Fusion protein, and preparation method and use thereof
Disclosed are a fusion protein, and a preparation method and use thereof, which belong to the field of biomedicine technologies. The fusion protein comprises a polypeptide specifically bound to a KRas protein, a specific tumor-cell-penetrating peptide and a lysosome recognition peptide. The fusion protein with tumor targeting, penetrability and specific protein degradation is designed and constructed direct to an undruggable protein for the first time, thus providing a new idea for development of an anti-cancer targeted drug. Different from the prior art in which one molecule can only target one target protein, the fusion protein can simultaneously induce degradation of wild-type and mutant-type KRas proteins. Meanwhile, the fusion protein can improve the sensitivity of the KRas mutant-type tumor to the tumor-targeted drug by inducing degradation of KRas, thus expanding an application range of existing anti-cancer targeted drugs and having important significance in tumor clinic treatment.
1 . A fusion protein, comprising an anti-KRas protein nanobody, a ganglioside binding peptide and a lysosome recognition peptide; and
wherein the amino acid sequence of the anti-KRas protein nanobody comprises:
(SEQ ID NO: 1)
DVQLQESGGGLVQAGGSLRLSCVASGRTFSTYPTGWFRQA
PGKEREFVARINLSGGITNYADSVKGRFTISRDNAKNTVY
LQMNSLKPEDTAVYYCGGGSTTWAGGIPTNFDYWGQGTQV
TVSSGR;
wherein the amino acid sequence of the ganglioside binding peptide comprises:
(SEQ ID NO: 2)
RAGLQFPVGRLLRRLLR;
and
wherein the amino acid sequence of the lysosome recognition peptide comprises:
(SEQ ID NO: 3)
KFERQKILDQRFFE.
2 . A nucleic acid molecule encoding the fusion protein according to claim 1 .
3 . A vector, comprising the nucleic acid molecule according to claim 2 .
4 . A host cell, comprising the vector according to claim 3 .
5 . A preparation method of the fusion protein according to claim 1 , comprising the step of: culturing the host cell according to claim 4 to obtain the fusion protein.
6 . A drug, comprising the fusion protein according to claim 1 and a pharmaceutically acceptable excipient.
7 . A combination drug, comprising a fusion protein and an epidermal growth factor receptor (EGFR)-targeted drug, wherein the fusion protein comprises an anti-KRas protein nanobody, a ganglioside binding peptide and a lysosome recognition peptide; and
wherein the amino acid sequence of the anti-KRas protein nanobody comprises:
(SEQ ID NO: 1)
DVQLQESGGGLVQAGGSLRLSCVASGRTFSTYPTGWFRQA
PGKEREFVARINLSGGITNYADSVKGRFTISRDNAKNTVY
LQMNSLKPEDTAVYYCGGGSTTWAGGIPTNFDYWGQGTQV
TVSSGR;
wherein the amino acid sequence of the ganglioside binding peptide comprises:
RAGLQFPVGRLLRRLLR (SEQ ID NO: 2); and
wherein the amino acid sequence of the lysosome recognition peptide comprises:
KFERQKILDORFFE (SEQ ID NO: 3).
8 . The host cell according to claim 4 , wherein the host cell is selected from the group consisting of prokaryotic cell and eukaryotic cell.
9 . The drug according to claim 6 , wherein the drug is any one selected from the group consisting of a preparation for degrading a KRas protein, an anti-tumor drug, and a drug for improving a sensitivity of a KRas mutant-type colorectal cancer to a tumor-targeted drug.
10 . The drug according to claim 9 , the KRas protein is selected from wild-type KRas protein or mutant-type KRas protein.
11 . The drug according to claim 10 , the mutant-type KRas protein is the KRas protein mutated at one or more amino acid positions of G12, G13, S17, P34, A59, Q61 and A146.
12 . The drug according to claim 9 , wherein the KRas mutant-type tumor is a tumor related to a mutant-type KRas protein.
13 . The drug according to claim 9 , wherein the tumor-targeted drug is a human epidermal growth factor receptor (EGFR)-targeted drug.
14 . The drug according to claim 13 , wherein the tumor-targeted drug is gefitinib.
15 . A method for treating or ameliorating colorectal cancer, comprising administering to a subject in need thereof an effective amount of the fusion protein according to claim 1 and gefitinib.