Method of treating endotoxemia
A technology of endotoxemia and endotoxin, applied in gene therapy, biochemical equipment and methods, viruses, etc., can solve the problem of low responsiveness
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Embodiment 1
[0063] C3H / HeJ mouse Lps d Isolation and identification of genes
[0064] Studies by Kang et al. (Infection and Immunity, 64, 4612-4617 (1996)) showed that Lps n Gene restores lipopolysaccharide-mediated B cell responses in splenic B cells of C3H / HeJ mice.
[0065] To confirm that the Lps gene is defective in the genome of C3H / HeJ mice, and this defect leads to low responsiveness to LPS stimulation, the Lps gene was isolated from the cDNA library of pCD-HeJ.
[0066] RNA from C3H / HeJ splenic B cells was used to construct an Okayama-Berg cDNA expression library (Okayama, H. and Berg, P. et al., Mol. Cell Biol., 2, 161-170 (1982)). Spread the pCD-HeJ cDNA library on 8 flat plates (5.8×10 4 clones), with the Lps of LPS responder mouse C3H / HeOuJ n The library was probed with a 0.9 kb BamHI-PstI fragment in the cDNA (Infection and Immunity, 64, 4612-4617 (1996)). 25 positive clones were identified.
[0067] Of the 25 independent clones analyzed, 8 were full-length cDNAs, as ...
Embodiment 2
[0072] Lps n and Lps d Construction of retroviral vectors
[0073] The Lps of embodiment 1 n and Lps d The cDNA was cloned into the XhoI site of the N2 retroviral vector (Wong et al., Molecular Cell Biology, 9, 798-808 (1989); Wong et al., Genes Dev. 1, 358-365 (1987)). The N2 retroviral vector includes 5' and 3' MMLV LTRs, splice donor and splice acceptor sites, and a neomycin resistance marker gene.
[0074] Will Lps n cNDA was isolated from the XhoI restriction fragment of pCD-LPS (Infection and Immunity, 64, 4612-4617 (1996)) and ligated into the site of the XhoI restriction fragment of the N2 retroviral vector to generate pN2-Lps n . Use the same method to generate pN2-Lps d vector, except that the mutant LPS used d cDNA.
[0075] To generate carrying Lps n and Lps d Retroviral stocks of sequences, respectively, with pN2-Lps n and pN2-Lps d The plasmid DNA was transfected into GP / E virus packaging cells by calcium phosphate DNA co-precipitation method (Mar...
Embodiment 3
[0079] Lps n and Lps dGene transfer into splenic B cells of C3H / HeJ mice via retrovirus
[0080] A) Retroviral gene transfer
[0081] Splenic B cells from C3H / HeJ mice were depleted of red blood cells, retrovirally infected in the presence of 100 μg / ml LPS, and cultured in S17 mesenchymal cell conditioned medium (known to stimulate pre-B cell growth) (Collins et al., Journal of Immunology, 138, 1082-1087 (1987); Dorshkind et al., Journal of Immunology Methods (J.Immunol.Meathods), 123, 93-101 (1989); Narendran et al., European Journal of Immunology (Eur . J. Immunol.), 22, 1001-1006 (1992)). In order to maximize the infection efficiency of retroviruses, the culture medium was repeatedly refreshed with fresh stock solutions of concentrated viral supernatants, as described in the flowchart in Figure 2. N2 parental vector, N2-Lps n or N2-Lps d The virus titers of the concentrated stock solution of the vector were all normalized to 2×10 6 G418 colonies / ml.
[0082] B) MTT ...
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