Pegylated tetanus neurotoxins and treatment of hypotonia
A technology of PEGylation and neurotoxins, applied in chemical instruments and methods, neuromuscular system diseases, biochemical equipment and methods, etc., can solve problems such as unused
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Embodiment 1
[0171] Embodiment 1: the preparation of PEG-TeNT-c ( figure 1 A)
[0172] In this example, TeNT-c was generated by mutating the surface serine residues to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes. figure 1 A molecule. The mutations will be: S963C, S1041C, S1155C and S1187C.
[0173] Genes for TeNTs with surface serine→cysteine substitutions S963C, S1041C, S1155C and S1187C will be synthesized by a commercial provider such as Integrated DNA Technologies. The gene was subcloned into the pRSET-A expression vector by restriction digestion to add a 6x histidine tag from the vector to the N-terminus of the mutein.
[0174] TeNTs comprising S963C, S1041C, S1155C and S1187C were expressed, PEGylated and purified according to Example 5 to generate PEG-TeNT-c.
Embodiment 2
[0175] Embodiment 2: the preparation of PEG-TeNT-HC ( figure 1 B)
[0176]In this example, TeNT-HC was generated by mutating the surface serine residues to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes. figure 1 Molecule of B. The mutations will be: S600C, S963C, S1041C, S1155C and S1187C (Figure 24, SEQ ID NO: 16).
[0177] Genes for TeNTs with surface serine→cysteine substitutions S600C, S963C, S1041C, S1155C and S1187C will be synthesized by a commercial provider such as Integrated DNA Technologies. The gene was subcloned into the pRSET-A expression vector (FIG. 25, SEQ ID NO: 17, Figure 26 ) to add a 6×histidine tag from the vector to the N-terminus of the mutein.
[0178] TeNTs comprising S600C, S963C, S1041C, S1155C and S1187C were expressed, PEGylated and purified according to Example 5 to generate PEG-TeNT-HC.
Embodiment 3
[0179] Embodiment 3: the preparation of PEG-TeNT-LC-c ( figure 1 c)
[0180] In this example, LC and c were generated by mutating the surface serine residues to surface cysteine residues to facilitate directed PEG conjugation at immunogenic epitopes. figure 1 Molecules of C. The mutations are: S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C (Figure 21 , SEQ ID NO: 14).
[0181] Genes for TeNTs with surface serine → cysteine substitutions S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C in LC and c were synthesized by a commercial provider (Integrated DNA Technologies). The gene was subcloned into the pRSET-A expression vector (FIG. 22, SEQ ID NO: 15, Figure 23 ) to add a 6×histidine tag from the vector to the N-terminus of the mutein.
[0182] TeNTs comprising S81C, S120C, S144C, S248C, S335C, S428C, S963C, S1041C, S1155C and S1187C were expressed, PEGylated and purified according to Example 5 to generate PEG-TeNT-LC-...
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