Preparation method of canine interferon alpha 2 recombinant protein
A technology of canine interferon and recombinant protein, which is applied in the field of preparation of canine interferon-α2 recombinant protein, can solve the problems such as the expression of canine interferon-α2 recombinant protein, etc., and achieves the effect of improving activity
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Embodiment 1
[0038] Example 1 Design and Synthesis of Gene Sequence and Primers
[0039] Referring to the CaIFN-α2 gene sequence (M28625.1) in Genbank, codon optimization was performed according to the codon preference of the Escherichia coli expression system. The optimized gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. BamH I and Xho I restriction sites were added to the ' and 3' ends to obtain the CaIFN-α2 gene fragment. See SEQ ID NO:1 for the sequence. Using the synthetic CaIFN-α2 gene sequence as a template, the signal peptide was excised, and only the coding gene of the mature peptide (SEQ ID NO: 2) was retained, and a pair of specific primers were designed using Primer 5 software, among which the upstream primer CaIFN-α2-F The Nde I restriction enzyme cutting site was introduced, and the BamH I restriction enzyme cutting site was introduced into the downstream primer CaIFN-α2-R. Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence o...
Embodiment 2
[0040] Example 2 Construction of pET-32a-CaIFN-α2 recombinant expression plasmid
[0041] The pET-32a vector and the optimally synthesized CaIFN-α2 gene fragment were double-digested with BamH I and Xho I, respectively, and the digested expression vector fragment and target fragment were gel-recovered. T4 ligase was used to connect the CaIFN-α gene fragment and the pET-32a expression vector to construct the pET-32a-CaIFN-α2 recombinant expression plasmid. The bands of 5900bp and 528bp are consistent with the size of the expression vector pET-32a and the target gene band of CaIFN-α2, such as figure 1 shown. PCR identification was carried out on the selected plasmid, and a 528bp band was amplified, which was consistent with the theoretical size, see figure 2 . It indicated that the recombinant plasmid pET-32a-CaIFN-α2 was constructed successfully. The positive plasmid was sent to Jinweizhi Company for sequencing.
Embodiment 3
[0042] Example 3 Construction of pET-21a-CaIFN-α2 recombinant expression plasmid
[0043] Using the pET-32a-CaIFN-α2 recombinant expression plasmid as a template and CaIFN-α2-F / CaIFN-α2-R as primers, the CaIFN-α2 mature peptide gene fragment was amplified by polymerase chain reaction (PCR). 25 μL PCR system: PreMix 12.5 μL, upstream and downstream primers 1 μL, template 1 μL, double distilled water to make up to 25 μL. The PCR reaction conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 30 s, and 30 cycles; final extension at 72°C for 10 min. After PCR was completed, the product was subjected to 1% agarose gel electrophoresis ( image 3 ), according to the DNA recovery kit instructions for recovery and purification.
[0044] The pET-21a vector was digested with Nde I / BamH I, and recovered and purified according to the instructions of the DNA recovery kit. Add 1 μL of linearized pET-...
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