Results
Recombinant E. coli
Construction of plasmid pTRC99K-5HT
1. Preparation of plasmid vector pTRC99K
Figure 1. DNA concentration comparing with the pure water
The DNA concentrations of four tubes plasmid pTRC99K are 325.5ng/μl, 386.6ng/μl, 234.3ng/μl, 225.1ng/μl, respectively.
2. Acquisition of linearized vectors pTRC99K and inserts TDC-TPH
- Line 1: pTRC99K-vector, control plasmid
- Line 2: linearized pTRC99K-vector, correct
- Line 3: the PCR product of TDC, correct
- Line 4: the PCR product of TPH, correct
- Line 5: the over-lap PCR product of TDC-TPH, correct
- Line 6: the over-lap PCR product of TDC-TPH, correct
3. Gel electrophoresis of recombinant plasmid pTRC99K- TDC-TPH
- Line 1: pTRC99K-vector, control plasmid, 4167bp
- Line 2: recombinant plasmid pTRC99K-TDC-TPH-1, incorrect
- Line 3: recombinant plasmid pTRC99K-TDC-TPH-2, 7077bp, correct
- Line 4: control plasmid, 6379bp
Gel electrophoresis of recombinant plasmid pTRC99K- TDC-TPH shows that the size of pTRC99K-TDC-TPH-2 is correct.
4. Rcombinant plasmid pTRC99K- TDC-TPH sequencing analysis
Global sequence alignment of sequencing:
The details of TPH sequence alignment:
The details of TDC sequence alignment:
The sequencing results show that both TPH and TDC are inserted into pTRC99K successfully.
5. Expression of TPH and TDC proteins
Figure 2: SDS-PAGE analysis for TPH and TDC proteins.
- Lane 1: Culture supernatant with induction (40 μL)；
- Lane 2: Culture supernatant without induction (40 μL)；
- Lane 3: Without samples
- Lane 4: Supernatant of cell lysate with induction (40 μL)；
- Lane 5: Supernatant of cell lysate without induction (40 μL)；
- Lane 6: Pellet of cell lysate with induction (40 μL)；
- Lane 7: Pellet of cell lysate without induction (40 μL)；
- Lane 8: Supernatant of cell lysate with induction (20 μL)；
- Lane 9: Supernatant of cell lysate without induction (20 μL)；
We got two bands 43.53kDa (TPH) and 53.55kDa (TDC) in both supernatant of cell lysate and pellet of cell lysate. These results suggest that TPH and TDC proteins expressed successfully.
Rin 14b cell screening platform
Patch Clamp Results
After administration, the membrane potential became stable and the release frequency of AP increased, suggesting that this compound may have the ability to stably activate RIN14B cells.
After administration, the membrane potential decreased, the release frequency of AP increased, and the amplitude increased significantly, and the effect could not be eluted, which may indicate that this compound has a strong activation ability for RIN14B cells.
The membrane potential and AP release frequency increased after administration, suggesting that the compound may have the ability to activate RIN14B cells.