A codon-optimized blood coagulation factor VIII gene and its construct
A technology of codon optimization and blood coagulation factor, which is applied in the field of blood coagulation factor VIII gene and its construct, can solve problems affecting gene transcription efficiency and other issues
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Embodiment 1
[0073]Example 1 Capency Factor VIII Gene Expression Frame Design
[0074]According to the injection of the injection of Pfizer Pharmaceutical Co., Ltd., the amino acid sequence of recombinant human coagulation factor VIII (Zejie XYntha) as a foundation expression framework as a wild-type expression frame, which mainly includes A1, A2, B, A3, A3, C1, C2 regions. Among them, 887 amino acids were missing after B Domain, with only 21 amino acids, which were numbered P0001.
[0075]On the basis of the expression framework P0001, the additional three expression frames of P0002, P0003, and P0004 are designed, respectively, respectively, and the codon optimization of different conditions. The optimization principle is to preferentially select mammalian cells, and avoid hidden shear sites, shear donors, and receptor sequences, unriped end-end signals, strong mRNA secondary structures, RNA unstable sequences, Transcription termination signal, etc.
[0076]P0002 is based on these conditions, as far as ...
Embodiment 2
[0079]Example 2 Construction of a cycpoic factor VIII gene expression
[0080]The coagulation factor VIII gene expression frame P0001 designed in Example 1 was driven by TTR promoter, and the other expression frames were driven by two promoters of TTR and EF1A, and cloned to a slow viral carrier, and the slow viral skeleton originated from Kanglin Biotechnology (Hangzhou) Co., Ltd. Self-inactivated fake envelope chronic viral skeleton based on HIV-1-based third-generation reciprocating defective type -PKL-CCL, nucleotide sequence SEQ ID NO: 01, Pictureimage 3 . The slow viral skeleton contains a chimeric LTR promoter, a HIV-1 packaging signal (ψ), a central pondix area (CPPT), REV response element (RRE), polymetate fragment (PPT), after the transcription of Hepatitis B virus Control elements (WPRE), SV40P40 viral polyadenylation signals (SV40PA Signal), SV40 V140P40 V140 V140P40ori (SV40ori), sequence of sequences from the end of the active long end.
[0081]Example 1 Designed to Capital ...
Embodiment 3
[0105]Example 3: Clipporation Factor VIII Gene Expression Slow Virus Packaging
[0106]The condensation factor VIII gene was constructed in Embodiment 2. PKL-CCl-TTR-P0001, PKL-CCl-TTR-P0002, PKL-CCl-TTR-P0003, PKL-CCl-TTR-P0004, PKL-CCL -EF1A-P0002, PKL-CCL-EF1A-P0004), packet granules (PKL-KAN-VSVG, whose nucleotide sequences such as SEQ ID NO: 17) and packaging plasmids PKL-KAN-REV, its nucleotide sequence, such as SEQ ID NO: 18, PKL-KAN-GAGPOL, whose nucleotide sequence such as SEQ ID NO: 19 is also transfected with 293T cells (purchased from the United States The Mode Steel Collection Center (ATCC), the depositary number CRL-3216), and the coagulation factor VIII gene in the 293T cell line was treated with a slow viral vector. The transfection method is a transient transfection of PEI cationic polymer-mediated eukaryotic cells, and the PEI cationic polymer is PEI-Max transfection reagent (purchased from PolySciences, Item No. 24765-1), transfected operation It is recommended to pe...
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