Special target medicine and its use
A targeted and specific technology, applied in the direction of drug combination, application, antineoplastic drugs, etc., can solve the problems of not being able to effectively reduce the adverse reactions of IFN, and not being able to improve the curative effect of IFN at the dose
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[0051] Example 1 Design of specific targeting interferon amino acid sequence
[0052] 1. The X sequence selects the 1st to 50th amino acid sequence (SEQ ID NO: 3) of the surface large protein of HBV strain FMC#97, which includes the widely confirmed specific binding to the surface of hepatocytes and mediates HBV infection of hepatocytes. Key sequence.
[0053] 2. L selects a sequence composed of flexible amino acids (SEQ ID NO: 4) to reduce the interaction between X and Y on protein conformation and function.
[0054] 3. The Y sequence selects the full-length amino acid sequence of IFNα-2b (SEQ ID NO: 5).
[0055] 4. X and Y are located at the N-terminus and C-terminus of the L sequence respectively, and the overall sequence formed is shown in SEQ ID NO:6.
[0056] 5. Carry out glycosylation site analysis on SEQ ID NO: 6 (see Figure 1), where the 15th amino acid aspartyl is a possible N-linked glycosylation modification site, which is replaced by similar amino acid mutations ( 15N...
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[0057] Example 2 Design of DNA sequence encoding specific targeting interferon
[0058] 1. Select Pichia pastoris as the expression system of specific targeting interferon. Pichia pastoris is one of the most successful eukaryotic expression systems for foreign protein expression (Cereghino JL, Cregg J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris[J]. FEMS Microbiol Rev, 2000, 24(1): 45 -66.). It not only has the characteristics of rapid propagation, low cost and convenient operation of the prokaryotic expression system, but also has the advantages of a eukaryotic expression system with correct processing and folding of exogenous proteins and moderate glycosylation. Therefore, it is more and more widely used for the large-scale expression of proteins. At present, more than 200 proteins have been expressed in this expression system (Cregg JM, Cereghino JL, Shi JY, et al. Recombinant protein expression in Pichia pastoris[J].Mol Biotechnol, 2000, 16(1)...
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[0061] Example 3 Construction of specific targeting interferon yeast expression vector
[0062] 1. Choose pPIC9K as a yeast expression vector specifically targeting interferon. pPIC9K is a multi-copy secretory expression vector launched by Invitrogen, with a size of 9.3kb (Figure 3A). The vector contains the E. coli origin of replication Col E1 and two resistance genes, ampicillin and kanamycin. The kanamycin resistance gene can also make Pichia pastoris resistant to G418, which can be used for multi-copy insertion screening. . The promoter of the alcohol oxidase gene (pAOX1) is used as an inducible promoter in the vector. The promoter is inhibited by glucose and only starts transcription and translation after induction by methanol, which is very suitable for the expression of foreign genes. pPIC9K uses the secretion signal of the lead sequence of a mating factor to secrete the target gene product outside the cell. Since Pichia itself only secretes 5% of the total protein to the o...
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