A method for extending half-life of a protein
A protein and half-life technology, applied in the protein field, can solve the problems of increasing the induction of thyroid cancer and the risk of breast cancer, and achieve the effect of prolonging the half-life
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0164] Example 1 Analysis of β-opsonin ubiquitination and half-life extension and detection of signal transduction in cells
[0165] 1. β-opsonin expression vector cloning and protein expression
[0166] (1) Cloning of β-opsonin expression vector
[0167] RNA was extracted and purified from HepG2 (ATCC, HB-8065) cells using phenol and chloroform to clone β-opsonins. So, by using SuperScript TM Single-stranded DNA was synthesized with the First-Strand cDNA Synthesis System (Invitrogen, Grand Island, NY). The cDNA synthesized above was then used as a template to amplify β-opsonin. The obtained β-opsonin DNA amplification product was treated with BamHI and EcoRI, and then connected to the pcDNA3-myc (5.6kb) vector digested with the same enzyme ( figure 1 , β-opsonin amino acid sequence: SEQ No.1). Then, after restriction enzyme digestion of the cloning vector, the presence of the DNA insert was confirmed by agarose gel electrophoresis ( figure 2 ). The conditions o...
Embodiment 2
[0184] Example 2 Analysis of growth hormone ubiquitination and half-life extension, and analysis of signal transduction in cells
[0185] 1. GH expression vector cloning and protein expression
[0186] (1) Cloning of GH expression vector
[0187] The GH DNA amplified by PCR was treated with EcoRI, and then ligated to the pCS4-flag (4.3kb, Oncotarget., 7(12), 14441-14457, 2016) vector digested with the same enzyme in advance ( Figure 8 , growth hormone amino acid sequence: SEQNo.10). Then, after restriction enzyme digestion of the cloning vector, the presence of the DNA insert was confirmed by agarose gel electrophoresis ( Figure 9 ). The conditions of PCR are as follows: the first step: 94°C for 3 minutes (1 cycle); the second step: 94°C for 30 seconds; 60°C for 30 seconds; 72°C for 30 seconds (25 cycles); the third step: 72 °C for 10 minutes (1 cycle), then stored at 4°C. Figure 8 The underlined black nucleotide sequence indicates the primer set used for PCR and t...
Embodiment 3
[0203] Example 3 Analysis of insulin ubiquitination and half-life extension, and analysis of signal transduction in cells
[0204] 1. Insulin expression vector cloning and protein expression
[0205] (1) Cloning of insulin expression vector
[0206] The amplified product of insulin DNA by PCR was treated with BamHI and EcoRI, and then ligated to the pcDNA3-myc (5.6kb) vector digested with the same enzyme in advance ( Figure 15 , insulin amino acid sequence: SEQ No.17). Then, after restriction enzyme digestion of the cloning vector, the presence of the DNA insert was confirmed by agarose gel electrophoresis ( Figure 16 ). The conditions of PCR are as follows: the first step: 94°C for 3 minutes (1 cycle); the second step: 94°C for 30 seconds; 60°C for 30 seconds; 72°C for 30 seconds (25 cycles); the third step: 72 °C for 10 minutes (1 cycle), then stored at 4°C. Figure 15 The underlined black nucleotide sequence indicates the primer set used for PCR and to determine ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More