2. in the PCR product that 1. step obtains, add the freezing dehydrated alcohol of 2.5 times of volumes (adding simultaneously 0.1 times, the 3M sodium-acetate of pH5.6) precipitation, centrifugation, this throw out cooperates by following reaction system:
The PCR throw out
10 * L enzyme cutting buffering liquid, 2 μ l
KpnI(is available from TAKARA) 1 μ l
Sterile purified water to 20 μ l;
37 ℃ of processing of above reaction system 2 hours, electrophoresis reclaims goal gene RANK cross-film zone and bag inner compartment gene fragment.
Cooperate the endonuclease reaction system according to following system:
37 ℃ of processing of above reaction system 2 hours add alkaline phosphatase CIAP(available from TAKARA), process 30min for 37 ℃, electrophoresis reclaims carrier segments.
PENTR-2B-HG carrier after enzyme cut is with RANK cross-film zone after enzyme is cut and be connected the inner compartment gene fragment and be connected, and obtains recombinant vectors pENTR-2B-HG-RANK cross-film zone and wraps inner compartment (hereinafter to be referred as pENTR-2B-HG-RANK).
Linked system is as follows: double chain DNA fragment 100ng, pENTR-2B-HG carrier 20ng, DNALigation Kit Ver.2.0(are available from TAKARA company) 5 μ l, distilled water is mended to 10 μ l;
16 ℃ connect 12 hours, then transform competent escherichia coli cell DH5 α (available from TAKARA company) with 1 μ l connecting fluid, are incubated at the LB solid medium according to the molecular cloning formulated, and the kantlex that wherein contains 50 μ g/ml is selected.The clone who grows out is positive colony, the picking positive colony is incubated in the LB liquid nutrient medium that contains kantlex according to the molecular cloning formulated, extracting, obtain recombinant vectors, by order-checking, determine to obtain containing the recombinant plasmid pENTR-2B-HG-RANK of HA sequence and Gyrase B sequence, RANK cross-film zone and bag inner compartment sequence (as follows).RANK cross-film zone and bag inner compartment are positioned at 3 ' end of Gyrase B sequence.
The base sequence (1242bp) of mouse RANK cross-film zone and bag inner compartment, as follows:
CCCAGTCTCATCGTTCTGCTCCTCTTCATCTCTGTGGTAGTAGTGGCTGCCATCATCTTCGGCGTTTACTACAGGAAGGGAGGGAAAGCGCTGACAGCTAATTTGTGGAATTGGGTCAATGATGCTTGCAGTAGTCTAAGTGGAAATAAGGAGTCCTCAGGGGACCGTTGTGCTGGTTCCCACTCGGCAACCTCCAGTCAGCAAGAAGTGTGTGAAGGTATCTTACTAATGACTCGGGAGGAGAAGATGGTTCCAGAAGACGGTGCTGGAGTCTGTGGGCCTGTGTGTGCGGCAGGTGGGCCCTGGGCAGAAGTCAGAGATTCTAGGACGTTCACACTGGTCAGCGAGGTTGAGACGCAAGGAGACCTCTCGAGGAAGATTCCCACAGAGGATGAGTACACGGACCGGCCCTCGCAGCCTTCGACTGGTTCACTGCTCCTAATCCAGCAGGGAAGCAAATCTATACCCCCATTCCAGGAGCCCCTGGAAGTGGGGGAGAACGACAGTTTAAGCCAGTGTTTCACCGGGACTGAAAGCACGGTGGATTCTGAGGGCTGTGACTTCACTGAGCCTCCGAGCAGAACTGACTCTATGCCCGTGTCCCCTGAAAAGCACCTGACAAAAGAAATAGAAGGTGACAGTTGCCTCCCCTGGGTGGTCAGCTCCAACTCAACAGATGGCTACACAGGCAGTGGGAACACTCCTGGGGAGGACCATGAACCCTTTCCAGGGTCCCTGAAATGTGGACCATTGCCCCAGTGTGCCTACAGCATGGGCTTTCCCAGTGAAGCAGCAGCCAGCATGGCAGAGGCGGGAGTACGGCCCCAGGACAGGGCTGATGAGAGGGGAGCCTCAGGGTCCGGGAGCTCCCCCAGTGACCAGCCACCTGCCTCTGGGAACGTGACTGGAAACAGTAACTCCACGTTCATCTCTAGCGGGCAGGTGATGAACTTCAAGGGTGACATCATCGTGGTGTATGTCAGCCAGACCTCGCAGGAGGGCCCGGGTTCCGCAGAGCCCGAGTCGGAGCCCGTGGGCCGCCCTGTGCAGGAGGAGACGCTGGCACACAGAGACTCCTTTGCGGGCACCGCGCCGCGCTTCCCCGACGTCTGTGCCACCGGGGCTGGGCTGCAGGAGCAGGGGGCACCCCGGCAGAAGGACGGGACATCGCGGCCGGTGCAGGAGCAGGGTGGGGCGCAGACTTCACTCCATACCCAGGGGTCCGGACAATGTGCAGAATGA。
The HG-RANK double chain DNA fragment that (3) will be positioned on the recombinant vectors pENTR-2B-HG-RANK by the Gateway technology is transferred to the Physicochemical research of lentiviral vectors CSII-EF-RfA-IRES2-Venus(Japan, www.brc.riken.jp/lab/cfm/) on, obtain recombinant plasmid CSII-EF-RfA-IRES2-Venus-HG-RANK(and be designated hereinafter simply as LV-HG-RANK).
The HG double chain DNA fragment that same method will be positioned on the recombinant plasmid pENTR-2B-HG is transferred on the lentiviral vectors CSII-EF-RfA-IRES2-Venus, obtains CSII-EF-RfA-IRES2-Venus-HG recombinant plasmid (being designated hereinafter simply as LV-HG).
Specific operation process is as follows:
25 ℃ of lower reactions 6 hours add 1 μ l Proteinase K (concentration is 2 μ g/ μ l), and 37 ℃ are reacted 10 minutes to remove unnecessary LR clonase II enzyme.Then get the above-mentioned reaction solution of 1 μ l and transform bacillus coli DH 5 alpha competent cell (available from TAKARA company), cultivated 22～24 hours in 30 ℃ of LB solid mediums that containing 100 μ g/ml penbritins, the clone who grows out is positive colony.The picking positive colony is incubated in the LB liquid nutrient medium that contains 100 μ g/ml penbritins, cultivates plasmid purification 20 hours down for 30 ℃.Detect by BamHI and NotI digested plasmid, obtained correct recombinant plasmid, through order-checking, the sequence of HG-RANK is defined as this recombinant plasmid shown in SEQ ID NO.2: LV-HG-RANK(or LV-HG) the slow virus plasmid.Extract in a large number test kit with the plasmid of Qiagen company and extract this recombinant plasmid.
(4) detection of expression of LV-HG-RANK recombinant plasmid.
A, usefulness calcium-phosphorus plasmid introductory technique, with LV-HG-RANK, pCMV-VSV-G-RSV-Rev(available from Japanese RIKEN, http://www.brc.riken.jp/lab/cfm) and pCAG-HIVgp(available from Japanese RIKEN) import in the human embryo kidney (HEK) 293T cell (available from ATCC) according to the ratio of mass ratio 5:2:2, be experimental group; Import to human embryo kidney (HEK) 293T cell with LV-HG, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp according to the ratio of mass ratio 5:2:2, be control group; The supernatant liquor of collecting cell cultures namely obtains containing LV-HG-RANK(or LV-HG) virus of slow virus plasmid.
B, usefulness contain the virus infected mice NIH3T3 cell (available from ATCC) of LV-HG-RANK plasmid.The infection step is as follows: with 5 * 10
4Individual NIH3T3 cell and 1 * 10
6The iu virus liquid is suspended in the DMEM substratum, consumption preparation by 200 μ l mixed solution/holes, per 200 μ l mixed solutions also comprise the foetal calf serum (FBS) of volume percent 10%, penicillin and the Streptomycin sulphate of 100iu/ml, be put in behind the mixing in the dull and stereotyped culture dish of 48well, 37 ℃ of lower 1.5hr that cultivate, add 500 μ l nutrient solutions (DMEM+10%FBS+100iu/ml penicillin+100iu/ml Streptomycin sulphate), continue to cultivate 24hr, the cultivation of going down to posterity, with the virus infection NIH3T3 cell that contains the LV-HG plasmid in contrast.Cultivate after 10 days (long-term observation), utilize hematimeter to detect the importing rate (after the plasmid importing, cell expressing Venus) of plasmid under the fluorescent microscope, be more than 90%.Collect these cells, (step is seen Developmental stage-dependent collaboration between the RANK and lymphotoxin pathways for B-cell follicle organization in secondary lymphoid organs.J Immunol.2007 to western blotting technology by standard, 179 (10): 6799-6807), utilize HA antibody (available from SantaCruz company) and RANK antibody (available from SantaCruz company) to detect the expression of HG-RANK in the NIH3T3 cell, the result as shown in Figure 1.This presentation of results LV-HG-RANK recombinant plasmid successfully imports NIH 3 T 3 cells in vitro and expresses the HG-RANK fusion rotein.
B, use ultimate density are Notomycin (the coumermycin A1 of 0.05 μ M, available from Sigma company) stimulate to import NIH3T3 cell 5min, 15min, the 30min of LV-HG-RANK plasmid, utilize p-I κ B Alpha antibodies (available from CellSignaling company) to detect the phosphorylation that the coumermycin A1 of this concentration as can be known can stimulate I κ B α in the NIH3T3 cell that imports the LV-HG-RANK plasmid, the positive control of the dendritic cell that stimulates with RANK aglucon (RANKL) is identical; And be that the coumermycin A1 of 0.05 μ M stimulate to import the phosphorylation (as shown in Figure 2) that NIH3T3 cell 5min, 15min, the 30min of LV-HG plasmid can not induce I κ B α with ultimate density.Confidential reference items are α-tubulin (available from SantaCruz company).But these results show the interdependent signal path of coumermycin A1 targeted activation RANK.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. but the fusion rotein of a targeted activation RANK signal path, it is characterized in that: its aminoacid sequence is shown in SEQ ID NO.1.
2. but the fusion rotein of targeted activation RANK signal path according to claim 1, it is characterized in that: the nucleotide sequence of the described fusion rotein of encoding is shown in SEQ ID NO.2.
3. recombinant vectors is characterized in that: but contain the gene of the fusion rotein of the targeted activation RANK signal path as claimed in claim 1 of encoding, and nucleotide sequence is shown in SEQ ID NO.2.
4. recombinant vectors according to claim 3, it is characterized in that: described recombinant vectors is for to obtain gene clone to the lentiviral vectors shown in the SEQID NO.2.
5. recombinant vectors according to claim 4, it is characterized in that: described lentiviral vectors is CSII-EF-RfA-IRES2-Venus.
6. recombinant vectors according to claim 5, it is characterized in that: described recombinant vectors is the CSII-EF-RfA-IRES2-Venus-HG-RANK recombinant plasmid, and it prepares by the following method:
(1) take mycobacterium tuberculosis dna as template, take FP and RP as primer, the PCR reaction obtains the HA-GyraseB gene;
FP:CGGGATCCACCATGTACCCATACGATGTTCCAGATTACGCTATGTCGAACTCTTATGACTCCTCC
RP：CGGAATTCTTAGGTACCGCCTTCATAGTGGAAGTGGTCTTC；
(2) by EcoR I enzyme and the BamH I enzyme HA-Gyrase B gene that obtains of double digestion pENTR-2B carrier and step (1) respectively, enzyme is cut rear purifying, obtains the pENTR-2B carrier of double digestion and the HA-Gyrase B gene of double digestion; The HA-Gyrase B gene of again the pENTR-2B carrier of double digestion being connected with double digestion connects, and obtains recombinant vectors pENTR-2B-HA-Gyrase B; With Kpn I enzyme single endonuclease digestion recombinant vectors pENTR-2B-HA-Gyrase B, dephosphorylation obtains the pENTR-2B-HA-Gyrase B behind the single endonuclease digestion;
(3) take mouse thymus cDNA as template, FP2 and RP2 are primer, and PCR reaction amplification obtains RANK cross-film zone and bag inner compartment gene; With Kpn I enzyme single endonuclease digestion RANK cross-film zone and bag inner compartment gene, obtain RANK cross-film zone and bag inner compartment gene behind the single endonuclease digestion;
FP2：CGGGTACCCCCAGTCTCATCGTTCTGCTC；
RP2：CCGGGTACCTCATTCTGCACATTGTCCGGAC；
(4) the pENTR-2B-HA-Gyrase B behind the single endonuclease digestion that step (2) is obtained is connected 3 with step) RANK cross-film zone behind the single endonuclease digestion that obtains and be connected the connection of inner compartment gene, obtain recombinant vectors pENTR-2B-HG-RANK;
The HG-RANK double chain DNA fragment that (5) will be positioned on the recombinant vectors pENTR-2B-HG-RANK by the Gateway technology is transferred on the lentiviral vectors CSII-EF-RfA-IRES2-Venus, obtains the CSII-EF-RfA-IRES2-Venus-HG-RANK recombinant plasmid.
7. recombinant vectors according to claim 6 is characterized in that:
The condition of the PCR reaction described in step (1) and (2) is: 94 ℃ of 2min; 94 ℃ of 15sec, 55 ℃ of 15sec, 68 ℃ of 30sec, 30 circulations; 72 ℃ of 10min.
8. recombinant vectors according to claim 6 is characterized in that:
The reaction system of the described Gateway technology of step (5) is:
Wherein, TE consists of 10mM Tris-HCl, 1mMEDTA, and the pH value is 8;
Reaction conditions is: 25 ℃ of lower reactions 6 hours, add the Proteinase K that 1 μ l concentration is 2 μ g/ μ l, and 37 ℃ were reacted 10 minutes.
9. each described recombinant vectors application in the RANK functional study of claim 3～8.
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| CN201210251406.8A CN102851262B (en) | 2012-07-18 | 2012-07-18 | Fusion protein capable of targeting activation of RANK signal pathway and recombinant plasmid and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001036637A1 (en) * | 1999-11-17 | 2001-05-25 | Immunex Corporation | Receptor activator of nf-kappa b |
| US20030073097A1 (en) * | 2001-10-11 | 2003-04-17 | Chen Zhijian J. | TRAF6-regulated IKK activators (TRIKA1 and TRIKA2) and their use as anti-inflammatory targets |
| CN101434656A (en) * | 2007-11-16 | 2009-05-20 | 王炜 | Preparation and use of OPG-HSP70 fusion protein |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001036637A1 (en) * | 1999-11-17 | 2001-05-25 | Immunex Corporation | Receptor activator of nf-kappa b |
| US20030073097A1 (en) * | 2001-10-11 | 2003-04-17 | Chen Zhijian J. | TRAF6-regulated IKK activators (TRIKA1 and TRIKA2) and their use as anti-inflammatory targets |
| CN101434656A (en) * | 2007-11-16 | 2009-05-20 | 王炜 | Preparation and use of OPG-HSP70 fusion protein |
Non-Patent Citations (6)
| Title |
|---|
| 《Plos one》 20081229 Walsh et al TRAF6 Autoubiquitination-Independent Activation of the NFkB and MAPK Pathways in Response to IL-1 and RANKL 全文 1－9 第3卷, 第12期 * |
| 《中国生物工程杂志》 20110331 秦俊文等 shRNA慢病毒质粒的构建技巧 全文 1－9 第31卷, 第3期 * |
| SUZUKI ET AL: "Reconstruction of Monocyte Transcriptional Regulatory Network Accompanies Monocytic Functions in Human Fibroblasts", 《PLOS ONE》, vol. 7, no. 3, 13 March 2012 (2012-03-13) * |
| WALSH ET AL: "TRAF6 Autoubiquitination-Independent Activation of the NFkB and MAPK Pathways in Response to IL-1 and RANKL", 《PLOS ONE》, vol. 3, no. 12, 29 December 2008 (2008-12-29) * |
| 秦俊文等: "shRNA慢病毒质粒的构建技巧", 《中国生物工程杂志》, vol. 31, no. 3, 31 March 2011 (2011-03-31) * |
| 陈华群等: "TRAF6在NF-kB信号通路中的修饰与降解", 《南京医科大学学报(自然科学版)》, vol. 24, no. 1, 31 January 2004 (2004-01-31) * |
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