Recombinant porcine long-acting interferon alpha, fusion protein for preparing recombinant porcine long-acting interferon alpha, and preparation method of fusion protein
A technology of fusion protein and porcine interferon, applied in the field of biological genetic engineering, can solve the problems of small molecular weight of interferon, high cost of interferon, short half-life, etc., and achieve the effects of avoiding denaturation and renaturation, improving immune response and improving half-life.
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Embodiment 1
[0071] A fusion protein composed of porcine interferon gamma and porcine interferon alpha, the preparation method of which is as follows:
[0072] 1. Acquisition and amplification of porcine interferon gamma (IFN-gamma) and porcine interferon alpha (IFN-α) target genes
[0073] Primer design:
[0074] According to the target gene sequence reported in Genebank, the synthetic primers were designed and synthesized as shown in Table 1. The EcoRI restriction site and Linker sequence were introduced into the upstream primer and downstream primer of porcine interferon gamma respectively, and the upstream primer and downstream primer of porcine interferon alpha The Linker sequence and the HindⅢ enzyme cutting site were respectively introduced in.
[0075] Table 1 PCR amplification primers
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[0077]
[0078] RT-PCR to obtain the target gene:
[0079] RNA was extracted from pig liver tissue, and the target genes of IFN-γ and IFN-α were obtained by reverse transcription....
Embodiment 2
[0111] A fusion protein composed of porcine interferon gamma and porcine interferon alpha, 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 . The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 62.5KD in the supernatant is thicker, indicating that after the introduction of the molecular chaperone pGro7, the expression of the target protein in the supernatant is better, and it is 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.
[0112] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd. / Simb...
Embodiment 3
[0114] A fusion protein composed of porcine interferon gamma and porcine interferon alpha, the preparation method of which is as follows:
[0115] 1. Acquisition and amplification of porcine interferon gamma (IFN-gamma) and porcine interferon alpha (IFN-α) target genes
[0116] The IFN-γ and IFN-α in Example 1 are optimized, and the IFN-γ and IFN-α 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 .
[0117] 1.1 Codon optimization
[0118] 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 cod...
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