Double-function amalgamation protein, preparation method and application thereof
A fusion protein and dual-function technology, applied in the fields of bioengineering and structural fusion proteins, to achieve the effect of inhibiting tumor angiogenesis and enhancing the level of anti-tumor cell immune response
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Embodiment 1
[0050] Example 1: Design, Construction, Identification and Expression of ITIP Bifunctional Molecules
[0051] Such as figure 1 As shown, using the InsightII molecular simulation software and database prediction method, combined with the relevant reports of the existing ITAC and IP-10 function research, the N-terminal and N-LOOP regions of ITAC and the C-terminal of IP-10 were screened out, and the An EcoRI restriction site was introduced upstream of the N-terminal and N-LOOP region of ITAC, and a HindIII restriction site was introduced downstream; a HindIII restriction site was introduced upstream of the C-terminal of IP-10, and an Xho I restriction site was introduced downstream. The genes encoding the N-terminal and N-LOOP region of mouse ITAC and the C-terminal of IP-10 were respectively cloned by RT-PCR. The N-terminal and N-LOOP region gene fragments of ITAC and the C-terminal gene fragment of IP-10 were respectively digested with Hind III, and 1 μL of digested products...
Embodiment 2
[0076] Example 2 Effect of ITIP gene transfection on growth kinetics of 4T1 cells
[0077] 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 in BALB / c mice. When the tumor grew to be palpable, the maximum long diameter and wide diameter of the tumor were measured every other day, and the average value of the two was used as the index of tumor growth. Continuous observation for 30 days. Results The tumor formation rates were 4T1 (10 / 10), 4T1-pcDNA3 (6 / 6), 4T1-IP-10 (7 / 10), 4T1-ITAC (6 / 10) and 4T1-ITIP (3 / 10) ,Such as Figure 7 As shown in A and 7B, the 4T1-ITIP tumor grew slowly, and its weight was significantly lighter than that of 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...
Embodiment 3
[0078] Example 3 Analysis of the degree and type of lymphocyte infiltration in 4T1-ITIP tumors
[0079] Since the constructed bifunctional molecule contains the N-terminal and N-LOOP regions where ITAC has a significant interaction with the receptor, and the supernatant of 4T1-ITIP cells we constructed has no effect on CXCR3 + The cells had significant chemotactic activity. Further, we studied the local lymphocyte infiltration of 4T1-ITIP tumors. The tumor tissues 14 days after tumor formation were taken out, digested with type IV collagenase, and the tumor-infiltrating lymphocytes were separated by Ficoll density-gradient centrifugation and counted to compare the number of infiltrating lymphocytes per unit weight of tumor tissue. The tumor-infiltrating lymphocytes were reacted with goat anti-mouse CXCR3 antibody at 4°C for 30 minutes, washed once with PBS, then reacted with FITC-labeled donkey anti-goat IgG for 30 minutes at 4°C, washed twice with PBS, and detected by FACS. ...
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