Derivative of endostatin resistant to tumor proteins
A technology of endostatin and its derivatives, which can be applied in the fields of antineoplastic drugs, peptide/protein components, animal/human proteins, etc., and can solve problems such as instability, short half-life of endostatin, and affecting the effect of endostatin on antitumor therapy , to achieve the effect of high anti-tumor biological activity and long half-life
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Embodiment 1
[0049] Example 1 A derivative of the anti-tumor protein endostatin, the derivative includes a fusion protein of endostatin linked to the constant region of antibody IgG, its anti-tumor activity and the half-life of the fusion protein in mice.
[0050] protein sample
[0051] Sample 1: pre-sequence-endostatin; the front of endostatin shown in SEQ ID NO.4 is connected with the pre-sequence shown in SEQ ID NO.2, and the signal peptide in the pre-sequence is to allow expressed endostatin can be secreted extracellularly.
[0052] Sample 2: fusion protein of pre-sequence-endostatin-antibody IgG partial constant region CH1 (shown in SEQ ID NO.8, the nucleic acid sequence encoding SEQ ID NO.8 is shown in SEQ ID NO.7).
[0053] Sample 3: pre-sequence-endostatin-antibody IgG partial constant region CH1CH2 fusion protein (shown in SEQ ID NO.10, the nucleic acid sequence encoding SEQ ID NO.10 is shown in SEQ ID NO.9).
[0054] Sample 4: pre-sequence-endostatin-antibody IgG constant regi...
Embodiment 2
[0061] Example 2 Evaluation of the newly introduced Arg in front of the N-terminus of endostatin derivatives
[0062] Select the pre-sequence-endostatin-antibody IgG constant region fusion protein (SEQ ID NO.11 and SEQ ID NO.12) of Example 4 of Example 1 as the experimental material, and illustrate it with the amino acid sequence (SEQ ID NO.12) , 1-23 amino acids are derived from albumin, 1-18 is the signal peptide of albumin, 19-23 are the 5 amino acids at the N-terminal of mature albumin, of which there are two Arg, 24-206 are endostatin, 207-536 is the constant region of antibody IgG, which consists of CH1, CH2 and CH3. Various experimental proteins:
[0063] Sample 1: pre-sequence-endostatin-antibody IgG constant region fusion protein, shown by SEQ ID NO.12, wherein 19-23 are Arg-Gly-Val-Phe-Arg.
[0064] Sample 2: pre-sequence-endostatin-antibody IgG constant region fusion protein point mutant 1, in which 19-23 is Ala-Gly-Val-Phe-Arg, the agg point mutation of the 19th ...
Embodiment 3
[0071] Example 3 Checking the Effect of Glycosylation Sites on the Activity of Endostatin Fusion Protein
[0072] The pre-sequence-endostatin-antibody IgG constant region CH1CH2CH3 fusion protein (SEQ ID NO.11 and SEQ ID NO.12) of Example 1 was used as the experimental material to construct derivatives, that is, the N-terminal front of endostatin or A glycosylated amino acid short peptide Gly-Ala-Ser-Asn-Ser-Thr-Gly-Ala-Ser is added behind the C-terminus, where Asn-Ser-Thr is the position of glycosylation, and the sugar chain is connected to Asn. Select embodiment 1 to express the plasmid (pHEK293 of the front sequence-endostatin-antibody IgG constant region CH1CH2CH3 fusion protein (shown in SEQ ID NO.12, encoding the nucleic acid sequence SEQ ID NO.11 of SEQ ID NO.12) Ultra Expression Vector II) for various gene manipulations, and the gene fragments of these mutants can also be directly synthesized, and then inserted into the plasmid (pHEK293 Ultra Expression Vector II) to e...
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