Method of producing antibodies
a technology of antibody and recombinant protein, which is applied in the field of producing antibodies, can solve the problems of difficult to obtain dna expression in cells, alterations and/or damage to the genomic dna of host cells, etc., and achieve the effects of increasing the efficiency of recombinantly expressed protein translation, and increasing the production of recombinantly expressed protein
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example 1
Synthesis of Modified mRNA
[0170]Modified mRNAs (modRNAs) according to the invention were made using standard laboratory methods and materials. The open reading frame (ORF) of the gene of interest is flanked by a 5′ untranslated region (UTR) containing a strong Kozak translational initiation signal and an alpha-globin 3′ UTR terminating with an oligo(dT) sequence for templated addition of a polyA tail. The modRNAs were modified with pseudouridine (ψ) and 5-methyl-cytidine (5meC) to reduce the cellular innate immune response. Kariko K et al. Immunity 23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson B R et al. NAR (2010).
[0171]The cloning, gene synthesis and vector sequencing was performed by DNA2.0 Inc. (Menlo Park, Calif.). Vector sequences and insert sequences are set forth in SEQ ID NOs: 5-8. The ORFs were restriction digested using XbaI or HindIII and used for cDNA synthesis using tailed-PCR. This tailed-PCR cDNA product was used as the template for the modif...
example 2
De novo Generation of a Mammalian Commercial Production Cell Line Expressing Human G-CSF as a Therapeutic Agent in Model Bioreactor
[0176]The nucleic acid sequence for the precursor of human granulocyte colony stimulating factor (G-CSF) is set forth in SEQ ID NO: 1:
(SEQ ID NO: 1)agcttttggaccctcgtacagaagctaatacgactcactatagggaaataagagagaaaagaagagtaagaagaaatataagagccaccatggccggtcccgcgacccaaagccccatgaaacttatggccctgcagttgctgctttggcactcggccctctggacagtccaagaagcgactcctctcggacctgcctcatcgttgccgcagtcattccttttgaagtgtctggagcaggtgcgaaagattcagggcgatggagccgcactccaagagaagctctgcgcgacatacaaactttgccatcccgaggagctcgtactgctcgggcacagcttggggattccctgggctcctctctcgtcctgtccgtcgcaggctttgcagttggcagggtgcctttcccagctccactccggtttgttcttgtatcagggactgctgcaagcccttgagggaatctcgccagaattgggcccgacgctggacacgttgcagctcgacgtggcggatttcgcaacaaccatctggcagcagatggaggaactggggatggcacccgcgctgcagcccacgcagggggcaatgccggcctttgcgtccgcgtttcagcgcagggcgggtggagtcctcgtagcgagccaccttcaatcatttttggaagtctcgtaccgggtgctgagacatcttgcgcagccgtgaagcgctgccttctg...
example 3
De novo Generation of a Mammalian Commercial Production Cell Line Expressing Humanized IgG Antibodies (Trastuzumab and Rituximab) as a Therapeutic Agent in Model Bioreactor
[0180]The nucleic acid sequence for the Heavy Chain of Rituximab is set forth in SEQ ID NO: 4:
(SEQ ID NO: 4)CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGTGATGGCGCCGAGGACCCTGGTGCTCTTGCTCACGGGTGCCTTGGCCCTCACGCAAACATGGGCGGGACAGGCGTACTTGCAGCAGTCAGGGGCAGAACTCGTAAGGCCCGGAGCGTCGGTGAAGATGTCGTGTAAAGCGTCGGGCTATACTTTCACATCGTACAACATGCACTGGGTCAAACAGACGCCCCGACAAGGGCTGGAGTGGATTGGAGCTATCTACCCCGGTAACGGGGATACGTCGTACAACCAGAAGTTTAAGGGGAAGGCGACTCTTACTGTCGACAAGTCGTCCTCCACCGCCTATATGCAGCTGTCGAGCCTGACTTCGGAAGATTCAGCGGTGTACTTTTGTGCGCGCGTGGTCTATTACTCAAATTCGTATTGGTATTTCGATGTGTGGGGTACGGGGACCACTGTGACCGTGTCAGGACCCTCGGTATTCCCCCTCGCGCCTAGCTCAAAGTCCACCTCCGGGGGAACAGCCGCCTTGGGTTGCTTGGTAAAGGACTATTTCCCCGAGCCCGTCACAGTGAGCTGGAACTCCGGGGCACTGACATCGGGAGTGCACACGTTTCCCGCGGTACTTCAGTCATCAGGACTCTACTCGCTGTCAAGCGTGGTCACGGTGC...
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