Framework for encoding double-specificity molecules of mediated secretion effector cells and killing target cells as well as virus
A bispecific, mediated technology, applied in the direction of viruses/phages, medical preparations containing active ingredients, microorganisms, etc., can solve the problems of high preparation cost, short half-life, complex fusion protein expression and purification, etc. High, low toxicity, and the effect of reducing the number of treatments
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Embodiment 1
[0035] Example 1, construction and identification of recombinant adenovirus
[0036] 1. Preparation of recombinant adenovirus
[0037] 1. Insert the double-stranded DNA molecule shown in Sequence 1 of the sequence listing between the EcoRI and SalI restriction sites of plasmid pDC316 to obtain a recombinant plasmid.
[0038] The double-stranded DNA molecule shown in sequence 1 of the sequence listing encodes the protein shown in sequence 2 of the sequence listing (in sequence 1, nucleotides 1-51 encode a signal peptide, which will be excised eventually). The protein shown in Sequence 2 of the sequence listing is named F3 protein, also known as Fcab-anti-CD3 protein. In sequence 2, the 1-243rd is an anti-CD3 fragment (the anti-CD3 fragment targets T cells), the 246-483rd is an Fcab fragment (the Fcab fragment targets the HER2 antigen on the surface of tumor cells), and the 484-493 bit for the myc tag.
[0039] 2. Spread 293A cells in a six-well plate (about 6×10 per well 5 ...
Embodiment 2
[0079] Embodiment 2, the construction of recombinant adenovirus
[0080] 1. Insert the double-stranded DNA molecule shown in sequence 3 in the sequence listing between the EcoRI and SalI restriction sites of plasmid pDC316 to obtain a recombinant plasmid.
[0081] The double-stranded DNA molecule shown in sequence 3 of the sequence listing encodes the protein shown in sequence 4 of the sequence listing (in sequence 3, nucleotides 1-51 encode a signal peptide, which will be excised eventually). The protein shown in sequence 4 in the sequence listing is named H3 protein, also known as HER2-anti-CD3 protein. In Sequence 4, the 1-242nd is the HER2 fragment (HER2 fragment targets the HER2 antigen on the surface of tumor cells), the 260-502 is the anti-CD3 fragment (the anti-CD3 fragment targets T cells), and the 505-514 The first bit is the myc tag, and the 516th-520th bit is the His tag.
[0082] Other steps are the same as in Example 1.
Embodiment 3
[0083] Embodiment 3, the construction of recombinant adenovirus
[0084] 1. Insert the double-stranded DNA molecule shown in Sequence 5 of the sequence listing between the EcoRI and SalI restriction sites of plasmid pDC316 to obtain a recombinant plasmid.
[0085] The double-stranded DNA molecule shown in sequence 5 of the sequence listing encodes the protein shown in sequence 6 of the sequence listing (in sequence 5, nucleotides 1-51 encode a signal peptide, which will be excised eventually). The protein shown in sequence 6 in the sequence listing is named M3 protein, also known as Mart1-anti-CD3 protein. In Sequence 6, positions 1-117 are Mart1 fragments (Mart1 fragments target the HLA-A2-MART1 polypeptide complex on the surface of tumor cells), and positions 137-379 are anti-CD3 fragments (anti-CD3 fragments target T cells) , the 382-391th position is the myc tag, and the 393-397th position is the His tag.
[0086] Other steps are the same as in Example 1.
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