Recombinant canine long-acting interferon gamma as well as fusion protein preparing long-acting interferon gamma as well as preparation method of fusion protein
A technology of fusion protein and canine interferon, which is applied in the field of biogenetic engineering, can solve the problems of unfavorable clinical application, small molecular weight of interferon, and high cost of interferon, so as to avoid denaturation and renaturation, shorten the time of expression, and improve immunity The effect of answering
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Embodiment 1
[0073] A fusion protein composed of canine interleukin 2 and canine interferon gamma, the preparation method of which is as follows:
[0074] 1. Acquisition and amplification of canine interleukin 2 (IL-2) and canine interferon gamma (IFN-gamma) target genes
[0075] Primer design:
[0076] Design synthetic primers according to the target gene sequence reported in Genebank, see Table 1, introduce BamHI restriction site and Linker sequence into the upstream primer and downstream primer of canine interleukin 2, respectively, and introduce the upstream primer and downstream primer of canine interferon gamma Linker sequence and XhoI restriction site were introduced respectively.
[0077] Table 1PCR amplification primers
[0078]
[0079] RT-PCR to obtain the target gene:
[0080] RNA was extracted from canine liver tissue, and the target genes of IL-2 and IFN-γ were obtained by reverse transcription. The gene sequences of the two are shown in SEQUENCE LISTING 400 and SEQUENC...
Embodiment 2
[0113] A fusion protein composed of canine interleukin 2 and canine interferon gamma, the others are the same as in Example 1, except that the Escherichia coli BL21 (DE3) competent cells are replaced by BL21 (DE3) competent cells with pGro7 plasmid cell. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 54.7KD in the supernatant is relatively thick, indicating that after the introduction of molecular chaperone pGro7, the expression of the target protein in the supernatant is better, and the obtained The amount of fusion protein is higher. Most of the proteins expressed in E. coli exist in inclusion bodies; by introducing molecular chaperones into the expression strains, the co-expressed proteins can be correctly folded to achieve protein soluble expression.
[0114] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Si...
Embodiment 3
[0116] A fusion protein composed of canine interleukin 2 and canine interferon gamma, the preparation method of which is as follows:
[0117] 1. Acquisition and amplification of canine interleukin 2 (IL-2) and canine interferon gamma (IFN-gamma) target genes
[0118] IL-2 and IFN-γ in Example 1 are optimized, and IL-2 and IFN-γ target genes are artificially synthesized. After optimization, the nucleotide sequences of the two are as SEQUENCE LISTING 400 and SEQUENCE LISTING 400 respectively As shown in .
[0119] 1.1 Codon optimization
[0120] There are 64 genetic codes, but most organisms tend to use a subset of these. Those that are most frequently used are called optimal codons, and those that are not frequently used are called rare or low-usage codons. Virtually every organism commonly used for protein expression or production (including E. coli, yeast, mammalian cells, plant cells, and insect cells) exhibits some degree of difference or bias in codon usage. The expres...
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