Antitumor vaccine, encoding gene, expression vector, expression engineering bacteria and application of targeted VEGF and mucin1

An expression vector, anti-tumor technology, applied in the field of anti-tumor vaccine, can solve the problem of lack of VEGF protein research, and achieve the effect of convenient affinity chromatography and inhibiting tumor angiogenesis

Active Publication Date: 2017-01-04
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research on the use of VEGF protein as a tumor epitope carrier

Method used

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  • Antitumor vaccine, encoding gene, expression vector, expression engineering bacteria and application of targeted VEGF and mucin1
  • Antitumor vaccine, encoding gene, expression vector, expression engineering bacteria and application of targeted VEGF and mucin1
  • Antitumor vaccine, encoding gene, expression vector, expression engineering bacteria and application of targeted VEGF and mucin1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The anti-tumor vaccine fusion protein mVEGF165b-muc1 targeting VEGF and mucin1 uses mutated VEGF165b as the carrier of the epitope peptide of the tumor-associated antigen mucin1, and a piece of epitope peptide of mucin1 is fused at its C-terminus. Linked by GGSAAY, its amino acid sequence is as follows:

[0035]

[0036] Among them, the normal part is the amino acid sequence of mVEGF165b, the underlined part is the amino acid sequence of the connecting peptide GGSAAY, and the italic part is the amino acid sequence of the epitope peptide of the tumor-associated antigen mucin1.

[0037] The nucleotide sequence encoding the above amino acid sequence is as follows:

[0038]

[0039]

[0040]Wherein, the normal part is the nucleotide sequence of mVEGF165b, the underlined part is the nucleotide sequence of the connecting peptide GGSAAY, and the italic part is the nucleotide sequence of the epitope peptide of the tumor-associated antigen mucin1.

Embodiment 2

[0042] The anti-tumor vaccine expression vector targeting VEGF and mucin1, its construction steps are:

[0043] 1) Using restriction endonucleases Not I and EcoR I to double-enzyme pPIC9K vector and mVEGF165b-muc1 gene sequence (as shown in SEQ ID NO. The protected base, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.), the enzyme digestion reaction system and reaction conditions are as follows:

[0044] Enzyme digestion reaction system of pPIC9K vector (100μl): pPIC9K vector (75ng / μl) 26.7μl, endonuclease Not I (10U / μl) 2μl, endonuclease EcoR I (10U / μl) 2μl, 10×H Buffer ( Enzyme digestion buffer) 10 μl, 0.1% BSA (bovine serum albumin) 10 μl, sterilized distilled water 49.3 μl; enzyme digestion reaction conditions: react at 37° C. for 3 hours;

[0045] Enzyme digestion reaction system of mVEGF165b-muc1 gene sequence (100μl): mVEGF165b-muc1 gene fragment (149ng / μl) 13.4μl, endonuclease Not I (10U / μl) 2μl, endonuclease EcoR I (10U / μl) 2μl , 10×H Buffer (digestion buffe...

Embodiment 3

[0056] The anti-tumor vaccine expression engineering bacteria targeting VEGF and mucin1, the construction process is as follows: use the restriction endonuclease Sal I to digest the above pPIC9K-mVEGF165b-muc1 expression vector to obtain the linearized pPIC9K-mVEGF165b-muc1 expression vector, and then Transform Pichia pastoris GS115 competent cells, screen positive colonies, and cultivate high-copy positive clone colonies. The specific operations are as follows:

[0057] 1) Linearization of pPIC9K-mVEGF165b-muc1 expression vector

[0058] The pPIC9K-mVEGF165b-muc1 expression vector was digested with the restriction endonuclease Sal I, and the digested product was precipitated by adding 20 μl of 3mol / L NaAc solution (pH=5.2) and 400 μl of absolute ethanol, and at 4° C. Centrifuge at 12000rpm for 10 minutes, wash the precipitate with 70% (v / v) alcohol, then centrifuge at 12000rpm for 10 minutes, dissolve the precipitate in 20 μl of sterile deionized water after drying, and take ...

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Abstract

The invention discloses an antitumor vaccine, an encoding gene, an expression vector, an expression engineering bacteria and application of targeted VEGF and mucin1, and belongs to the technical field of biology. An antitumor vaccine fusion protein adopts VEGF165, VEGF165b or corresponding modified VEGF mutants as an epitope vector, at least one section of epitope peptide of tumor related antigen mucin1 is connected to an end N or an end C of the vector to form the antitumor vaccine having a double target effect. The VEGF in the vaccine can be used as the epitope vector to improve the antigenicity of the mucin1 epitope connected with the VEGF, the over-expressed mucin1 tumor cells are killed, the VEGF can also be used as an antigen to produce an antibody for VEGF so as to inhibit the generation of tumor vessels and cut off the nutrient supply of the tumor. Meanwhile, the epitope vector has a heparin-binding domain which can be bond with heparin sepharose, thereby facilitating the subsequent protein purification.

Description

technical field [0001] The invention relates to an anti-tumor vaccine targeting VEGF and mucin1, and also relates to a gene encoding the anti-tumor vaccine, an anti-tumor vaccine expression vector, an expression engineering bacterium and its application, and belongs to the field of biotechnology. Background technique [0002] At present, malignant tumors have become the greatest threat to national health. Conventional tumor treatment methods, such as surgery, radiotherapy and chemotherapy, have their own limitations, which makes immunotherapy a supplementary therapy after conventional therapy. In immunotherapy, various mAbs targeting tumor-associated antigens or immune checkpoints play a vital role in tumor treatment. However, it is expensive, requires long-term administration, and has problems such as easy resistance of tumor cells. In theory, tumor vaccines are highly specific and utilize the patient's own immune system to generate an anti-tumor response, which can gener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19A61K39/00A61K47/42A61P35/00C12R1/84
CPCA61K39/0011A61K47/42C07K14/4748C07K14/49C07K2319/00A61K2300/00
Inventor 张会勇夏文姣贾恩朝闫影苏静路臣桂朱武凌
Owner XINXIANG MEDICAL UNIV
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