CN104892770A - Lentiviral vector with efficient infectivity and multiplication capacity promoting effect for T cells and hematopoietic stem cells - Google Patents
Lentiviral vector with efficient infectivity and multiplication capacity promoting effect for T cells and hematopoietic stem cells Download PDFInfo
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Abstract
The invention provides a method for constructing a lentiviral vector with efficient transfection capacity and efficient bioactivity expression capacity. Experimental results show that the viral infection efficiency can be increased by 30-70% by using the method; in addition, the inventor unexpectedly finds that the proliferation of the T cells can also be effectively stimulated by using the technical scheme, and furthermore the treatment process of CAR-T cells is simplified, and the cost is reduced.
Description
Technical field
The invention belongs to medical biotechnology field, specifically, the present invention relates to a kind of for building the lentiviral vectors method that there is high-efficiency transfection ability and express efficient bio-active.
Background technology
Along with the development of biotechnology, genetically engineered (genetic engineering) application is medically more and more extensive.Genetically engineered take molecular genetics as theoretical basis, with molecular biology and microbiological modernism for means, by the gene of different sources by the blueprint designed in advance, build hybrid DNA molecule in vitro, then viable cell is imported, with the technology changing biological original hereditary property, obtain new variety, produce product innovation.Genetic engineering technique is that the research of the structure and function of gene provides strong means.
For the genetic modification of mammalian cell, need to use suitable carrier, foreign gene to be imported in host cell and to realize its due function.Conventional carrier comprises lentiviral vectors, adenovirus carrier and retroviral vector etc.But, still there is the problem that efficiency of infection is low, experiment flow is complicated.Therefore, it is high that those skilled in the art are devoted to develop new efficiency of infection, and the mammalian cell genetic engineering techniques that biological activity is strong.
Summary of the invention
The object of the present invention is to provide a kind of for building the lentiviral vectors method and the application thereof that there is high-efficiency transfection ability and express efficient bio-active.
A first aspect of the present invention, provide a kind of recombinant virus envelope glycoprotein, described albumen has the structure shown in formula I:
N holds ' B-A C hold ' (I)
Wherein,
Element A is the protein component derived from wild type viral envelope glycoprotein;
Element B is accessory protein element, and described accessory protein element can specific recognition surface of cell membrane albumen;
"-" represents the peptide bond or peptide linker that connect above-mentioned each element.
In another preference, described virus is slow virus, adenovirus or adeno-associated virus.
In another preference, described slow virus is blister Stomatovirus.
In another preference, described wild type viral envelope glycoprotein is blister Stomatovirus envelope glycoprotein.
In another preference, described element A is VSVG (blister stomatitis viral glycoprotein).
In another preference, the aminoacid sequence of described element A is as shown in SEQ ID NO.:2, and preferably, the encoding polynucleotide sequence of described element A is as shown in SEQ ID NO.:1.
In another preference, described surface of cell membrane albumen is the albumen being expressed in T cell surface of cell membrane; Preferably, described surface of cell membrane albumen is selected from: VLA-4, VLA-5 or its combination.
In another preference, described surface of cell membrane albumen is the albumen being expressed in hemopoietic stem cell cell film surface.
In another preference, described element B is derived from fibronectin, VCAM-1 or its active fragments (can with the polypeptide fragment of VLA-4 and/or VLA-5 specific binding).
In another preference, described element B is selected from: FN-p and active fragments, CS1 and active fragments thereof or its combination.
In another preference, the sequence of described element B is as shown in SEQ ID NO.:4 or SEQ ID NO.:6.
In another preference, the encoding polynucleotide sequence of described element B is as shown in SEQ ID NO.:3 or SEQ ID NO.:5.
In another preference, described recombinant virus envelope glycoprotein is selected from lower group:
I () has the polypeptide of aminoacid sequence shown in SEQ ID NO:19,21;
(ii) have with aminoacid sequence >=80% homology shown in SEQ ID NO:19,21 (preferably, >=
The homology of 90%; Deng the homology of preferably >=95%; Most preferably, the homology of >=97%,
As more than 98%, more than 99%) polypeptide, and described polypeptide has the work similar to polypeptide in (A)
Property;
(iii) by aminoacid sequence shown in arbitrary in (i) through the replacement of 1-5 amino-acid residue, disappearance or
Add and formed, and retaining the derivative polypeptide of polypeptide active in (i).
In another preference, described recombinant virus envelope glycoprotein has the one or more characteristics being selected from lower group:
A) there is the activity of specific recognition surface of cell membrane albumen;
B) there is the activity promoting T cell propagation.
A second aspect of the present invention, provides a kind of polynucleotide of separation, the described recombinant virus envelope glycoprotein described in polynucleotide encoding first aspect present invention.
A third aspect of the present invention, provides a kind of carrier, and it contains the polynucleotide described in second aspect present invention.
In another preference, the polynucleotide sequence of the fusion rotein containing structure shown in coding type I in described carrier,
B-A (I)
Wherein,
A is VSVG (blister stomatitis viral glycoprotein) protein component;
B is accessory protein element, and described accessory protein element can specific recognition surface of cell membrane albumen;
"-" represents the peptide bond or peptide linker that connect above-mentioned each element.
In another preference, the aminoacid sequence of described element A is as shown in SEQ ID NO.:2, and preferably, the encoding polynucleotide sequence of described element A is as shown in SEQ ID NO.:1.
In another preference, described surface of cell membrane albumen is the albumen being expressed in T cell surface of cell membrane; Preferably, described surface of cell membrane albumen is selected from: VLA-4, VLA-5 or its combination.
In another preference, described element B is derived from fibronectin or VCAM-1.
In another preference, described element B is selected from: FN-p and active fragments, CS1 and active fragments thereof or its combination.
In another preference, the aminoacid sequence of described element B is as shown in SEQ ID NO.:4 or SEQ ID NO.:6.
In another preference, the encoding polynucleotide sequence of described element B is as shown in SEQ ID NO.:3, SEQ ID NO.:5.
In another preference, described carrier is plasmid.
In another preference, described carrier is lentiviral vectors, adenovirus carrier or adenovirus related vector.
A fourth aspect of the present invention, provides a kind of genetically engineered recombinant virus, and described virus has the recombinant virus envelope glycoprotein described in first aspect present invention.
In another preference, described virus is slow virus, adenovirus or adeno-associated virus.
In another preference, in the genome of described virus, there is foreign gene.
In another preference, described foreign gene comprises the foreign gene of expressing CAR (Chimeric antigen receptor).
In another preference, described virus has at least two kinds of recombinant virus envelope glycoproteins according to claim 1 (preferably having identical element A and different element B).
In another preference, there is in the envelope glycoprotein of described virus the polypeptide of the aminoacid sequence shown in SEQ ID NO:19 and SEQ ID NO.:21.
A fifth aspect of the present invention, provides a kind of host cell, and it contains in carrier described in third aspect present invention or genome the polynucleotide be integrated with described in second aspect present invention.
In another preference, described host cell is prokaryotic cell prokaryocyte or eukaryotic cell (as Chinese hamster ovary celI, NS0 cell or 293 cells).
A sixth aspect of the present invention, provide a kind of test kit, described test kit comprises recombinant virus envelope glycoprotein, the polynucleotide described in second aspect present invention, the carrier described in third aspect present invention, the recombinant virus described in fourth aspect present invention and/or the host cell described in fifth aspect present invention described in first aspect present invention.
In another preference, described test kit comprises the carrier of the recombinant virus envelope glycoprotein of coding described in first aspect present invention; With, wild-type carrier, the wild type viral envelope glycoprotein that described wild-type vector encoded is corresponding with described recombinant virus envelope glycoprotein or its fragment.
In another preference, described test kit comprises the recombinant virus described in fourth aspect present invention.
A seventh aspect of the present invention, provides the purposes of the recombinant virus envelope glycoprotein described in first aspect present invention, the polynucleotide described in second aspect present invention, the carrier described in third aspect present invention, for the packaging of slow virus or adenovirus.
A eighth aspect of the present invention, provides a kind of Packaging Method of recombinant virus, and described method comprises step:
(1) packing cell is provided;
(2) prepare rotaring redyeing system, described rotaring redyeing system comprises the carrier of the recombinant virus envelope glycoprotein of expressing described in first aspect present invention; With
(3) Transfection of packaging cells, carries out virus packaging.
In another preference, in described step (2), described rotaring redyeing system comprises the recombinant type carrier that at least two kinds are expressed different recombinant virus envelope glycoproteins; Preferably, in described rotaring redyeing system, also comprise wild-type carrier, the wild type viral envelope glycoprotein that described wild-type vector encoded is corresponding with described recombinant virus envelope glycoprotein or its fragment.
In another preference, described recombinant type carrier is FN-p-VSVG, and/or CS1-VSVG; Described wild-type carrier is wild-type carrier H2.
In another preference, described step (2) described rotaring redyeing system comprises recombinant type carrier
FN-p-VSVG, and CS1-VSVG; And wild-type carrier H2; Preferably, described recombinant type carrier
(FN-p-VSVG and CS1-VSVG sum) is 7:3-6 with the number ratio of described wild-type carrier.
In another preference, in described recombinant type carrier, FN-p-VSVG:CS1-VSVG is 1:1.
In another preference, described packing cell is HEK-293T cell.
A ninth aspect of the present invention, provides the purposes of the recombinant virus described in fourth aspect present invention, for the preparation of the reagent producing CAR-T cell.
A tenth aspect of the present invention, provides a kind of preparation, and described preparation contains recombinant virus described in fourth aspect present invention and optional vehicle.
In another preference, described preparation is pharmaceutical preparation.
In another preference, described vehicle is pharmaceutically acceptable vehicle.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 shows the structure collection of illustrative plates of wild-type H2 plasmid.
Fig. 2 shows the structure collection of illustrative plates of the FN-p – VSVG plasmid through restructuring.
Fig. 3 shows the structure collection of illustrative plates of the CS1-VSVG plasmid through restructuring.
In Fig. 4, A figure shows in rotaring redyeing system that dissimilar H2 plasmid proportioning is on the impact of viral yield, and B figure shows the immune-blotting method result of the virion of successful expression.
Fig. 5 show in the present invention through transformation slow virus to the detected result of the efficiency of infection of T cell.
Fig. 6 shows in the present invention can promote that T cell increases significantly through the slow virus of transformation.
Fig. 7 shows in the present invention and all shows higher efficiency of infection efficiency through the slow virus of transformation to dissimilar cell.
Embodiment
The present inventor is by extensive and deep research, obtain a kind of can the technology of high-efficiency transfection mammalian cell, experimental result shows, this technology can improve virus infection efficiency and reach 30-70%, and contriver is surprised to find that, use technical scheme of the present invention can also the propagation of effective stimulus T cell, and then simplify the flow process of CAR-T cell therapy, reduce costs.
These class methods and condition before describing the present invention, be to be understood that and the invention is not restricted to described concrete grammar and experiment condition, because can change.It should also be understood that its object of term used herein is only to describe specific embodiments, and be not intended to be restrictive, scope of the present invention will only be limited by appending claims.
Unless otherwise defined, otherwise whole technology used herein and scientific terminology all have identical meanings as those skilled in the art understand usually.As used herein, mention use in the numerical value specifically enumerated time, term " about " mean this value can from enumerate value variation no more than 1%.Such as, as used herein, statement " about 100 " comprise 99 and 101 and between whole values (such as, 99.1,99.2,99.3,99.4 etc.).
Although any method similar or of equal value to described in the present invention and material can be used in enforcement of the present invention or test, exemplify preferred method and material herein herein.
Particularly, the present invention relates to a kind of novel slow virus envelope plasmid, can be applicable to the packaging of slow virus and the infection experiment of mammalian cell, comprise CAR-T cell therapy T cell activation and infection experiment.The slow virus that current normal experiment uses is all use VSVG as envelope glycoprotein, but the slow virus of this type envelope glycoprotein is when infecting the mammalian cell of some difficult infection, and efficiency is lower.By the cell-binding domain of recombinant human fibronectin polypeptide or CS1 functional domain in the present invention, with VSVG amalgamation and expression, thus make novel slow virus obtain the ability of selectively targeted VLA-5, VLA-4 antigen, improve virus infection efficiency further.Meanwhile, the fibronectin of restructuring can also stimulate the lymphocytic propagation of T.In the CAR-T cell therapy T cell activation stage, use this new lentiviral while activating T cell, namely can proceed to foreign gene, simplify experiment flow, reduce costs.And common slow virus there is no this function.
By the cell recognition domain of fibronectin and CS1 functional domain respectively with the VSVG gene fusion expression of the envelope plasmid H2 of slow virus, with vector plasmid, helper plasmid H1 cotransfection 293T cell, thus obtain can the new lentiviral particle of VLA-4, VLA-5 antigen of target cell surface.Compared with traditional method, greatly can improve the efficiency (can 30-70% be reached) of slow virus infection mammalian cell, the propagation of thousands of times of T lymphocyte can also be impelled in addition.
VSVG
In the present invention, term VSVG refers to vesicular stomatitis virus glycoprotein, of the present invention one preferred embodiment in, the aminoacid sequence of described VSVG is as follows:
mKCLLYLAFLFIGVNCkFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADG WMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQ SCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWH SDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYC KHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPA FTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGD TGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQ IYTDIEMNRLGK (SEQ ID NO.:2, contain signal peptide alternatively),
Its encoding polynucleotide sequence is:
atgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaa(SEQ ID NO.:1)。
Fibronectin
Fibronectin (Fibronectin, FN), also claims fibronectin, is a kind of polymer glycoprotein, has various biological function.FN is extensively present in animal tissues and tissue juice, and molecular weight is about 450KD, has multiple biological activity.
FN-p: fibronectin precursor (Fibronectin precursor), can be combined with the VLA-5 of cell surface, of the present invention one preferred embodiment in, the aminoacid sequence of FN-p is
PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS(SEQ ID NO.:4)，
Its encoding polynucleotide sequence is:
cccactgacctgcgattcaccaacattggtccagacaccatgcgtgtcacctgggctccacccccatccattgatttaaccaacttcctggtgcgttactcacctgtgaaaaatgaggaagatgttgcagagttgtcaatttctccttcagacaatgcagtggtcttaacaaatctcctgcctggtacagaatatgtagtgagtgtctccagtgtctacgaacaacatgagagcacacctcttagaggaagacagaaaacaggtcttgattccccaactggcattgacttttctgatattactgccaactcttttactgtgcactggattgctcctcgagccaccatcactggctacaggatccgccatcatcccgagcacttcagtgggagacctcgagaagatcgggtgccccactctcggaattccatcaccctcaccaacctcactccaggcacagagtatgtggtcagcatcgttgctcttaatggcagagaggaaagtcccttattgattggccaacaatcaacagtttctgatgttccgagggacctggaagttgttgctgcgacccccaccagcctactgatcagctgggatgctcctgctgtcacagtgagatattacaggatcacttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctgggagcaagtctacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgctgtcactggccgtggagacagccccgcaagcagcaagccaatttccattaattaccgaacagaaattgacaaaccatcc(SEQ ID NO.:3)。
CS1: another functional domain of fibronectin, can be combined with the VLA-4 of cell surface, of the present invention one preferred embodiment in, the aminoacid sequence of CS1 is
DELPQLVTLPHPNLHGPEILDVPST(SEQ ID NO.:6)，
Its encoding polynucleotide sequence is:
gacgagcttccccaactggtaacccttccacaccccaatcttcatggaccagagatcttggatgttccttccaca(SEQ ID NO.:5)。
VLA-5: Vla-4-5, is distributed in thymocyte, monocyte, thrombocyte, T cell, and part is fibronectin.
VLA-4: Vla-4-4, is distributed in thymocyte, monocyte, B cell, T cell, NK cell, and part is fibronectin and VCAM-1.
Recombinant virus envelope glycoprotein
Of the present invention one preferred embodiment in, FN-p and CS1 is packed respectively the VSVG amalgamation and expression in helper plasmid with slow virus, form different recombinant plasmid FN-p – VSVG, CS1-VSVG, by these two kinds of plasmids respectively or common with corresponding wild-type helper plasmid (as H2 plasmid, purchased from Addgene, i.e. pVSV-G, Fig. 1) carry out slow virus packaging, the efficiency of infection to mammalian cell can be increased, the function promoting T cell amplification can be played simultaneously.
Preferably scheme is as follows:
1. by FN-p and VSVG amalgamation and expression, structure is: SS-6His-FN-p-VSVG, this recombinant plasmid called after FN-p-VSVG; (SS is signal peptide, and its aminoacid sequence is MKCLLYLAFLFIGVNC, SEQ ID NO.:7)
2. by CS1 and VSVG amalgamation and expression, structure is: SS-6His-CS1-VSVG, this recombinant plasmid called after CS1-VSVG;
3. by vector plasmid (foreign gene-carrying, as EGFP EGFP), helper plasmid H1 is (purchased from Addgene, gag/pol gene containing HIV-1), helper plasmid FN-p – VSVG, helper plasmid CS1-VSVG, cotransfection 293T cell obtain virion i;
4. by viral i mammalian cell-infecting, thus foreign gene is imported in mammalian cell.
In the present invention preferably embodiment, the aminoacid sequence of SS-6His-FN-p-VSVG fusion rotein is:
MKCLLYLAFLFIGVNCHHHHHHPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSGGGSGGGSSGGGSKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK SEQ ID NO.:19，
Its encoding polynucleotide sequence is:
atgaagtgccttttgtacttagcctttttattcattggggtgaattgccatcatcatcatcat catcccactgacctgcgattcaccaacattggtccagacaccatgcgtgtcacctgggctccacccccatccattgatttaaccaacttcctggtgcgttactcacctgtgaaaaatgaggaagatgttgcagagttgtcaatttctccttcagacaatgcagtggtcttaacaaatctcctgcctggtacagaatatgtagtgagtgtctccagtgtctacgaacaacatgagagcacacctcttagaggaagacagaaaacaggtcttgattccccaactggcattgacttttctgatattactgccaactcttttactgtgcactggattgctcctcgagccaccatcactggctacaggatccgccatcatcccgagcacttcagtgggagacctcgagaagatcgggtgccccactctcggaattccatcaccctcaccaacctcactccaggcacagagtatgtggtcagcatcgttgctcttaatggcagagaggaaagtcccttattgattggccaacaatcaacagtttctgatgttccgagggacctggaagttgttgctgcgacccccaccagcctactgatcagctgggatgctcctgctgtcacagtgagatattacaggatcacttacggagaaacaggaggaaatagccctgtccaggagttcactgtgcctgggagcaagtctacagctaccatcagcggccttaaacctggagttgattataccatcactgtgtatgctgtcactggccgtggagacagccccgcaagcagcaagccaatttccattaattaccgaacagaaattgacaaaccatccggaggcggttcaggaggtggctcgagcggaggcggttcaaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaa SEQ ID NO.:18。
In the present invention preferably embodiment, the aminoacid sequence of SS-6His-CS1-VSVG fusion rotein is:
MKCLLYLAFLFIGVNCHHHHHHDELPQLVTLPHPNLHGPEILDVPSTGGGSGGGSSGGGSKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK SEQ ID NO.:21，
Its encoding polynucleotide sequence is:
atgaagtgccttttgtacttagcctttttattcattggggtgaattgccatcatcatcatcatcatgacgagcttccccaactggtaacccttccacaccccaatcttcatggaccagagatcttggatgttccttccacaggaggcggttcaggaggtggctcgagcggaggcggttcaaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccat aactctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaa SEQ ID NO.:20。
In the present invention, " fusion rotein ", " recombinant protein ", " albumen of the present invention ", " fusion rotein of the present invention " are used interchangeably, and refer to have structure described in formula Ia or Ib, namely containing the protein component comprised derived from wild type viral envelope glycoprotein; With can specific recognition surface of cell membrane albumen protein component merge polypeptide.Representational example comprises FN-p-VSVG fusion rotein and CS1-VSVG fusion rotein.In addition, should be understood that described term also comprises active fragments and the derivative of fusion rotein, these active fragmentss and derivative energy can specific binding surface of cell membrane albumen.
As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.
Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding and the present invention have protein fragments, the sum analogous to general Dedekind sum of identical aminoacid sequence.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function changing in fact its coded polypeptide.
As used herein, term " primer " refers to and is matching with template, can with it for starting point carries out synthesizing the general name with the oligonucleotide of the DNA chain of template complementation under the effect of archaeal dna polymerase.Primer can be natural RNA, DNA, also can be any type of natural nucleotide.Primer can be even that non-natural Nucleotide is as LNA or ZNA etc.The complementary that primer " haply " (or " substantially ") is special with on a chain in template.Primer could must start to extend with the abundant complementation of the chain of in template, but the sequence of primer need not with the sequence complete complementary of template.Such as, hold at 3' end and the 5' of the primer of template complementation and adds the preceding paragraph and the not complementary sequence of template, such primer still haply with template complementation.As long as there is sufficiently long primer can be combined fully with template, the primer of non-fully complementation also can form primer-template complex with template, thus increases.
The Nucleotide full length sequence of fusion rotein of the present invention or its element (as VSVG) or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.
Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.
In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.
The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR suitably can be selected according to sequence information of the present invention disclosed herein, and using conventional procedures synthesis.Using conventional procedures is as the DNA/RNA fragment increased by gel electrophoresis abstraction and purification.
The present invention also relates to the carrier comprising polynucleotide of the present invention, and with the host cell that carrier of the present invention or fusion rotein encoding sequence produce through genetically engineered, and produce method of protein of the present invention through recombinant technology.
By the recombinant DNA technology of routine, polynucleotide sequence of the present invention can be utilized to can be used to express or Restruction albumen.In general following steps are had:
(1) with the polynucleotide (or varient) of code book invention albumen of the present invention, or transform or suitable host cell of transduceing with the recombinant expression vector containing these polynucleotide;
(2) host cell cultivated in suitable substratum;
(3) separation, protein purification from substratum or cell.
Method well-known to those having ordinary skill in the art can be used for building the DNA sequences encoding containing albumen of the present invention and the suitable expression vector of transcribing/translating control signal.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, synthesizes to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as Tetrahydrofolate dehydrogenase, neomycin resistance and green fluorescent protein (GFP) that eukaryotic cell is cultivated, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell is as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast etc. of CHO, NS0, COS7 or 293 cells.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses RbCl.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.
The transformant obtained can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.
Should be understood that described term also comprises the derivative of fusion rotein of the present invention, refer to that fusion rotein of the present invention is through 1-3 aminoacid addition or replacement, a 1-2 aminoacid deletion still have the polypeptide of tumors inhibition activity.These conservative variation's polypeptide preferably carry out amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | Preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Once qualification obtains relevant peptide sequence, just related peptide sequences can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains related peptides (fusion rotein).
In addition, go back using chemical methods and directly synthesize related peptide sequences.
Peptide linker
The invention provides a kind of fusion rotein, it is optionally containing peptide linker.Peptide linker size and complicacy may affect the activity of albumen.Usually, peptide linker should have enough length and snappiness, to ensure that two albumen connected spatially have enough degree of freedom to play its function.Avoid in peptide linker, forming the impact on the stability of fusion rotein such as α spiral or β-pleated sheet structure simultaneously.
The length of connection peptides is generally 0-20 amino acid, preferably 1-15 amino acid.In order in order to be effective, the connection peptides that the present invention uses is 13 amino acid.
Major advantage of the present invention is:
(1) technical scheme of the present invention can improve the efficiency of infection for mammalian cell significantly;
(2) technical scheme of the present invention can greatly promote the propagation of T cell.
(3) Late Cambrian merge FN-p and/or CS1 on lentiviral particle surface can the function of significant stimulation T cell propagation.
Below in conjunction with specific embodiment, further detailed old the present invention.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted detailed conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.Experiment material used in following examples and reagent all can obtain from commercially available channel if no special instructions.
Embodiment 1: build recombined lentivirus vector
The structure of recombined lentivirus vector carries out based on virus packaging helper plasmid H2 (purchased from Addgene, i.e. pVSV-G) carrying VSVG gene.
FN-p-VSVG recombined lentivirus vector builds:
A) FN-p gene fragment is obtained:
(signal peptide nucleotide sequence information is SEQ ID NO.:8 to the flexible peptide fragment of full genome composite signal peptide-6His-FN-p-, 6His nucleotide sequence information is SEQ ID NO.:9, FN-p nucleotide sequence information is SEQ ID NO.:3, flexible peptide nucleotide sequence information is SEQ ID NO.:10), with full genome synthetic plasmid for template, design FN-p gene primer, forward primer 1 (5'TTCCTCGACGGATCC
cTCGAG aTGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGG3', SEQ ID NO.:11) and reverse primer 2 (5'TGGTGAACTTTGAACCGCCTCCGCTCGAGCCAC3', SEQ ID NO.:12), pcr amplification, PCR condition is: 98 DEG C of sex change 3 minutes; 98 DEG C of sex change 10 seconds, 60 DEG C of annealing 10 seconds, 72 DEG C extend 1 minute, 30 circulations; 72 DEG C 8 minutes; Obtain FN-p gene fragment thus.
B) VSVG gene fragment is obtained:
With virus packaging helper plasmid H2 (with reference to plasmid Fig. 1, SEQ ID NO.:1) be template, design VSVG gene primer, forward primer 3 (5'GAGGCGGTTCAAAGTTCACCATAGTTTTTCCAC 3', SEQ ID NO.:13) and reverse primer 4 (5'TCCTCGACGGATCC
cTCGAGtTACTTTCCAAGTCGGTTCATCTC3', SEQ ID NO.:14), pcr amplification, PCR condition is: 98 DEG C of sex change 3 minutes; 98 DEG C of sex change 10 seconds, 60 DEG C of annealing 10 seconds, 72 DEG C extend 1 minute 30 seconds, 30 circulations; 72 DEG C 8 minutes; Obtain VSVG gene fragment thus.
C) signal peptide-6His-FN-p-flexible peptide-VSVG recombination fragment is obtained:
With the gene fragment of step a, b acquisition for template, forward primer 1 (5'TTCCTCGACGGATCC
cTCGAG aTGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGG3', SEQ ID NO.:11) and reverse primer 4 (5'TCCTCGACGGATCC
cTCGAGtTACTTTCCAAGTCGGTTCATCTC 3', SEQ ID NO.:14), Overlap pcr amplification, PCR condition is: 98 DEG C of sex change 3 minutes; 98 DEG C of sex change 10 seconds, 60 DEG C of annealing 10 seconds, 72 DEG C extend 2 minutes 30 seconds, 30 circulations; 72 DEG C 8 minutes; Obtain signal peptide-6His-FN-p-flexible peptide-VSVG recombination fragment thus.
D) process of virus packaging helper plasmid H2:
Cut H2 plasmid (purchased from Addgene, i.e. pVSV-G) with XhoI enzyme, obtain 4.8Kb and 1676bp two fragments, the carrier segments of gel purification 4.8Kb.
E) In-Fusion (restructuring enzyme process) builds FN-p-VSVG recombinant retroviral vector
The enzyme that step c acquisition recombination fragment and steps d obtain is cut carrier and does In-Fusion homologous recombination, obtain FN-p-VSVG recombinant retroviral vector, its In-Fusion homologous recombination system (purchased from Suzhou Shenzhou Gene Co., Ltd.) and condition as follows:
In-Fusion Enzyme：1μl
In-Fusion Buffer：2μl
Recombination fragment: 300ng
Enzyme cuts carrier segments: 100ng
Distilled water: cumulative volume mends 10 μ l
37 DEG C 30 minutes.
CS1-VSVG recombined lentivirus vector builds:
F) CS1 gene fragment is obtained:
(signal peptide nucleotide sequence information is SEQ ID NO.:8 to the flexible peptide fragment of full genome composite signal peptide-6His-CS1-, 6His nucleotide sequence information is SEQ ID NO.:9, CS1 nucleotide sequence information is SEQ ID NO.:5, flexible peptide nucleotide sequence information is SEQ ID NO.:10), with full genome synthetic plasmid for template, design CS1 gene primer, forward primer 5 (5'CTTAGCCTTTTTATTCATTGGGGTGAATTGCCATCATCATCATCATCATGACGA GCTTCCCCAACTGGT 3', SEQ ID NO.:15) and reverse primer 6 (5'GGTTGTGTGGAAAAACTATGGTGAAC 3', SEQ ID NO.:16), pcr amplification, PCR condition is: 98 DEG C of sex change 3 minutes, 98 DEG C of sex change 10 seconds, 60 DEG C of annealing 10 seconds, 72 DEG C extend 20 seconds, 30 circulations, 72 DEG C 8 minutes, obtain CS1 gene fragment thus.
G) VSVG gene fragment is obtained:
With virus packaging helper plasmid H2 (with reference to plasmid Fig. 1, VSVG genes of SEQ ID NO.:1) be template, design VSVG gene primer, forward primer 7 (5'GTTCACCATAGTTTTTCCACACAACC 3', SEQ ID NO.:17) and reverse primer 4 (5'TCCTCGACGGATCCCTCGAGTTACTTTCCAAGTCGGTTCATCTC 3', SEQ ID NO.:14), pcr amplification, PCR condition is: 98 DEG C of sex change 3 minutes; 98 DEG C of sex change 10 seconds, 60 DEG C of annealing 10 seconds, 72 DEG C extend 1 minute 30 seconds, 30 circulations; 72 DEG C 8 minutes; Obtain VSVG gene fragment thus.
H) signal peptide-6His-CS1-flexible peptide-VSVG recombination fragment is obtained:
The gene fragment obtained with step a, b is for template, forward primer 1 (5'TTCCTCGACGGATCCCTCGAGCGCCACCATGAAGTGCCTTTTGTACTTAGCCTT TTTATTCATTGG3', SEQ ID NO.:11) and reverse primer 4 (5'TCCTCGACGGATCCCTCGAGTTACTTTCCAAGTCGGTTCATCTC 3', SEQ ID NO.:14), Overlap pcr amplification, PCR condition is: 98 DEG C of sex change 3 minutes; 98 DEG C of sex change 10 seconds, 60 DEG C of annealing 10 seconds, 72 DEG C extend 1 minute 50 seconds, 30 circulations; 72 DEG C 8 minutes; Obtain signal peptide-6His-CS1-flexible peptide-VSVG recombination fragment thus.
I) process of virus packaging helper plasmid H2:
Cut H2 plasmid with XhoI enzyme, obtain 4.8Kb and 1676bp two fragments, the carrier segments of gel purification 4.8Kb.
J) In-Fusion builds CS1-VSVG recombinant retroviral vector
The enzyme that step c acquisition recombination fragment and steps d obtain is cut carrier and does In-Fusion homologous recombination, obtain CS1-VSVG recombinant retroviral vector, its In-Fusion homologous recombination system (purchased from Suzhou Shenzhou Gene Co., Ltd.) and condition as follows:
In-Fusion Enzyme：1μl
In-Fusion Buffer：2μl
Recombination fragment: 300ng
Enzyme cuts carrier segments: 100ng
Distilled water: cumulative volume mends 10 μ l
37 DEG C 30 minutes.
K) transform
A peek TOP10 (100 μ l) competence is placed on ice, after it melts, add step e, j homologous recombination product, ice bath 30 minutes, and 42 DEG C of heat shocks 90 seconds continue ice bath 2-3 minute; Add the LB substratum 500 μ l of antibiotic-free, 37 DEG C of 220rpm cultivate 1 hour; Centrifugal 1 minute of 5000rpm, discards 450 μ l supernatant liquors, blows even by residue substratum and thalline, be coated on the LB solid medium of corresponding resistant in Bechtop; 37 DEG C of incubated overnight.
L) positive clone identification
Identify positive colony by the method for bacterium colony PCR and send order-checking, after compare of analysis result, finally determining the clone with correct sequence.
M) plasmid extraction
Use alkaline lysis or buy slow virus plasmid FN-p-VSVG, CS1-VSVG that plasmid extraction test kit extracts restructuring, after measuring concentration ,-20 DEG C of Refrigerator stores.
The present invention's gene order used is described as follows:
Signal peptide nucleotide sequence SEQ ID NO.:8:
atgaagtgccttttgtacttagcctttttattcattggggtgaattgc
6His nucleotide sequence SEQ ID NO.:9:CATCATCATCATCATCAT
Flexible peptide nucleotide sequence SEQ ID NO.:10:
GGAGGCGGTTCAGGAGGTGGCTCGAGCGGAGGCGGTTCA
H2 plasmid is with CMV promoter, and this promotor can start downstream gene expression in a large number in eukaryotic cell, inorganization specificity.Between CMV promoter and VSVG, also have β-Globin sequence, also have one section of β-Globin sequence in VSVG downstream, the existence of this sequence can strengthen the transcriptional level of VSVG gene in eukaryotic cell to a certain extent.But it should be appreciated by those skilled in the art, PN-p and the CS1 gene of insertion can also be obtained by molecular cloning method well known in the art, such as, from sample tissue, extract total serum IgE, design Auele Specific Primer, carries out RT-PCR and can obtain object Insert Fragment.
Embodiment 2: recombinant slow virus is packed
1. go down to posterity cycle supply HEK-293T cell (purchased from ATCC) according to 24h, before transfection, change liquid, often coil cell electric pipettor and change the DMEM substratum of 5ml containing 2%FBS.
2. prepare rotaring redyeing system, add plasmid H1 successively (purchased from Addgene company, 12 μ g/ dishes, dish refer under 10cm Tissue Culture Dish with), (total amount of H2 plasmid is 10 μ g/ dishes to plasmid H2, in the present embodiment, recombinant type and wild-type H2 plasmid are arranged in pairs or groups in varing proportions), vector plasmid (comprise the lentiviral vectors of foreign gene, purchased from Addgene company, 24 μ g/ dishes), CaCl
2(50 μ l/ dish), finally dropwise add 2 × HBS (500 μ l/ dish) on concussion limit, vortex oscillator top, rotaring redyeing system is 1ml/ dish.The H2 that wherein recombinates is that FN-p-VSVG recombinant plasmid and CS1-VSVG recombinant plasmid 1:1 mix.
3. carefully blow and beat rotaring redyeing system, get 1000 μ l and dropwise add 293T cell after mixing, operation keeps stable makes rotaring redyeing system be uniformly distributed in 10cm plate.
4. keep plate level, and make liquid in plate respectively rock ten times respectively all around, blending process is wanted fully, but liquid can not be had to spill or flow to outside plate wall, is then placed in 37 DEG C, 5%CO
2cultivate in incubator.After transfection 8 hours, abandoning supernatant, changed containing DMEM substratum with electric pipettor.
5. transfection terminates to start between rear 28-30 hour to collect supernatant, polishing 10ml DMEM substratum for the first time.Within 48-50 hour after transfection terminates, start second time and collect supernatant.Ultracentrifugation, is resuspended in 100ul DMEM for future use.
FN-p-VSVG and CS1-VSVG adds with the ratio of 1:1, arranges in pairs or groups in varing proportions and corresponding lentiviral particle output (strictly using the Lenti-X p24Rapid titer kit of clontech to detect), the results are shown in Figure 4A with the H2 of wild-type.Visible, when to account for H2 total amount used be 30% to FN-p-VSVG and CS1-VSVG, slow virus output is the highest.Adopt the condition gained virion of this group to carry out the method inspection of immunoblotting (Western blot), VSVG normal expression modified as seen, is shown in Fig. 4 B.
Embodiment 3: recombinant lentiviral Retroviral infects T lymphocyte
Infection experiment carries out according to ordinary method well known by persons skilled in the art.Summary infection step is as follows:
1. the acquisition of peripheral blood mononuclear lymphocyte (PBMC), obtains >1x10 by blood list extraction system
7cell.
2. use perfect medium (autoserum of TexMACS substratum+5%) adjustment member cell suspension to make its final concentration be 0.7 × 10
6/ ml, and adding interleukin-22 (IL-2), penicillin (penicillin) and Streptomycin sulphate (streptomycin), OKT3 and bovine serum albumin (BSA), make its final concentration be respectively 600IU/ml, 100U/ml, 100 μ g/ml and 0.2%.
3. resuspended good cell is mixed gently, take out cell suspension (total cell concentration 0.28 × 10 of 0.4ml
6) be transferred in 1.5mL centrifuge tube.
4. adding required virus, is 10 to calculate the virus quantity of interpolation according to MOI.
5. the virus added and cell suspension are mixed gently, then cell and viral mixed solution are transferred in 24-orifice plate.
6. cell plate are placed in centrifugal 30 minutes of the board-like whizzer 1000g of 32 DEG C of preheatings.
7. be then put in 37 DEG C, 5%CO
2incubator cultivate.
After 8.8-12 hour, perfect medium (the interleukin-22 (IL-2) of the autoserum+600IU/ml of TexMACS substratum+5% of 1mL is added to the PBMC cell infecting virus, the penicillin (penicillin) of 100U/ml, the Streptomycin sulphate (streptomycin) of 100 μ g/ml and 0.2% bovine serum albumin (BSA).
9. every other day carried out changing liquid by cell with perfect medium in cell cultivation process, the density maintaining cell maintains 0.5-2 × 10 at maintenance cell density
6/ ml.
10. born of the same parents are put in 37 DEG C, 5%CO
2incubator cultivate in cultivate after 4 days and carry out effect survey.
Embodiment 4: the effect detection of recombinant slow virus
1. efficiency of infection detects
1.1 collect 1 × 10 after cell counting
6individual cell, the centrifugal 5min of 400g.
1.2 discard substratum, add 500 μ l streaming washing lotions (PBS, PH7.2 ,+2%BSA, 0.22 μm of membrane filtration), and washing once.
1.3 remove the streaming washing lotions bullet gently that formed sediment by heavy for cell finger falls apart.
1.4 add FACS washing lotion, and 4 DEG C of 400g 5min are centrifugal, abandon supernatant;
1.5 to add 400ul FACS washing lotion resuspended by cell, then transfer to 400ul cell suspension in 96-orifice plate, and machine testing in preparation, detected result is shown in Fig. 5.
As seen from Figure 5, to the infection of T cell, the slow virus infection efficiency of wild-type VSVG bag quilt is obviously weaker than the VSVG through transformation.
Embodiment 5: the slow virus through transformation can promote that T cell is bred
Cell in embodiment 4, except carrying out streaming, also within continuous many days, carry out cell counting, its growth curve as shown in Figure 6.
As seen from Figure 6, improved T Lymphocyte expansion multiple significantly improves, and is about more than 2 times of wild-type.
Embodiment 6: the multiple mammalian cell of improved slow virus infection
Experiment is carried out according to ordinary method well known by persons skilled in the art.Infect h CD34+, hMSC and hADSC cell respectively.Directly to adding wild-type or reworked VSVG slow virus in cell culture fluid respectively, judge efficiency of infection by streaming counting afterwards.Result as shown in Figure 7.
Result shows, for different cells, the slow virus of reworked all can raising efficiency of infection in various degree.Wherein, the efficiency of infection for h CD34+ cell improves about 55%, improves about 25% to the efficiency of infection of hMSC cell, improves about 70% to the efficiency of infection of hADSC cell.
Sum up
1) by FN-p and CS1 respectively with VSVG protein fusion expression, obtain can be interactional with cell surface protein VLA-4 and VLA-5 VSVG albumen, and then obtain the slow virus with more wide range system cell interaction.
2), in wrapping process, by hybrid packed for the helper plasmid of VLA-4 and VLA-5 and wild-type, the virus that can significantly improve efficiency of infection can be obtained while guaranteeing viral yield.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Reference:
[01]N Engl J Med.2013Apr 18；368(16):1509-18.doi:10.1056/NEJMoa1215134.Epub 2013Mar 25.Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.Grupp SA1,Kalos M,Barrett D,Aplenc R,Porter DL,Rheingold SR,Teachey DT,Chew A,Hauck B,Wright JF,Milone MC,Levine BL,June CH.
[02]Biomaterials.2014Apr；35(13):4204-12.doi:10.1016/j.bioma-terials.2014.01.051.Systematic improvement of lentivirus transduction protocols by antibody fragments fused to VSV-G asenvelope glycoprotein.
I1,Barth S2,Salomon M3,Jagusch V3,Atkinson MJ1,Anastasov N1,Thirion C4.
[03]Leukemia.2015Feb；29(2):415-22.doi:10.1038/leu.2014.215.Epub2014Jul 9.IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia.Pegram HJ1,Purdon TJ1,van Leeuwen DG1,Curran KJ2,Giralt SA3,Barker JN3,Brentjens RJ4.
[04]Blood.2013Oct 24；122(17):2965-73.doi:10.1182/blood-2013-06-506741.Epub 2013Sep 12.Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant:a phase 1study.Cruz CR1,Micklethwaite KP,Savoldo B,Ramos CA,Lam S,Ku S,Diouf O,Liu E,Barrett AJ,Ito S,Shpall EJ,Krance RA,Kamble RT,Carrum G,Hosing CM,Gee AP,Mei Z,Grilley BJ,Heslop HE,Rooney CM,Brenner MK,Bollard CM,Dotti G. 。
Claims (10)
1. a recombinant virus envelope glycoprotein, is characterized in that, described albumen has the structure shown in formula I:
N holds ' B-A C hold ' (I)
Wherein,
Element A is the protein component derived from wild type viral envelope glycoprotein;
Element B is accessory protein element, and described accessory protein element can specific recognition surface of cell membrane albumen;
"-" represents the peptide bond or peptide linker that connect above-mentioned each element;
Preferably, described wild type viral envelope glycoprotein is blister Stomatovirus envelope glycoprotein;
More preferably, described element B is derived from fibronectin, VCAM-1 or its active fragments (can with the polypeptide fragment of VLA-4 and/or VLA-5 specific binding);
Most preferably, described recombinant virus envelope glycoprotein is selected from lower group:
I () has the polypeptide of aminoacid sequence shown in SEQ ID NO:19,21;
(ii) have and the aminoacid sequence >=80% homology (homology of preferably, >=90% shown in SEQ ID NO:19,21; Deng the homology of preferably >=95%; Most preferably, the homology of >=97%, as more than 98%, more than 99%) polypeptide, and described polypeptide has the activity similar to polypeptide in (A);
(iii) aminoacid sequence shown in arbitrary in (i) is formed through the replacement of 1-5 amino-acid residue, disappearance or interpolation, and retain the derivative polypeptide of (i) middle polypeptide active.
2. the polynucleotide be separated, is characterized in that, described polynucleotide encoding recombinant virus envelope glycoprotein according to claim 1.
3. a carrier, is characterized in that, it contains polynucleotide according to claim 2.
4. a genetically engineered recombinant virus, is characterized in that, described virus has recombinant virus envelope glycoprotein according to claim 1.
5. a host cell, is characterized in that, it contains in carrier according to claim 3 or genome and is integrated with polynucleotide according to claim 2.
6. a test kit, it is characterized in that, described test kit comprises recombinant virus envelope glycoprotein according to claim 1, polynucleotide according to claim 2, the carrier described in claim 3, recombinant virus according to claim 4 and/or host cell according to claim 5.
7. the purposes of polynucleotide, carrier according to claim 3 described in recombinant virus envelope glycoprotein according to claim 1, claim 2, for the packaging of slow virus or adenovirus.
8. a Packaging Method for recombinant virus, is characterized in that, described method comprises step:
(1) packing cell is provided;
(2) prepare rotaring redyeing system, described rotaring redyeing system comprises the carrier of expressing recombinant virus envelope glycoprotein according to claim 1; With
(3) Transfection of packaging cells, carries out virus packaging.
9. the purposes of recombinant virus according to claim 4, is characterized in that, for the preparation of the reagent producing CAR-T cell.
10. a preparation, is characterized in that, described preparation contains recombinant virus according to claim 4 and optional vehicle.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978442A (en) * | 2016-01-18 | 2017-07-25 | 爱康得生物医学技术（苏州）有限公司 | A kind of preparation method of Chimeric antigen receptor T cell |
| CN112646928A (en) * | 2020-12-25 | 2021-04-13 | 康美润源(北京)生物技术有限公司 | Detection kit and detection method for VSVG gene residual quantity |
| CN114702571A (en) * | 2022-04-24 | 2022-07-05 | 安博威生物科技(厦门)有限公司 | Fibronectin with function of promoting stem cell colonization and preparation method thereof |
| CN117645664A (en) * | 2024-01-29 | 2024-03-05 | 英特菲尔(成都)生物制品有限责任公司 | Recombinant human fibronectin standard substance, preparation method, identification method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007095201A2 (en) * | 2006-02-15 | 2007-08-23 | The Regents Of The University Of California | Pseudotyped retroviral vectors and methods of use thereof |
| CN101532031A (en) * | 2009-03-03 | 2009-09-16 | 上海吉盛制药技术有限公司 | Non-integration slow virus vector system and preparation and application thereof |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007095201A2 (en) * | 2006-02-15 | 2007-08-23 | The Regents Of The University Of California | Pseudotyped retroviral vectors and methods of use thereof |
| CN101532031A (en) * | 2009-03-03 | 2009-09-16 | 上海吉盛制药技术有限公司 | Non-integration slow virus vector system and preparation and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978442A (en) * | 2016-01-18 | 2017-07-25 | 爱康得生物医学技术（苏州）有限公司 | A kind of preparation method of Chimeric antigen receptor T cell |
| CN112646928A (en) * | 2020-12-25 | 2021-04-13 | 康美润源(北京)生物技术有限公司 | Detection kit and detection method for VSVG gene residual quantity |
| CN114702571A (en) * | 2022-04-24 | 2022-07-05 | 安博威生物科技(厦门)有限公司 | Fibronectin with function of promoting stem cell colonization and preparation method thereof |
| CN114702571B (en) * | 2022-04-24 | 2023-07-25 | 安博威生物科技(厦门)有限公司 | Fibronectin capable of promoting stem cell colonization and preparation method thereof |
| CN117645664A (en) * | 2024-01-29 | 2024-03-05 | 英特菲尔(成都)生物制品有限责任公司 | Recombinant human fibronectin standard substance, preparation method, identification method and application thereof |
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