Recombinant fusion protein with targeted cell for killing tumor
A technology of fusion protein and Pseudomonas, which is applied in the field of recombinant fusion protein with the ability to kill tumor cells, can solve the problems of high dosage and low biological activity, and achieve the effect of improving activity
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example 1
[0009] According to the protein sequence of fusion protein 1 and the principle of codon preference of hosts in different expression systems, the prokaryotic expression gene sequence and eukaryotic expression gene sequence of fusion protein 1 were designed, and synthesized by the method of whole gene synthesis combined with PCR. After the gene sequence is verified by sequencing, the target gene is connected into the expression vector. The prokaryotic system mainly uses the improved pET plasmid, and the eukaryotic system mainly uses the pIC9K plasmid series. The verified recombinant plasmid is transferred into the host bacteria. After expression, The target bacteria were obtained by fermentation, and then the bacteria were broken by mechanical method or osmotic pressure method, and the supernatant was collected by centrifugation, and the fusion protein 1 was obtained by purification by hydrophobic chromatography and ion exchange chromatography. The target protein was determined b...
example 2
[0011] According to the protein sequence of fusion protein 3 and the principle of codon preference of hosts in different expression systems, the prokaryotic expression gene sequence and eukaryotic expression gene sequence of fusion protein 1 were designed and synthesized by the method of whole gene synthesis combined with PCR. gene sequence. After sequencing and verification, the target gene is connected into the expression vector. The prokaryotic system mainly uses the improved pET plasmid, and the eukaryotic system mainly uses the pIC9K plasmid series. The verified recombinant plasmid is transferred into the host bacteria, and the target gene is obtained through expression and fermentation. The bacteria are broken by mechanical method or osmotic pressure method, centrifuged to obtain supernatant, and purified by hydrophobic chromatography and ion exchange chromatography to obtain fusion protein 3. The target protein was determined by electrophoresis, HPLC, mass spectrometry,...
example 3
[0013] On the basis of the gene sequence of fusion protein 1, the 4 amino acids Lys-Asp-Glu-Leu at its C-terminus were removed by PCR mutation method and replaced with Arg-Glu-Asp-Leu-Lys, similar to Example 1 The method obtained the fusion protein GnRH-PE40, which is the reference substance. The target protein was determined by electrophoresis, HPLC, mass spectrometry, N-terminal amino acid sequence determination and other methods. Finally, the cell viability test was performed.
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