TUMOR VASCULAR DISRUPTING AGENT POLYPEPTIDE, GENE, EXPRESSION VECTOR, AND USE THEREOF

Abstract

The present invention relates to a tumor vascular disrupting agent polypeptide, gene, expression vector, and use thereof. The tumor vascular disrupting agent polypeptide has the amino acid sequence as shown by SEQ ID NO: 1. The polypeptide comprises a truncated tissue factor (tTF) and a tumor-targeting molecule (pHLIP); the factor and the molecule are connected by 5 amino acids, thereby ensuring the function of each not being affected by the other; the fusion protein can be positioned to the surface of a tumor vascular endothelial cell by means of the pHLIP, and provides the blood coagulation feature of the tTF in a tumor vessel and forms a thrombus, thereby disrupting the blood supply to the tumor area and treating tumor. The polypeptide of the present invention is significant to the treatment of tumor, and can be used in medicines for treating tumors.

Claims

1. A tumor blood vessel blocker polypeptide, characterized in that said polypeptide comprises amino acid sequence as SEQ ID NO: 1.

2. A tumor blood vessel blocker gene, characterized in that said gene comprises nucleotide sequence encoding said polypeptide according to claim 1.

3. The tumor blood vessel blocker gene according to claim 2, characterized in that said gene comprises nucleotide sequence as SEQ ID NO: 5.

4. A expression vector of tumor blood vessel blocker, characterized in that said expression vector comprises nucleotide sequence encoding said polypeptide according to claim 1.

5. The expression vector according to claim 4, characterized in that said expression vector comprises nucleotide sequence as SEQ ID NO: 5.

6. The expression vector according to claim 4, characterized in that said expression vector is constructed by pET30a plasmid vector.

7. The use of the tumor blood vessel blocker polypeptide according to claim 1 in preparing medicine for tumor treatment.

8. The use of the tumor blood vessel blocker polypeptide according to claim 1 in treating tumor.

9. The expression vector according to claim 5, characterized in that said expression vector is constructed by pET30a plasmid vector.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is the structural diagram (A) of the tumor blood vessel blocker polypeptide (fusion protein) of this invention and identification results of SDS-PAGE electrophoresis (B) of the fusion protein. Wherein, the tumor blood vessel blocker polypeptide comprises three domains: an activity domain, a connection domain and a target domain; M means protein standard molecular weight; 1 means lane of the fusion protein, and location pointed out by arrow is the fusion protein.

[0029] FIG. 2 shows the therapy effects of 12 hours after injecting tumor blood vessel blocker of the invention into the mammary cancer tumor model of nude mice through tail vein, wherein, (A) is the in-vitro morphology of the tumor-bearing mice which is injected with physiological saline (control), (B) is the control tumor, (C) is the control tumor pathological section (the arrow means the tumor blood vessel without thrombopoiesis), (D) is the in-vitro morphology of the tumor-bearing mice which is injected with tumor blood vessel blocker, (E) is the tumor after injecting tumor blood vessel blocker, and (F) is the tumor pathological section after injecting tumor blood vessel blocker (the arrow directs to the tumor blood vessel with clear thrombopoiesis).

EMBODIMENTS

[0030] The following describes the implementation plan of the invention in detail by combining with embodiments. A person skilled in the art shall understand that, the following examples are only the preferable embodiments of the invention, it's only for better understanding the invention but shall not be deemed as to limit the scope of the invention.

[0031] All experiment methods used in the following embodiments are conventional methods, unless other specified; and all experiment materials are purchased from the conventional biochemical reagent manufacturers unless other specified.

Example 1: Construction of Fusion Protein Expression Vector

[0032] Firstly, the gene sequence of tissue factor extracellular 219 amino acids (shown as SEQ ID NO:2) is found by searching from NCBI website, and secondly, the amino acid sequences of the tumor targeting peptide (shown as SEQ ID NO:3) and the connection part are translated to get the gene sequence, to get the gene sequence of the fusion protein shown by SEQ ID NO: 5. Gene of the fusion protein is synthesized by the full-gene synthesis method, and restriction enzyme cutting sites Nde I and Xho I are respectively designed at both ends of the gene, and then, the fusion protein gene is connected into the pET30a vector through the above restriction enzyme cutting sites so as to get the fusion protein expression vector.

Example 2: Expression and Purification of Fusion Protein

(1) Expression of Fusion Protein

[0033] The above expression vector of fusion protein is transformed into BL21 E. coli.; firstly, 5 μL of bacterial fluid was inoculated into 5 mL of LB liquid nutrient medium, and incubated by shake cultivation for 16 h at 37° C., 200×rpm. The cultivated bacterial fluid was innoculated into 500 mL of LB liquid nutrient medium, and incubated by shake cultivation for 16 h at 37° C., 200×rpm, to 0D=0.6˜0.8, and then induced expression is conducted for 4 h by using IPTG (0.5 mM).

(2) Purification of Fusion Protein

[0034] Above IPTG induced-expressed bacterial fluid was centrifuged (6000×rpm, 5 min); the liquid supernatant was discarded, and the bacteria was collected. precipitate was, blown off with the 25 mL 10 mM Tris-HCl (pH=8.0) solution; the bacteria were broken with ultrasound, and centrifuged for 10 min at 12000×rpm with liquid supernatant removed; the precipitate obtained by ultrasonic centrifugation was re-suspended with 25 mL of 10 mM Tris-HCl (pH=8.0) solution, and placed for 10 min. Above operation was repeated for one time again and get the precipitate. The precipitate was re-suspended by adding little amount of 10 mM Tris-HCl (pH=8.0) solution, and then 8 mL of 10 mM Tris-HCl (pH=8.0) solution containing 8M carbamide was added to dissolve the protein, and was centrifuged for 10 min at 12000×rpm, and then to collect the liquid supernatant.

[0035] The fusion protein was identified with SDS-PAGE electrophoresis, with results shown as FIG. 1 (B); clear and pure bands which are higher than 30 KDa can be seen, which is consistent with expectation.

Example 3: Effect Experiments of Tumor Blood Vessel Blocker

[0036] The tumor blood vessel blocker polypeptide provided by embodiment 2 is injected into the mammary cancer tumor model of nude mice by the tail vein, according to the amount of 833 ug/kg body weight or 20 ug/per mouse; the therapy effects were observed after 12 hours and mice injecting physiological saline were taken as the control group. The results are shown as FIG. 2.

[0037] Compared with the tumor-bearing mice (A) injecting the physiological saline, the tumor blood vessel blocker of the invention can cause the tumor part to turn on the deep red (D) which can be observed by naked eyes; and after dissection, the normal flesh pink can be observed in the tumor (B) of the control group while the tumor injecting the tumor blood vessel blocker (E) appears the dark red which is clearly caused by thrombus; it's shown by tumor pathological sections that, compared with the control group (C), and the group (F) injecting the tumor blood vessel blocker forms clear thrombus (indicated by the arrow) in the tumor blood vessel.

[0038] The applicants hereby declare that, the invention explains the detailed features and detailed methods through above embodiments, but the invention is not limited by above detailed features and detailed methods, i.e., it does not mean that, it's necessary to implement the invention only by depending on above detailed features and detailed methods. A person skilled in the art shall understand that, any improvement of the invention, equivalent substitution of components used by the invention and adding of auxiliary components, selection of detailed ways, etc., are all in the protection scope and disclosure scope of the invention.