Application in adenovirus expression carrier of humen immunoglobulin storter
A technology for human immunoglobulin and expression vectors, which is applied in the application field of human immunoglobulin promoters in adenovirus expression vectors, and can solve problems such as inactive p53 protein, amino acid changes, and loss of tumor suppressor effect
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Embodiment 1
[0039] Example 1. Immunohistochemical method proves that immunoglobulin genes are expressed in various tumor cells
[0040] Tumor tissue chips containing various epithelial sources, including lung cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, kidney cancer, etc.; and tumor tissue chips of various mesenchymal sources (purchased from Chaoying Tissue Chip Company, Xi'an ), including 214 tumor tissues such as fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma and osteosarcoma and corresponding normal tissues. The tissue chip was first dewaxed, hydrated, and the antigen was heat-restored, and then pre-cleared endogenous peroxidase with 1% hydrogen peroxide, blocked with 10% normal sheep serum, and then added rabbit anti-human IgG antibody (working solution, Dako company, Denmark) at 37°C for 30 minutes; after washing three times with 0.01M PBS, add goat anti-rabbit IgG-HRP (1:100; DaKO Company, Denmark) at 37°C for 45 minutes; after washing three times with...
Embodiment 2
[0042] Example 2, proved that cancer cells can initiate the expression of immunoglobulin genes
[0043] First, using the tumor cell line Hela-S3 genomic DNA as a template, the PCR method was used to amplify the gene sequences of -1200bp and -293bp upstream of the V region gene of the immunoglobulin heavy chain (including the complete VH Promoter and TATA box), and then the The two gene fragments and the pGL3 reporter gene carrier were constructed as recombinant plasmids. After sequencing, the plasmids were mixed with 10ug / 2×10 6 The concentration of cells was transfected into Hela-S3 (cervical cancer cell line) and HT-29 (colorectal cancer cell line) to detect its activation activity. At the same time, the tumor cell line Raji derived from the B cell line was used as a positive control; the tumor cell line Jurkat derived from the T cell line was used as a negative control (the above cell lines were all purchased from ATCC). One of the viral promoters, the SV40 promoter, was u...
Embodiment 4
[0054] Example 4, Ig promoter can more effectively promote the expression of HSG
[0055] HeLa or HT-29 cells were cultured in a culture dish in DMEM medium containing 10% fetal bovine serum. After culturing for 24 h, the supernatant was discarded, and adenovirus (empty adenovirus, Ig / HSG / adenovirus and CMV / HSG / adenovirus), after culturing for 48 hours, discard the supernatant, scrape the cells of each group, wash with PBS, collect the cells, and use protein lysate (10mM Tris, pH8.0, 10mMNaCl, 0.3M sucrose, 3mM MgCl 2 , 0.5% NP-40, mixed protease inhibitor Protease inhibitor cocktail) to lyse the cells, centrifuge to get the supernatant after the lysis is sufficient, after the protein is quantified, carry out SDS-PAGE, then, transfer the protein to the nylon membrane, and then degrease with 5% Blocked with milk powder, reacted with rabbit anti-HSG C-terminal and N-terminal antibodies (purchased from Tokyo Institute of Biotechnology), respectively; after washing with PBS, rea...
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