Use of autophagy (B-cell apoptosis) inhibitor
A technology of inhibitors and cells, applied in the field of drugs for lung injury in mammals, preparation of drugs for preventing and/or treating avian influenza in mammals, and can solve problems such as unclear specific mechanisms
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Embodiment 1
[0127] Codon optimization and humanization gene optimization methods are used to replace the rarely used codons in the expression host of the H5 protein gene coding with frequently used codons. Find the amino acid sequence corresponding to the wild-type H5N1 virus H5 protein (A / Thailand / 4(SP-528) / 2004(H5N1)) through NCBI, and replace the corresponding codons with codons with a high proportion of expression host usage frequency , so as to obtain several optimized H5 protein gene sequences, use DNAMAN software to remove the sequences that may form complex secondary structures, select the optimized H5 genes, remove the transmembrane sequence, and perform gene synthesis in Beijing Qingke Biological Co., Ltd. as Construct the target gene of the plasmid.
[0128] The synthetic sequence is shown as SEQ ID NO:1.
Embodiment 2
[0130] Plasmid construction and detection (see the third edition of > for the experimental method).
[0131] Carrier: Peak13 CD5L TEV human IgG (preserved in our laboratory).
[0132] Insert: Optimized H5 gene with transmembrane removed.
[0133] After the enzyme digestion, electrophoresis, purification, ligation, and transformation processes shown in molecular cloning, the insert fragments were identified, and finally the correct plasmids detected by enzyme digestion (Nhe I / BamH I) were sequenced (Beijing Qingke Biological Co., Ltd. ) for further confirmation.
[0134] The experimental results are attached figure 1 As shown, the three lanes 1, 2, and 3 are λ-Hind III Marker, Peak13 CD5LH5 TEV human IgG, and D2000 Marker. λ-Hind III Marker from small to large (from bottom to top) is 564bp (difficult to distinguish from the picture), 2027bp, 2322bp, 4361bp, 6557bp, 9416bp, 23130bp; D2000Marker from small to large (from bottom to top) The order is 100bp, 250bp, 500bp, 750bp,...
Embodiment 3
[0135] Example 3 Large-scale extraction of plasmids
[0136] A large number of recombined plasmids were extracted by CsCl density gradient centrifugation (see the third edition of > for the experimental method).
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