公开(公告)号：US11884974B2

公开(公告)日：2024-01-30

申请号：US16919500

申请日：2020-07-02

申请人： Shihezi University

发明人： Chuangfu Chen ,  Peng Wu

IPC分类号： C07K16/28 ,  C12Q1/686 ,  C12Q1/6886

CPC分类号： C12Q1/686 ,  C07K16/2803 ,  C12Q1/6886 ,  C07K2317/569 ,  C07K2317/76

摘要： The application belongs to the technical field of animal or human antibodies, and discloses a TIM-3 nanobody, a preparation method thereof and use thereof. The nanobody is a TIM-3 nanobody with sequence of SEQ ID NO:1. TIM-3 antigen is a transient transfection expression by mammalian cells. TIM-3 antigen is used to screen the nanobody library repeatedly, specific phage of nanobody is obtained, and the target fragment is conducted sequencing. The application uses the HEK293 cell line to express antigen. Using the mammalian expression system to express human protein may maximumly guarantee the original structure of the protein, guarantee the protein to have a post-translational modification and specific modifications of eukaryotic proteins such as glycosylation, which makes the obtained protein have high activity. This method maximizes the original structure and activity of the protein; the nanobodies screened by the application can efficiently and specifically bind to the target.

Shihezi University
TIM-3 nanobody, a preparation method thereof, and use thereof
3
1
1049
DNA
Artificial Sequence
1
GGTGGGCGGA CATTTCACAA GCTTAAGGAG ACAGTACATA TGAAATACCT ATTGCCTACG GCAGCCGCTG GATTGTTATT ACTCGCGGCC CAGCCGGCCA TGGCCCAGGT GCAGCTGCAG GAGTCTGGAG GAGGCTTGGT GCAGCCTGGG GGGTCTCTGA GACTCTCCTG TGCAGCCTCT GGGTTCACCT TCCGTAGATC TATTCTGAAA TGGCTCCGAC AGGCTCCAGG GAAGGAACTG GAGTGGGTGT CCACTATAGA TACATACTCT AATAACACAT ACTATGAAGA CTCCTTGAAG GGCCGATTCA CCATCTCCGC AGACAACGCC AAGAACACGC TGTATCTGCA AATGGACAGC CTGAAACCTG AGGACACGGC CGTGTATTAC TGTGCAAAGG GTGGAGGTGG TATCTACTCC CGCACGTATG ACTACCGGGG CCAGGGGACC CAGGTCACCG TCTCCTCAGC GGCCGCATAC CCGTACGACG TTCCGGACTA CGGTTCCCAC CACCATCACC ATCACTAGAC TGTTGAAAGT TGTTTAGCAA AACCTCATAC AGAAAATTCA TTTACTAACG TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGCTGT CTGTGGAATG CTACAGGCGT TGTCGTTTGT ACTGGTGACG AAACTCAGTG TTACGGTACA TGGGTTCCTA TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA GGGTGGCGGT TCTGAGGGTG GCGGTTCTGA GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT ATTCCGGGCT ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA AACCCCGCTA ATCCTAATCC CTTCTCTTGA GGAGTCTCAG CCTCTTATAC TTTCATGTTT CAGATATAGG TTCCGAAATA GGCAGGTGCA TTAACTGTTA TACGGGCACT GTACTCATGC ACTGACCCCG TTAAACTTAT TACCAGTACA CTCCTGTATC ATCAAAAGCC ATGTATGA
2
30
DNA
Artificial Sequence
2
GACACGAATTCGCCACCATGTTCAGCCACC
3
35
DNA
Artificial Sequence
3
GTGTCAAGCTTTCACTTGTCATCATCATCCTTGTA
4
4541
DNA
Unknown
4
GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTGAGCGCA ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCTATG ACCATGATTA CGCCAAGCTT GCATGCAAAT TCTATTTCAA GGAGACAGTC ATAATGAAAT ACCTATTGCC TACGGCAGCC GCTGGATTGT TATTACTCGC GGCCCAGCCG GCCATGGCCC AGGTGCAGCT GCAGGAGTCT AGAGGGGACC CAGGTCACCG TCTCCTCAGC GGCCGCATAC CCGTACGACG TTCCGGACTA CGGTTCCCAC CACCATCACC ATCACTAGAC TGTTGAAAGT TGTTTAGCAA AACCTCATAC AGAAAATTCA TTTACTAACG TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGCTGT CTGTGGAATG CTACAGGCGT TGTGGTTTGT ACTGGTGACG AAACTCAGTG TTACGGTACA TGGGTTCCTA TTGGGCTTGC TATCCCTGAA AATGAGGGTG GTGGCTCTGA GGTGGCGGT TCTGAGGGTG GCGGTTCTGA GGGTGGCGGT ACTAAACCTC CTGAGTACGG TGATACACCT ATTCCGGGCT ATACTTATAT CAACCCTCTC GACGGCACTT ATCCGCCTGG TACTGAGCAA AACCCCGCTA ATCCTAATCC TTCTCTTGAG GAGTCTCAGC CTCTTAATAC TTTCATGTTT CAGAATAATA GGTTCCGAAA TAGGCAGGGT GCATTAACTG TTTATACGGG CACTGTTACT CAAGGCACTG ACCCCGTTAA AACTTATTAC CAGTACACTC CTGTATCATC AAAAGCCATG TATGACGCTT ACTGGAACGG TAAATTCAGA GACTGCGCTT TCCATTCTGG CTTTAATGAG GACCCATTCG TTTGTGAATA TCAAGGCCAA TCGTCTGACC TGCCTCAACC TCCTGTCAAT GCTGGCGGCG GCTCTGGTGG TGGTTCTGGT GGCGGCTCTG AGGGTGGCGG CTCTGAGGGT GGCGGTTCTG AGGGTGGCGG CTCTGAGGGT GGCGGTTCCG GTGGCGGCTC CGGTTCCGGT GATTTTGATT ATGAAAAAAT GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT GAAAACGCGC TACAGTCTGA CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT GCTGCTATCG ATGGTTTCAT TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG CTGGCTCTAA TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA ATTTCCGTCA ATATTTACCT TCTTTGCCTC AGTCGGTTGA ATGTCGCCCT TATGTCTTTG GCGCTGGTAA ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA TTCCGTGGTG TCTTTGCGTT TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCGACG TTTGCTAACA TACTGCGTAA TAAGGAGTCT TAATAAGAAT TCACTGGCCG TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC TGTGCGGTAT TTCACACCGC ATATAAATTG TAAACGTTAA TATTTTGTTA AAATTCGCGT TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC AAAATCCCTT ATAAATCAAA AGAATAGCCC GAGATAGGGT TGAGTGTTGT TCCAGTTTGG AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA AACCGTCTAT CAGGGCGATG GCCCACTACG TGAACCATCA CCCAAATCAA GTTTTTTGGG GTCGAGGTGC CGTAAAGCAC TAAATCGGAA CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG ACGGGGAAAG CCGGCGAACG TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGC TAGGGCGCTG GCAAGTGTAG CGGTCACGCT GCGCGTAACC ACCACACCCG CCGCGCTTAA TGCGCCGCTA CAGGGCGCGT ACTATGGTTG CTTTGACGGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA GCCAGCCCCG ACACCCGCCA ACACCCGCTG ACGCGCCCTG ACGGGCTTGT CTGCTCCCGG CATCCGCTTA CAGACAAGCT GTGACCGTCT CCGGGAGCTG CATGTGTCAG AGGTTTTCAC CGTCATCACC GAAACGCGCG AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTATTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTC CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG AACTGAGATA CCTACAGCGT GAGCATTGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGAA

公开(公告)号：US11820811B2

公开(公告)日：2023-11-21

申请号：US17371241

申请日：2021-07-09

申请人： Shihezi University

发明人： Chuangfu Chen ,  Peng Wu ,  Chencheng Xiao ,  Chengyao Li ,  Yong Wang ,  Zhen Wang

IPC分类号： C07K16/10 ,  G01N33/569

CPC分类号： C07K16/10 ,  G01N33/56983 ,  C07K2317/569 ,  C07K2317/76 ,  G01N2333/165

摘要： A nanobody and its application based on SARS-CoV-2 S protein are provided, and the present disclosure relates to biomedical technology. The present disclosure chooses the Spike S1+S2 ECD of SARS-CoV-2 as a target, and screens the nanobody against of SARS-CoV-2 by using a nanobody library. After an ELISA test, the Spike S1+S2 ECD target of SARS-CoV-2 can be specifically identified while a SPIKE RBD target is identified, and a binding signal is relatively strong. The corresponding nanobody sequence is constructed into a prokaryotic expression vector for expression and purification to express the target nanobody successfully. After the purification, the purity is greater than 90%. The ELISA test of VHH nanobody showed that the purified nanobody has higher affinity to the two targets.

公开(公告)号：US11661448B2

公开(公告)日：2023-05-30

申请号：US17371155

申请日：2021-07-09

申请人： Shihezi University

发明人： Chuangfu Chen ,  Peng Wu ,  Chencheng Xiao ,  Chengyao Li ,  Yong Wang ,  Zhen Wang

IPC分类号： C07K16/10 ,  C12N15/70

CPC分类号： C07K16/10 ,  C12N15/70 ,  C07K2317/569 ,  C07K2317/92

摘要： A nanobody and its application based on SARS-CoV-2 S protein S1 subunit are provided, and the present disclosure relates to biomedical technology. The present disclosure chooses the Spike RBD of SARS-CoV-2 as a target, and screens the nanobody targeting SARS-CoV-2 by using a nanobody library. After an ELISA test, the Spike RBD target of SARS-CoV-2 can be specifically identification while a SPIKE S1+S2 ECD target is identification, and a binding signal is relatively strong. The corresponding nanobody sequence is constructed into a prokaryotic expression vector for expression and purification to express the target nanobody successfully. After purification, the purity is greater than 90%. The ELISA test of VHH nanobody showed that the purified nanobody has higher affinity to the two targets.