CN108530541A - A kind of recombination of Chimeric antigen receptor for CD22 is built and its application - Google Patents

A kind of recombination of Chimeric antigen receptor for CD22 is built and its application Download PDF

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CN108530541A
CN108530541A CN201710119499.1A CN201710119499A CN108530541A CN 108530541 A CN108530541 A CN 108530541A CN 201710119499 A CN201710119499 A CN 201710119499A CN 108530541 A CN108530541 A CN 108530541A
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recombinant protein
antigen receptor
chimeric antigen
dna
seq
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张同存
柳浥时
顾潮江
廖兴华
汤杰
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Wuhan Ruida Biotechnology Co Ltd
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Wuhan Ruida Biotechnology Co Ltd
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Abstract

The present invention discloses structure and the application of a kind of third generation Chimeric antigen receptor gene for CD22 molecules.The recombinant protein of the antigen receptor gene code includes such as lower part：Extracellular region, by the series connection single chain variable fragment V for CD22 antigensHAnd VLIt is formed with hinge area；Transmembrane region is originated from CD8 α；Intracellular region is made of with different permutation and combination methods tri- structures of ligand CD137 and CD80 of CD3 ζ and costimulatory molecules, is directly connected between three structures or is connected by Linker.By the third generation Chimeric antigen receptor gene fragment clone to plasmid vector, Lentiviral or retroviral vector, rotaring redyeing 293 cell system etc. is expanded, recombination is obtained after isolating and purifying, the T cell for transfecting from or separating in vivo is proliferated the CAR T cells being prepared to CD22 positive tumor cells with significant fragmentation effect after filtering out positive colony.

Description

A kind of recombination of Chimeric antigen receptor for CD22 is built and its application
Technical field
The invention belongs to biotechnologies, and present invention relates particularly to a kind of Chimeric antigen receptors for CD22（CAR）Weight Group is gene constructed and its applies.
Background technology
Chimeric antigen receptor (chimeric antigen receptor, CAR) recent years are rapidly developed, and Clinically it is applied to the treatment for kinds of tumors.The route of the technology mainly has 3 key steps：
1, the T cell of patient is detached, in vitro culture increment is carried out；
It 2, will be for the antibody variable region of tumor-cell antigen and transmembrane region and intracellular signal area weight using gene recombination technology Group constitutes new gene, is cloned into carrier system and is transfected the T cell for importeding into vitro culture, is then separated to positive gram Grand T cell；
3, patient's body is inputted after being proliferated positive colony T cell so that these T cell tumor cells, startup are subsequently exempted from Epidemic disease reacts to kill tumour cell；
CAR-T cell technologies are used to treat tumour, and most important feature is to be capable of the kill tumour cell of high degree of specificity, can A variety of difference CAR are developed with the specific antigen using tumor cell surface expression.The structure of CAR is broadly divided into extracellular anti- Former bonding land, hinge region, transmembrane region and intracellular signal area, anti-tumor effect dependent on extracellular antigen binding domain targeting, The flexibility of hinge region, intracellular signal area are for stimulation molecule and the synergistic effect of T cell activation sequence.
Invention content
The present invention is directed to the antigens c D22 of tumor cell specific, develops the CD22 Chimeric antigen receptor bases of third generation recombination Cause.
A kind of Chimeric antigen receptor recombinant protein for CD22, the recombinant protein are made of three sections in series, point It is not extracellular region, transmembrane region and intracellular region, wherein：
The extracellular region, by being connected in series for the antibody variable region of antigens c D22；
The transmembrane region is originated from CD8 α；
The intracellular region is connected in series by the polypeptide fragment permutation and combination of three separate sources.
Chimeric antigen receptor recombinant protein as described above, it is preferable that the nucleotides sequence for encoding the recombinant protein is classified as：
(A) recombinant DNA with the nucleotide sequence as shown in SEQ ID NO.1；Or
(B) there is the DNA sequence dna with the homology of nucleotide sequence shown in SEQ ID NO.1 >=75%；
Chimeric antigen receptor recombinant protein as described above, it is preferable that the amino acid sequence of the recombinant protein is：
(A) recombinant protein with the amino acid sequence as shown in SEQ ID NO.1；Or
(B) there is the polypeptide or recombinant protein with the homology of amino acid sequence shown in SEQ ID NO.1 >=85%.
Chimeric antigen receptor recombinant protein as described above, it is preferable that the coding nucleotide of the recombinant protein is to pass through It is artificial synthesized to obtain the nucleotide sequence of various pieces, it is then spliced and is cloned on expression vector using PCR amplification.
Chimeric antigen receptor recombinant protein as described above, it is preferable that the expression vector includes but not limited to：Plasmid carries Body, slow virus carrier or retroviral vector.
Chimeric antigen receptor recombinant protein as described above, it is preferable that connect the antigen receptor variable region of the extracellular region Unit can be made of the identical or different VhVl units of 1 to 3 groups.
Chimeric antigen receptor recombinant protein as described above, it is preferable that the nucleotide for encoding the recombinant protein, according to Its corresponding DNA sequence dna, the oligonucleotides for designing and synthesizing a plurality of staggeredly 60 to 80 complementary bases use after annealing T4DNA ligases link, and are obtained using PCR amplification.
Chimeric antigen receptor recombinant protein as described above, it is preferable that three polypeptide fragments of the intracellular region, are different rows Any type of row sequence, be directly connected between segment or using the polypeptide linker connections that less than 10 amino acid forms and At.
Chimeric antigen receptor recombinant protein as described above, it is preferable that three polypeptide fragments of the intracellular region, including signal Area, costimulation ligand and cell factor, wherein the signaling zone comes from CD3 δ, and the costimulation ligand one is matched from TNF classes Body, secondly coming from the Ig superfamily molecular cell factors；The cell factor includes IL-2, IL-3, IL-6, IL-7, IL-11, IL- 12, IL-15, IL-21 or GM-CSF.
Chimeric antigen receptor recombinant protein as described above, it is preferable that state TNF class ligands include 4-1BBL, OX40L, CD70, LIGHT and CD30L；The Ig superfamilies molecule is CD80 and CD86.
The present invention is directed to CD22 antigens, and the Chimeric antigen receptor full-length gene of structure uses the side of full genome de novo formation Method can efficiently and rapidly build the Chimeric antigen receptor of various various combinations.
Description of the drawings
Full-length gene structure three kind different arrangement mode schematic diagrames of the Fig. 1 for the Chimeric antigen receptor of CD22.
Specific implementation mode
Embodiment 1
The technology path that the present invention uses is：
The first step：Prepare the monoclonal antibody for CD22 antigens；
Second step：Variable region gene sequence of monoclonal antibody and the progress for CD18 antigens are cloned using the method for RT-PCR Sequencing；
Third walks：Variable region sequences VH, VL, hinge area, transmembrane region, Intracellular signals area are closed using full genome synthetic method At；
4th step：The full-length gene of synthesis is cloned into expression vector, and is proliferated in 293 cells.
The preparation of the monoclonal antibody is using conventional preparation method, and specific steps include mainly immune animal, carefully Born of the same parents are merged, and selectivity is cultivated, the screening and purifying of hybridoma positive colony.
The RT-PCR is obtained using the precious biological reverse transcription PCR kit in Dalian using conventional experimental method.
Three structures of the signaling zone sections in series, which is characterized in that one from CD3 δ, other two parts difference One kind in TNF classes ligand, Ig superfamilies molecule or cell factor；TNF class ligands include CD137, OX40L, CD70, LIGHT and CD30L；Ig superfamily molecules are CD80 and CD86；Cell factor include IL-2, IL-3, IL-6, IL-7, IL-11, IL-12、IL-15、IL-21。
Three structures of the signaling zone sections in series, which is characterized in that three structures can use various ways arrangement group It closes, by taking CD3 δ, CD137 and CD86 as an example, the arrangement mode of these three segments can be CD3 δ ~ CD137 ~ CD86, CD3 δ ~ CD86 ~ CD137, CD137 ~ CD3 δ ~ CD86, CD137 ~ CD86 ~ CD3 δ, CD86 ~ CD3 δ ~ CD137, CD86 ~ CD137 ~ CD3 δ's is any One kind, then the different arrangement mode schematic diagrames of three kinds of the full-length gene structure of the Chimeric antigen receptor of CD22 are as shown in Figure 1.
The present invention has synthesized the Chimeric antigen receptor recombinant protein for CD22, variable region albumen（Vh-Vl）Sequence is （SEQ ID NO.3）：
MESKYGPPCPPCPAPEFLGGPSVFVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK
It corresponds to the nucleotides sequence of coding and is classified as（SEQ ID NO.4）： ATGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCGTTAG AAGGGCCCCCCTCTCTGAAGGCCCCCACTCCCTGGGATGCTACAATCCAATGATGGAAGATGGCATTAGCTACACCA CCCTGCGCTTTCCAGAGATGAACATACCACGAACTGGGGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAG CAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAA GTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGC CCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTGAAGGGC TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCT TTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGTCCCTGAGCCTGGGCAAGATG
Transmembrane region CD8 α transmembrane regions, amino acid sequence are（SEQ ID NO.5）：
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS；
It corresponds to the nucleotides sequence of coding and is classified as（SEQ ID NO.6）：
TTCTGGGTGCTGGTCGTGGTGGGTGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCT TTTGGGTGAGGAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACC CGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGACTTCGCCGCCTACCGGAGC
Intracellular region sequence C D3 δ, amino acid sequence are（SEQ ID NO.7）：
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；
It corresponds to the nucleotides sequence of coding and is classified as（SEQ ID NO.8）：
CGGGTGAAGTTCAGCCGGAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGA ACCTGGGCCGGAGGGAGGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCGG AGAAAGAACCCTCAGGAGGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCAT GAAGGGCGAGCGGCGGAGGGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTACG ACGCCCTGCACATGCAGGCCCTGCCCCCCAGATGA
Intracellular region sequence C D137, amino acid sequence are（SEQ ID NO.9）：
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；
It corresponds to the nucleotides sequence of coding and is classified as（SEQ ID NO.10）：
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGG AAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG。
Intracellular region sequence C D86, amino acid sequence are（SEQ ID NO.11）：
SCRFPEEEEGGCELKRGRKKLLYIFKQPFMRPVQTTQEEDGC；
It corresponds to the nucleotides sequence of coding and is classified as（SEQ ID NO.12）：
GTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGGGGCAGAAAGAAACTCCTGTATAT ATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCT。
For the Chimeric antigen receptor recombination of CD22, its complete sequence is (SEQ ID NO.1)： ATGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCGTTAG AAGGGCCCCCCTCTCTGAAGGCCCCCACTCCCTGGGATGCTACAATCCAATGATGGAAGATGGCATTAGCTACACCA CCCTGCGCTTTCCAGAGATGAACATACCACGAACTGGGGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAG CAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAA GTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGC CCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTGAAGGGC TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCT TTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGTCCCTGAGCCTGGGCAAGATG TTCTGGGTGCTGGTCGTGGTGGGTGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTG GGTGAGGAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCACCCGGA AGCACTACCAGCCCTACGCCCCTCCCAGGGACTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCCGGAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACC AGCTGTACAACGAGCTGAACCTGGGCCGGAGGGAGGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAG ATGGGCGGCAAGCCCCGGAGAAAGAACCCTCAGGAGGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGC CTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGGGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCG CCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGATGA
The encoded protein sequence of Chimeric antigen receptor recombination for CD22 is (SEQ ID NO.2）：
MESKYGPPCPPCPAPEFLGGPSVFVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGKMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
The present invention synthesizes the preparation method of template used when the recombination：80 bases are designed to 90 according to gene order The oligonucleotides of a bases longs, covering gene sequence double-strand, interlaced complementation take each 0.1OD of synthetic oligonucleotides It is put into screw lid plastic miniature test tube, 5 μ L of sterilizing 5MNaCl solution are added, then use ddH2O polishings screw lid to 200 μ L, hang Float on containing in at least pot of 5L water, boils after ten minutes, cooled to room temperature, take templates of the 1 μ L as PCR amplification.
PCR sense primers are SEQ ID NO.13：ATGGAGAGCAAGTACGG, downstream primer are SEQ ID NO.14： TCATCTGGGGGGCAGGG.PCR amplification condition is：95 DEG C denaturation after five minutes, 93 DEG C 30 seconds, 61 DEG C 30 seconds, 72 DEG C 1 minute Second, 35 cycles, last 72 DEG C extend 11 minutes.
Reaction system is to gather specificity amplification primer, PCR buffer solutions, deoxynucleotide triphosphates mixture and Pfu DNA Synthase is added template DNA, is made into PCR reaction solution；
After PCR amplification, takes 5ul into row agarose gel electrophoresis, select the electrophoretic band of 1.3k to recycle and be tapped and recovered, make With Axygen gel reclaims kits.Rear clone is recycled to pucm-T carriers, 5 clones of picking carry out sequence verification, select 2 sequences are correctly cloned.
Embodiment 2
To carry out illustratively specific implementation mode for Vh-Vl-CD8 α-CD3 ζ-CD137-CD80 this structure：
Complete genome sequence is divided into the oligonucleotide sequence of several 80 bases or so and is synthesized by biotech firm complete by the first step Portion's oligonucleotides, each synthetic quantity are 1OD.Wherein anti-chain complete sequence start-up portion deletes 40 bases so that normal chain anti-chain Oligonucleotides can staggeredly complementation.
Complete genome sequence normal chain is：
Normal chain segment 1（SEQ ID NO.15）：
ATGGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCG TTAGAAGGGCCC
Normal chain segment 2（SEQ ID NO.16）：
CCCTCTCTGAAGGCCCCCACTCCCTGGGATGCTACAATCCAATGATGGAAGATGGCATTAGCTACACCACCCT GCGCTTTCCAGAGA
Normal chain segment 3（SEQ ID NO.17）：
TGAACATACCACGAACTGGGGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCAC CTACCGGGTGG
Normal chain segment 4（SEQ ID NO.18）：
TGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGG CCTGCCCAGCA
Normal chain segment 5（SEQ ID NO.19）：
GCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCA AGAGGAGATGA
Normal chain segment 6（SEQ ID NO.20）：
CCAAGAATCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAG CAACGGCCAGCCC
Normal chain segment 7（SEQ ID NO.21）：
GAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCG TGGACAAGAGCCG
Normal chain segment 8（SEQ ID NO.22）：
GTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGC CTGTCCCTGAGCC
Normal chain segment 9（SEQ ID NO.23）：
TGGGCAAGATGTTCTGGGTGCTGGTCGTGGTGGGTGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGC CTTCATCATCTTT
Normal chain segment 10（SEQ ID NO.24）：
TGGGTGAGGAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCCCGGAGGCCTGGCCCCA CCCGGAAGCAC
Normal chain segment 11（SEQ ID NO.25）：
TACCAGCCCTACGCCCCTCCCAGGGACTTCGCCGCCTACCGGAGCAAACGGGGCAGAAAGAAACTCCTGTATA TATTCAAACAACCA
Normal chain segment 12（SEQ ID NO.26）：
TTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTGCG
Normal chain segment 13（SEQ ID NO.27）：
GGTGAAGTTCAGCCGGAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAAC CTGGGCCGGAG
Normal chain segment 14（SEQ ID NO.28）：
GGAGGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCGGAGAAAGAAC CCTCAGGAG
Normal chain segment 15（SEQ ID NO.29）：
GGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGC GGAGGGGCAAG
Normal chain segment 16（SEQ ID NO.30）：
GGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCC TGCCCCCCAGATGA
Complementary strand
Anti-chain segment 1（SEQ ID NO.31）：
GTATCCTTGGTGGCGGTGCTCAGGCCCTGGTACAGGCCGTCGTGGCCCTTGCCCCTCCGCCGCTCGCCCTTCA TGCCGATCTCGCT
Anti-chain segment 2（SEQ ID NO.32）：
GTAGGCCTCGGCCATCTTGTCTTTCTGCAGTTCGTTATACAGGCCCTCCTGAGGGTTCTTTCTCCGGGGCTTG CCGCCCATCTCAGGG
Anti-chain segment 3（SEQ ID NO.33）：
TCCCGGCCTCTCCGCTTGTCCAGCACGTCGTACTCCTCCCTCCGGCCCAGGTTCAGCTCGTTGTACAGCTGGT TCTGGCCCTGCTGG
Anti-chain segment 4（SEQ ID NO.34）：
TAGGCAGGGGCGTCGGCGCTCCGGCTGAACTTCACCCGCAGTTCACATCCTCCTTCTTCTTCTTCTGGAAATC GGCAGCTACAGCCAT
Anti-chain segment 5（SEQ ID NO.35）：
CTTCCTCTTGAGTAGTTTGTACTGGTCTCATAAATGGTTGTTTGAATATATACAGGAGTTTCTTTCTGCCCCG TTTGCTCCGGTAGGCG
Anti-chain segment 6（SEQ ID NO.36）：
GCGAAGTCCCTGGGAGGGGCGTAGGGCTGGTAGTGCTTCCGGGTGGGGCCAGGCCTCCGGGGGGTCATGTTCA TGTAGTCGCT
Anti-chain segment 7（SEQ ID NO.37）：
GTGGCCGCCTCTGCTCCGCTTGCTCCTCACCCAAAAGATGATGAAGGCCACTGTCACCAGCAGGCTGTAGCAG GCCAGCACGCC
Anti-chain segment 8（SEQ ID NO.38）：
ACCCACCACGACCAGCACCCAGAACATCTTGCCCAGGCTCAGGGACAGGCTCTTCTGGGTGTAGTGGTTGTGC AGGGCCTCGTG
Anti-chain segment 9（SEQ ID NO.39）：
CATCACGGAGCAGCTAAAGACGTTGCCCTCCTGCCACCGGCTCTTGTCCACGGTCAGCCTGCTGTACAGGAAG AAGCTGCCGTC
Anti-chain segment 10（SEQ ID NO.40）：
GCTGTCCAGCACAGGGGGGGTGGTCTTGTAGTTGTTCTCGGGCTGGCCGTTGCTCTCCCACTCCACGGCGATG TCGCTGGGG
Anti-chain segment 11（SEQ ID NO.41）：
TAGAAGCCCTTCACCAGGCAGGTCAGGGACACCTGATTCTTGGTCATCTCCTCTTGGCTAGGGGGCAGGGTGT ACACCTGGG
Anti-chain segment 12（SEQ ID NO.42）：
GCTCCCGAGGCTGGCCCTTGGCCTTGCTGATGGTTTTCTCGATGCTGCTGGGCAGGCCCTTGTTGGACACCTT ACACTTGTAT
Anti-chain segment 13（SEQ ID NO.43）：
TCCTTGCCGTTCAGCCAGTCCTGGTGCAGCACGGTCAGCACGGACACCACCCGGTAGGTGCTATTGAACTGCT CCTCCCGG
Anti-chain segment 14（SEQ ID NO.44）：
GGCTTGGTCTTGGCGTTGTGCACCTCCACCCCAGTTCGTGGTATGTTCATCTCTGGAAAGCGCAGGGTGGTGT AGCTAATGCC
Anti-chain segment 15（SEQ ID NO.45）：
ATCTTCCATCATTGGATTGTAGCATCCCAGGGAGTGGGGGCCTTCAGAGAGGGGGGCCCTTCTAACGAACACG CTGGGTCCGCCCAGGAACTCGGGGGC
Second step prepares PCR amplification template, dissolves each primer with 50 μ L sterilizing distilled waters, every primer takes 5 μ L, i.e., 0.1OD is put into jointly in a screw lid plastic miniature test tube, 5 μ L of sterilizing 5MNaCl solution is added, then use ddH2O polishings are to 200 μ L screws lid, is suspended in containing in at least pot of 5L water, boils after ten minutes, cooled to room temperature, 1 μ L is taken to expand as PCR The template of increasing.
Third walks, and carries out DNA cloning full-length gene, and the PCR sense primers used are SEQ ID NO.46： ATGGAGAGCAAGTACGG, downstream primer are SEQ ID NO.47：TCATCTGGGGGGCAGGG.PCR amplification condition is：95℃ Denaturation after five minutes, 93 DEG C 30 seconds, 61 DEG C 30 seconds, 72 DEG C of 1 minute seconds, 35 cycle, it is last 72 DEG C extend 11 minutes.Amplification is anti- It is that mould is added in specificity amplification primer, PCR buffer solutions, deoxynucleotide triphosphates mixture and Pfu archaeal dna polymerases to answer system Plate DNA, is made into PCR reaction solution；
4th step after PCR amplification, takes 5ul into row agarose gel electrophoresis, selects the electrophoretic band of 1.3k to recycle and tap rubber back It receives, uses Axygen gel reclaims kits.Rear clone is recycled to pucm-T carriers, 20 clones of picking carry out sequence verification, This example has been selected 2 sequences and has correctly been cloned.
Recombination is used seamless Cloning Kit, is cloned into slow virus carrier by the 5th step.
Other different permutation and combination also take similar method, and DNA sequence dna is changed according to required arrangement mode Afterwards, this process is repeated using identical method.
The foregoing examples are only illustrative of the present invention, does not constitute the limitation to protection scope of the present invention, all Be with the present invention it is same or analogous design all belong to the scope of protection of the present invention within.
SEQUENCE LISTING
<110>Wuhan wave is farsighted to reach bio tech ltd
<120>A kind of recombination of Chimeric antigen receptor for CD22 is built and its application
<130>
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 1362
<212> DNA
<213>It is artificial synthesized
<400> 1
atggagagca agtacggccc tccctgcccc ccttgccctg cccccgagtt cctgggcgga 60
cccagcgtgt tcgttagaag ggcccccctc tctgaaggcc cccactccct gggatgctac 120
aatccaatga tggaagatgg cattagctac accaccctgc gctttccaga gatgaacata 180
ccacgaactg gggtggaggt gcacaacgcc aagaccaagc cccgggagga gcagttcaat 240
agcacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 300
gaatacaagt gtaaggtgtc caacaagggc ctgcccagca gcatcgagaa aaccatcagc 360
aaggccaagg gccagcctcg ggagccccag gtgtacaccc tgccccctag ccaagaggag 420
atgaccaaga atcaggtgtc cctgacctgc ctggtgaagg gcttctaccc cagcgacatc 480
gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 540
ctggacagcg acggcagctt cttcctgtac agcaggctga ccgtggacaa gagccggtgg 600
caggagggca acgtctttag ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 660
cagaagagcc tgtccctgag cctgggcaag atgttctggg tgctggtcgt ggtgggtggc 720
gtgctggcct gctacagcct gctggtgaca gtggccttca tcatcttttg ggtgaggagc 780
aagcggagca gaggcggcca cagcgactac atgaacatga ccccccggag gcctggcccc 840
acccggaagc actaccagcc ctacgcccct cccagggact tcgccgccta ccggagcaaa 900
cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 960
actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1020
ctgcgggtga agttcagccg gagcgccgac gcccctgcct accagcaggg ccagaaccag 1080
ctgtacaacg agctgaacct gggccggagg gaggagtacg acgtgctgga caagcggaga 1140
ggccgggacc ctgagatggg cggcaagccc cggagaaaga accctcagga gggcctgtat 1200
aacgaactgc agaaagacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 1260
cggcggaggg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggat 1320
acctacgacg ccctgcacat gcaggccctg ccccccagat ga 1362
<210> 2
<211> 453
<212> PRT
<213>It is artificial synthesized
<400> 2
Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Val Arg Arg Ala Pro Leu Ser Glu
20 25 30
Gly Pro His Ser Leu Gly Cys Tyr Asn Pro Met Met Glu Asp Gly Ile
35 40 45
Ser Tyr Thr Thr Leu Arg Phe Pro Glu Met Asn Ile Pro Arg Thr Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly Lys Met Phe Trp Val Leu Val Val Val Gly Gly
225 230 235 240
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
245 250 255
Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn
260 265 270
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
275 280 285
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys
290 295 300
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
305 310 315 320
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
325 330 335
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
340 345 350
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
355 360 365
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
370 375 380
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
385 390 395 400
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
405 410 415
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
420 425 430
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
435 440 445
Ala Leu Pro Pro Arg
450
<210> 3
<211> 230
<212> PRT
<213>It is artificial synthesized
<400> 3
Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Phe Leu Gly Gly Pro Ser Val Phe Val Arg Arg Ala Pro Leu Ser Glu
20 25 30
Gly Pro His Ser Leu Gly Cys Tyr Asn Pro Met Met Glu Asp Gly Ile
35 40 45
Ser Tyr Thr Thr Leu Arg Phe Pro Glu Met Asn Ile Pro Arg Thr Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly Lys
225 230
<210> 4
<211> 693
<212> DNA
<213>It is artificial synthesized
<400> 4
atggagagca agtacggccc tccctgcccc ccttgccctg cccccgagtt cctgggcgga 60
cccagcgtgt tcgttagaag ggcccccctc tctgaaggcc cccactccct gggatgctac 120
aatccaatga tggaagatgg cattagctac accaccctgc gctttccaga gatgaacata 180
ccacgaactg gggtggaggt gcacaacgcc aagaccaagc cccgggagga gcagttcaat 240
agcacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 300
gaatacaagt gtaaggtgtc caacaagggc ctgcccagca gcatcgagaa aaccatcagc 360
aaggccaagg gccagcctcg ggagccccag gtgtacaccc tgccccctag ccaagaggag 420
atgaccaaga atcaggtgtc cctgacctgc ctggtgaagg gcttctaccc cagcgacatc 480
gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg 540
ctggacagcg acggcagctt cttcctgtac agcaggctga ccgtggacaa gagccggtgg 600
caggagggca acgtctttag ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 660
cagaagagcc tgtccctgag cctgggcaag atg 693
<210> 5
<211> 68
<212> PRT
<213>Artificial sequence
<400> 5
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
20 25 30
Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
35 40 45
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
50 55 60
Ala Tyr Arg Ser
65
<210> 6
<211> 204
<212> DNA
<213>It is artificial synthesized
<400> 6
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 60
gccttcatca tcttttgggt gaggagcaag cggagcagag gcggccacag cgactacatg 120
aacatgaccc cccggaggcc tggccccacc cggaagcact accagcccta cgcccctccc 180
agggacttcg ccgcctaccg gagc 204
<210> 7
<211> 112
<212> PRT
<213>It is artificial synthesized
<400> 7
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 8
<211> 339
<212> DNA
<213>It is artificial synthesized
<400> 8
cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gaaccagctg 60
tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 120
cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 180
gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240
cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 300
tacgacgccc tgcacatgca ggccctgccc cccagatga 339
<210> 9
<211> 42
<212> PRT
<213>It is artificial synthesized
<400> 9
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 10
<211> 126
<212> DNA
<213>It is artificial synthesized
<400> 10
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 11
<211> 42
<212> PRT
<213>It is artificial synthesized
<400> 11
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Lys Arg
1 5 10 15
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
20 25 30
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
35 40
<210> 12
<211> 126
<212> DNA
<213>It is artificial synthesized
<400> 12
gtagctgccg atttccagaa gaagaagaag gaggatgtga actgaaacgg ggcagaaaga 60
aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag 120
atggct 126
<210> 13
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 13
atggagagca agtacgg 17
<210> 14
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 14
tcatctgggg ggcaggg 17
<210> 15
<211> 85
<212> DNA
<213>It is artificial synthesized
<400> 15
atggagagca agtacggccc tccctgcccc ccttgccctg cccccgagtt cctgggcgga 60
cccagcgtgt tcgttagaag ggccc 85
<210> 16
<211> 87
<212> DNA
<213>It is artificial synthesized
<400> 16
ccctctctga aggcccccac tccctgggat gctacaatcc aatgatggaa gatggcatta 60
gctacaccac cctgcgcttt ccagaga 87
<210> 17
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 17
tgaacatacc acgaactggg gtggaggtgc acaacgccaa gaccaagccc cgggaggagc 60
agttcaatag cacctaccgg gtgg 84
<210> 18
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 18
tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggaatac aagtgtaagg 60
tgtccaacaa gggcctgccc agca 84
<210> 19
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 19
gcatcgagaa aaccatcagc aaggccaagg gccagcctcg ggagccccag gtgtacaccc 60
tgccccctag ccaagaggag atga 84
<210> 20
<211> 86
<212> DNA
<213>It is artificial synthesized
<400> 20
ccaagaatca ggtgtccctg acctgcctgg tgaagggctt ctaccccagc gacatcgccg 60
tggagtggga gagcaacggc cagccc 86
<210> 21
<211> 86
<212> DNA
<213>It is artificial synthesized
<400> 21
gagaacaact acaagaccac cccccctgtg ctggacagcg acggcagctt cttcctgtac 60
agcaggctga ccgtggacaa gagccg 86
<210> 22
<211> 86
<212> DNA
<213>It is artificial synthesized
<400> 22
gtggcaggag ggcaacgtct ttagctgctc cgtgatgcac gaggccctgc acaaccacta 60
cacccagaag agcctgtccc tgagcc 86
<210> 23
<211> 86
<212> DNA
<213>It is artificial synthesized
<400> 23
tgggcaagat gttctgggtg ctggtcgtgg tgggtggcgt gctggcctgc tacagcctgc 60
tggtgacagt ggccttcatc atcttt 86
<210> 24
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 24
tgggtgagga gcaagcggag cagaggcggc cacagcgact acatgaacat gaccccccgg 60
aggcctggcc ccacccggaa gcac 84
<210> 25
<211> 87
<212> DNA
<213>It is artificial synthesized
<400> 25
taccagccct acgcccctcc cagggacttc gccgcctacc ggagcaaacg gggcagaaag 60
aaactcctgt atatattcaa acaacca 87
<210> 26
<211> 86
<212> DNA
<213>It is artificial synthesized
<400> 26
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 60
gaagaagaag gaggatgtga actgcg 86
<210> 27
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 27
ggtgaagttc agccggagcg ccgacgcccc tgcctaccag cagggccaga accagctgta 60
caacgagctg aacctgggcc ggag 84
<210> 28
<211> 82
<212> DNA
<213>It is artificial synthesized
<400> 28
ggaggagtac gacgtgctgg acaagcggag aggccgggac cctgagatgg gcggcaagcc 60
ccggagaaag aaccctcagg ag 82
<210> 29
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 29
ggcctgtata acgaactgca gaaagacaag atggccgagg cctacagcga gatcggcatg 60
aagggcgagc ggcggagggg caag 84
<210> 30
<211> 87
<212> DNA
<213>It is artificial synthesized
<400> 30
ggccacgacg gcctgtacca gggcctgagc accgccacca aggataccta cgacgccctg 60
cacatgcagg ccctgccccc cagatga 87
<210> 31
<211> 86
<212> DNA
<213>It is artificial synthesized
<400> 31
gtatccttgg tggcggtgct caggccctgg tacaggccgt cgtggccctt gcccctccgc 60
cgctcgccct tcatgccgat ctcgct 86
<210> 32
<211> 88
<212> DNA
<213>It is artificial synthesized
<400> 32
gtaggcctcg gccatcttgt ctttctgcag ttcgttatac aggccctcct gagggttctt 60
tctccggggc ttgccgccca tctcaggg 88
<210> 33
<211> 87
<212> DNA
<213>It is artificial synthesized
<400> 33
tcccggcctc tccgcttgtc cagcacgtcg tactcctccc tccggcccag gttcagctcg 60
ttgtacagct ggttctggcc ctgctgg 87
<210> 34
<211> 88
<212> DNA
<213>It is artificial synthesized
<400> 34
taggcagggg cgtcggcgct ccggctgaac ttcacccgca gttcacatcc tccttcttct 60
tcttctggaa atcggcagct acagccat 88
<210> 35
<211> 89
<212> DNA
<213>It is artificial synthesized
<400> 35
cttcctcttg agtagtttgt actggtctca taaatggttg tttgaatata tacaggagtt 60
tctttctgcc ccgtttgctc cggtaggcg 89
<210> 36
<211> 83
<212> DNA
<213>It is artificial synthesized
<400> 36
gcgaagtccc tgggaggggc gtagggctgg tagtgcttcc gggtggggcc aggcctccgg 60
ggggtcatgt tcatgtagtc gct 83
<210> 37
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 37
gtggccgcct ctgctccgct tgctcctcac ccaaaagatg atgaaggcca ctgtcaccag 60
caggctgtag caggccagca cgcc 84
<210> 38
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 38
acccaccacg accagcaccc agaacatctt gcccaggctc agggacaggc tcttctgggt 60
gtagtggttg tgcagggcct cgtg 84
<210> 39
<211> 84
<212> DNA
<213>It is artificial synthesized
<400> 39
catcacggag cagctaaaga cgttgccctc ctgccaccgg ctcttgtcca cggtcagcct 60
gctgtacagg aagaagctgc cgtc 84
<210> 40
<211> 82
<212> DNA
<213>It is artificial synthesized
<400> 40
gctgtccagc acaggggggg tggtcttgta gttgttctcg ggctggccgt tgctctccca 60
ctccacggcg atgtcgctgg gg 82
<210> 41
<211> 82
<212> DNA
<213>It is artificial synthesized
<400> 41
tagaagccct tcaccaggca ggtcagggac acctgattct tggtcatctc ctcttggcta 60
gggggcaggg tgtacacctg gg 82
<210> 42
<211> 83
<212> DNA
<213>It is artificial synthesized
<400> 42
gctcccgagg ctggcccttg gccttgctga tggttttctc gatgctgctg ggcaggccct 60
tgttggacac cttacacttg tat 83
<210> 43
<211> 81
<212> DNA
<213>It is artificial synthesized
<400> 43
tccttgccgt tcagccagtc ctggtgcagc acggtcagca cggacaccac ccggtaggtg 60
ctattgaact gctcctcccg g 81
<210> 44
<211> 83
<212> DNA
<213>It is artificial synthesized
<400> 44
ggcttggtct tggcgttgtg cacctccacc ccagttcgtg gtatgttcat ctctggaaag 60
cgcagggtgg tgtagctaat gcc 83
<210> 45
<211> 99
<212> DNA
<213>It is artificial synthesized
<400> 45
atcttccatc attggattgt agcatcccag ggagtggggg ccttcagaga ggggggccct 60
tctaacgaac acgctgggtc cgcccaggaa ctcgggggc 99
<210> 46
<211> 99
<212> DNA
<213>It is artificial synthesized
<400> 46
atcttccatc attggattgt agcatcccag ggagtggggg ccttcagaga ggggggccct 60
tctaacgaac acgctgggtc cgcccaggaa ctcgggggc 99
<210> 47
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 47
tcatctgggg ggcaggg 17

Claims (10)

1. a kind of Chimeric antigen receptor recombinant protein for CD22, which is characterized in that the recombinant protein is gone here and there by three parts Connection composition, is extracellular region, transmembrane region and intracellular region respectively, wherein：
The extracellular region, by being connected in series for the antibody variable region of antigens c D22；
The transmembrane region is originated from CD8 α；
The intracellular region is connected in series by the polypeptide fragment permutation and combination of three separate sources.
2. Chimeric antigen receptor recombinant protein as described in claim 1, which is characterized in that encode the core of the recombinant protein Nucleotide sequence is：
(A) recombinant DNA with the nucleotide sequence as shown in SEQ ID NO.1；Or
(B) there is the DNA sequence dna with the homology of nucleotide sequence shown in SEQ ID NO.1 >=75%.
3. Chimeric antigen receptor recombinant protein as described in claim 1, which is characterized in that the amino acid of the recombinant protein Sequence is：
(A) recombinant protein with the amino acid sequence as shown in SEQ ID NO.1；Or
(B) there is the polypeptide or recombinant protein with the homology of amino acid sequence shown in SEQ ID NO.1 >=85%.
4. Chimeric antigen receptor recombinant protein as claimed in claim 2, which is characterized in that the encoding nucleoside of the recombinant protein Acid is to obtain the nucleotide sequence of various pieces by artificial synthesized, is then spliced using PCR amplification and is cloned into expression On carrier.
5. Chimeric antigen receptor recombinant protein as claimed in claim 4, which is characterized in that the expression vector includes but unlimited In：Plasmid vector, slow virus carrier or retroviral vector.
6. Chimeric antigen receptor recombinant protein as described in claim 1, which is characterized in that the antigen receptor of the extracellular region can Becoming area's series unit can be made of the identical or different VhVl units of 1 to 3 groups.
7. Chimeric antigen receptor recombinant protein as claimed in claim 4, which is characterized in that the core for encoding the recombinant protein Thuja acid designs and synthesizes the oligonucleotides of a plurality of staggeredly 60 to 80 complementary bases, by moving back according to its corresponding DNA sequence dna After fire, is linked using T4DNA ligases, obtained using PCR amplification.
8. Chimeric antigen receptor recombinant protein as described in claim 1, which is characterized in that three polypeptide pieces of the intracellular region Section, is arranged differently any type of sequence, is directly connected between segment or using the polypeptide formed less than 10 amino acid Linker is formed by connecting.
9. Chimeric antigen receptor recombinant protein as claimed in claim 8, which is characterized in that three polypeptide pieces of the intracellular region Section, including signaling zone, costimulation ligand and cell factor, wherein the signaling zone comes from CD3 δ, the costimulation ligand one From TNF class ligands, secondly coming from the Ig superfamily molecular cell factors；The cell factor includes IL-2, IL-3, IL-6, IL- 7, IL-11, IL-12, IL-15, IL-21 or GM-CSF.
10. Chimeric antigen receptor recombinant protein as claimed in claim 9, which is characterized in that the TNF classes ligand includes 4- 1BBL, OX40L, CD70, LIGHT and CD30L；The Ig superfamilies molecule is CD80 and CD86.
CN201710119499.1A 2017-03-03 2017-03-03 A kind of recombination of Chimeric antigen receptor for CD22 is built and its application Pending CN108530541A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111254156A (en) * 2019-06-05 2020-06-09 南京艾德免疫治疗研究院有限公司 Chimeric antigen receptor targeting humanized CD22 and uses thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111254156A (en) * 2019-06-05 2020-06-09 南京艾德免疫治疗研究院有限公司 Chimeric antigen receptor targeting humanized CD22 and uses thereof

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