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Fused protein as well as preparation method and application thereof

A fusion protein and protein technology, applied in the biological field, can solve the problems of late or relapsed patients who cannot be effective, less than 50%, and fail to improve the prognosis of tumor patients, so as to achieve the effect of improving expression yield and accuracy, and improving expression efficiency

Active Publication Date: 2017-05-31
张紫寒
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Ovarian cancer, cervical cancer and endometrial cancer are three malignant tumors that seriously threaten women's life and health. The five-year survival rate is less than 50%. There are more than 10,000, and there is a clear trend of younger people. The country has widely carried out "two cancer screenings", but it still fails to improve the prognosis of cancer patients. Female reproductive system malignant tumors are malignant tumors that are tackled in my country's medium and long-term scientific and technological development outline plan one
The traditional treatment for malignant tumors of the female reproductive system is surgery, radiotherapy, and chemotherapy, but they only have a certain effect and cannot be effective for advanced or recurrent patients. It is urgent to find new treatment methods

Method used

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  • Fused protein as well as preparation method and application thereof
  • Fused protein as well as preparation method and application thereof
  • Fused protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 constructs and expresses a purified fusion protein

[0047] 1. Codon optimization and gene synthesis for CLIC1 and HSP27.

[0048] The designed target nucleotide sequence is shown in SEQ ID NO.2, which can encode the amino acid sequence shown in SEQ ID NO.1.

[0049] 2. Entrust Nantong University School of Medicine to use the method of whole gene synthesis to obtain the gene fragment shown in SEQ ID NO.2, named CLIC1-HSP27 gene.

[0050] 3. Using restriction endonucleases Nde I and Hind III to digest the CLIC1-HSP27 gene and the expression vector pET30a respectively, insert the CLIC1-HSP27 gene into the expression vector pET30a to obtain recombinant plasmids, named pET30aCLIC1-HSP27, pET30a CLIC1- The plasmid map of HSP27 is as follows Figure 5 shown. In plasmid construction, the ratio of gene to vector and some related conditions are as follows (20ul reaction system):

[0051] COMPONENT 20 μl REACTION 10X T4 DNA Ligase Buffer 2ul V...

Embodiment 2

[0067] Peripheral blood mononuclear cells were obtained through the standard procedure of leukocyte separation, and co-cultured with the recombinant protein CLIC1-HSP27 in vitro to obtain antigen-presenting cells (APCs) and killer lymphocytes activated by the recombinant protein CLIC1-HSP27, and the antigen-presenting cells will be The PAP protein is digested into a polypeptide and presented on its surface, which can be recognized by T cells of the immune system. After recognizing the antigen, the T cells can find and kill cancer cells expressing the CLIC1 antigen in vivo.

Embodiment 3

[0069] The TGF-β content in the supernatant of T lymphocytes activated by CLIC1-HSP27-loaded dendritic cells was detected by Elisa method (TGF-β is a cytokine secreted when T cells are activated), and the detection results were as follows: Figure 6 As shown, blood sample A, blood sample B and blood sample C come from peripheral blood of different people respectively. From Figure 6 It can be seen that, compared with the control group (blank control group) between CLIC1-HSP27, the content of TGF-β in the CLIC1-HSP27 stimulation group was significantly increased (pg / ml).

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Abstract

The invention provides a fused protein as well as a preparation method and application thereof. The fused protein comprises a CLIC1 protein, a connecting peptide and an Hsp27 (Heat Shock Proteins 27) protein; and the CLIC1 protein is connected with the Hsp27 protein through the connecting peptide. The fused protein provided by the invention can be applied to preparation of medicines for preventing and treating tumors and has the advantages of good specificity, high efficiency of antigens and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion protein, a preparation method and an application thereof. Background technique [0002] Ovarian cancer, cervical cancer and endometrial cancer are three malignant tumors that seriously threaten women's life and health. The five-year survival rate is less than 50%. There are more than 10,000, and there is a clear trend of younger people. The country has widely carried out "two cancer screenings", but it still fails to improve the prognosis of cancer patients. Female reproductive system malignant tumors are malignant tumors that are tackled in my country's medium and long-term scientific and technological development outline plan one. The traditional treatment for malignant tumors of the female reproductive system is surgery, radiotherapy, and chemotherapy, but they only have certain curative effects, and they are not effective for advanced or recurrent patients. At...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/00A61P35/00
CPCA61K39/0011C07K14/47C07K14/4702C07K2319/00C12N15/70C12N2800/101
Inventor 张紫寒于文涛张震宇刘崇东
Owner 张紫寒
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