Part:BBa_K4833003

This sequence can silence the gene expression of PD-L1 in tumor cells through the RNAi mechanism.

This sequence is a partial antisense sequence of the PD-L1 gene. PD-L1 is a molecule present on the surface of cancer cells that inhibits the immune pathway. Binding of PD-L1 to the programmed death receptor 1 (PD-1) on the surface of immune cells can initiate programmed cell death in T cells, inducing immune evasion of tumor cells. Silencing the PD-L1 gene can relieve the inhibition of cancer cells on immune cells, activate the immune system, and inhibit the occurrence and development of tumors. The shPD-L1 sequence in the plasmid can silence the gene expression of PD-L1 in tumor cells through the RNAi mechanism. This sequence transcribes and produces shPD-L1, which is the antisense sequence of PD-L1 mRNA. Inside the cell, shRNA is subsequently processed into siRNA, and the functional strand (guide strand) of these siRNAs is loaded into the RISC (RNA-induced silencing complex). The RISC then binds to the complementary sequence on PD-L1 mRNA and catalyzes the cleavage and degradation of the target mRNA. In conclusion, this sequence can silence the gene expression of PD-L1 in tumor cells, thereby activating the body's own immune response.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 22
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

1 Biological function validation

The shControl plasmid and shPD-L1 plasmid were transiently transfected into mouse colon cancer CT26 cells using Lipo3000 transfection reagent. Cells were collected 24 hours post-transfection for detection of PD-L1 gene transcription levels using RT-PCR. Cells were collected 48 hours post-transfection for detection of PD-L1 protein expression using Western blot analysis. The results in Figure 1 showed that compared to the control group, the shPD-L1 plasmid transfection group exhibited a significant decrease in PD-L1 mRNA expression (P<0.001). The results in Figure 2 showed that compared to the control group, the shPD-L1 plasmid transfection group showed a significant decrease in PD-L1 protein expression (P<0.001). These results indicate that the shPD-L1 plasmid was successfully transfected into CT26 cells and exerted its biological effect.

The shPD-L1 sequence we designed can inhibit the transcription and translation of the PD-L1 gene！

Fig.1 Quantification of PD-L1 mRNA expression levels in CT26 cells was performed using RT-PCR. The graph represents the quantification of PD-L1 mRNA expression.（Data are mean±SEM）

Fig.2 Western blot analysis was performed to detect the expression levels of PD-L1 protein in CT26 cells. The graph represents the quantification of PD-L1 protein expression.（Data are mean±SEM）