Double-function amalgamation protein, preparation method and application thereof

A fusion protein and dual-function technology, applied in the field of structural fusion protein and bioengineering, to achieve the effect of inhibiting tumor angiogenesis and enhancing the level of anti-tumor cell immune response

Inactive Publication Date: 2008-05-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the increase of IP-10 concentration, the proliferation of vascular endothelial cells was significantly i

Method used

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  • Double-function amalgamation protein, preparation method and application thereof
  • Double-function amalgamation protein, preparation method and application thereof
  • Double-function amalgamation protein, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design, construction, identification and expression of ITIP bifunctional molecule

[0052] Such as figure 1 As shown, using InsightII molecular simulation software and database prediction methods, combined with existing ITAC and IP-10 functional research reports, the N-terminal and N-LOOP regions of ITAC and the C-terminal of IP-10 were screened out, and then The upstream of the N-terminal and N-LOOP region of ITAC introduces EcoRI restriction site, and downstream of it introduces Hind III restriction site; the upstream of IP-10 C-terminal introduces HindIII restriction site, and downstream of it introduces Xho I restriction site. RT-PCR cloned the N-terminal and N-LOOP regions of mouse ITAC and the C-terminal coding genes of IP-10. Digest the N-terminal and N-LOOP region gene fragments of ITAC and the C-terminal gene fragment of IP-10 with Hind III respectively. Take 1μl of the digested products and mix them as templates. Use the N-terminal and N-LOOP region up...

Embodiment 2

[0073] Example 2 The effect of ITIP gene transfection on the growth motility of 4T1 cells

[0074] Female BALB / c mice were inoculated subcutaneously (5×10 4 Cells / only), including untransfected 4T1 cell group, 4T1-pcDNA3 cell group, 4T1-IP-10 cell group, 4T1-ITAC cell group and 4T1-ITIP cell group (n=6). Observe the tumor formation of BALB / c mice. When the tumor grows to palpable time, measure the maximum length and width of the tumor every other day, and use the average of the two as an indicator of tumor growth. Observe for 30 days. Results The tumor formation rate was 4T1 (10 / 10), 4T1-pcDNA3 (6 / 6), 4T1-IP-10 (7 / 10), 4T1-ITAC (6 / 10) and 4T1-ITIP (3 / 10) As shown in Figures 7A and 7B, the 4T1-ITIP tumor grew slowly and its weight was significantly reduced compared with the control (p5 Cells / only), including untransfected 4T1 cell group, 4T1-pcDNA3 cell group, 4T1-IP-10 cell group, 4T1-ITAC cell group and 4T1-ITIP cell group (n=3). Observe the tumor formation in mice. As shown in F...

Embodiment 3

[0075] Example 3 Analysis of the degree and type of lymphocyte infiltration in 4T1-ITIP tumor

[0076] Since the constructed bifunctional molecule contains the N-terminal and N-LOOP regions where ITAC and the receptor have a significant effect, and the 4T1-ITIP cell supernatant we constructed has a positive effect on CXCR3 + The cells have significant chemotactic activity. Further, we studied the infiltration of local lymphocytes in 4T1-ITIP tumors. The tumor tissue 14 days after tumor formation was taken out, and after type IV collagenase digestion, the tumor infiltrating lymphocytes were separated by Ficoll density-gradient centrifugation, and counted, and the number of lymphocytes infiltrating per unit weight of tumor tissue was compared. The tumor-infiltrating lymphocytes were treated with goat anti-mouse CXCR3 antibody at 4°C for 30 minutes, washed once with PBS, and then treated with FITC-labeled donkey anti-goat IgG at 4°C for 30 minutes, washed twice with PBS and detected ...

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Abstract

The invention belongs to the bioengineering field, and discloses a double-functional fusion protein which comprises two function structure areas which are formed by an N end of ITAC, an N-LOOP area and a C end of IP-10. The invention also discloses a carrier and a cell of a double-functional fusion protein gene with a code as well as the application of the double-functional fusion protein. Approved by checking mouse lymphocyte multiplication and killing experiment and tumor histomorphology, the double-functional fusion protein of the invention has the anti-tumor effect, thereby obviously strengthening the anti-tumor cell immunity response level, and synchronously stopping the producing of the tumor blood vessel.

Description

Technical field [0001] The present invention relates to bioengineering technology, in particular to structural fusion proteins, in particular to a bifunctional fusion protein and its preparation method and application. Background technique [0002] Studies have shown that ITAC is the strongest agonist of CXCR3. ITAC has a high affinity for CXCR3 receptor, has significant chemotactic activity, can rapidly induce receptor internalization and trigger cellular calcium influx. Compared with IP-10, ITAC's ability to mobilize intracellular calcium ions is twice that of IP-10. The activation of chemokine receptors inhibits its own reactivity, manifested in two forms of receptor desensitization and receptor internalization. This is the key mechanism for lymphocytes to keep responding to changes in small chemotactic components. Studies have shown that when IP-10 and ITAC are co-incubated with CXCR3 positive cells, the receptor internalization is most significant at 15 minutes, and the inte...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/16C12N15/09A61P35/00
Inventor 熊思东储以微杨秀利王缨
Owner FUDAN UNIV
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