Application of PDK3 gene and its expression product
A technology for expressing products and genes, which is applied in the field of application of PDK3 gene and its expression products, and can solve the problems of poor overall curative effect and poor pertinence
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0020] Example 1 RNAi screening of kinase genes essential for the growth of liver cancer cells within the scope of the whole kinase group
[0021] A library of siRNAs targeting all kinases and kinase-related genes on the human genome was obtained commercially (Stealth TM RNAi Human Kinase Collection, Invitrogen, Catalog no. 12938-200). Stealth per set TM The RNAi Collection consists of several 96-well plates. Stealth by 2 nmoles TM RNAi was dissolved in 100 μl of 1X RNA annealing / dilution solution (10 mM Tris-HCl, pH 8.0; 20 mM NaCl; 1 mM EDTA, pH 8.0) to a final concentration of 20 μM.
[0022] Use CellCounting Kit-8 (cell viability detection kit, referred to as CCK-8) to measure cell viability, the experimental steps are as follows figure 1 As shown, the absorbance is measured with a BioTek microplate reader in the United States to generate raw cell activity data. The raw data undergoes steps such as standardization, data correction, and Z value evaluation, such as ...
Embodiment 2
[0023] Example 2 RNA interference PDK3 gene inhibits the growth of liver cancer cells
[0024] 48 hours after the constructed plasmid pSUPER was transfected into liver cancer cells, the cell line RNA was extracted, and the effect of RNA interference was identified by quantitative PCR. For transfection, the cells were re-seeded in a 10cm large plate for culture, and neomycin G418 was added to the culture medium for selection and culture for 4 to 6 weeks. The culture medium was removed, rinsed twice with 1×PBS, stained with crystal violet, and photographed for counting.
Embodiment 3
[0025] Example 3 Immunoassay
[0026] 1. Acquisition of antigenic protein
[0027] Obtain the cDNA sequence of human PDK3 gene from Genebank in the United States, obtain the coding frame by PCR amplification, insert it into a prokaryotic or eukaryotic expression vector, express PDK3 protein, and purify the protein according to the purification system of genetic engineering expression products.
[0028] 2. Antibody Preparation
[0029] Antibodies can be prepared in several ways:
[0030] (1) Cell fusion method: immunize animals (including rabbits, goats, etc.) with the PDK3 protein prepared above, obtain spleen cells, fuse with myeloma cells, and prepare monoclonal antibodies according to conventional monoclonal antibody preparation techniques.
[0031] (2) Using the phage surface display library, cloning the spleen IgG variable region of the immunized animal and expressing it into a genetically engineered monoclonal antibody.
[0032] (3) Using the purified protein to immun...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More