Canine interferon-alpha6 alpha7 recombinant protein and preparation method and application thereof
A canine interferon and recombinant protein technology, which is applied in the fields of genetic engineering and biological products, and can solve the problems such as the expression of canine interferon tandem recombinant protein that has not yet been seen.
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Embodiment 1
[0034] The optimization of embodiment 1 gene sequence and the design and synthesis of primer
[0035] According to the cDNA sequence of canine interferon-α6 (NM_001007128.1) and the cDNA sequence of canine interferon-α7 (NM_001006654.1) in GenBank, after codon optimization, the target gene sequence was synthesized. The amplification primers designed for canine interferon-α6 are GN7PA-F and GN7PA-R1, and the sequences are shown in SEQ ID NO:7 and SEQ ID NO:8; the amplification primers GN7PA-F1 and GN7PA-R for canine interferon-α7, SEQ ID NO:9 and SEQ ID NO:10; Pichia pastoris general primer 5'AOX and 3'AOX sequences are shown in SEQ ID NO:11 and SEQ ID NO:12.
[0036] Table 1 Primer Sequence
[0037]
[0038] The optimization process of canine interferon-α6 and canine interferon-α7 gene sequences is as follows:
[0039] By analyzing the rare codons of Pichia pastoris in the canine interferon-α6 and canine interferon-α7 gene sequences, according to the codon usage preferenc...
Embodiment 2
[0040] Example 2 Construction of pPICZαA-CaIFN-α6-α7 Vector
[0041] Using the gene synthesized by the company as a template, the canine interferon-α6 gene fragment is amplified by polymerase chain reaction (PCR). The reaction system is as follows:
[0042]
[0043] PCR reaction program: pre-denaturation at 94°C for 8 min; denaturation at 94°C for 35 s, annealing at 52°C for 30 s, extension at 72°C for 45 s, a total of 35 cycles; final extension at 72°C for 5 min, and storage at 4°C. After mixing 5 μL of PCR product with 1 μL of loading buffer, use 1.0% agarose gel electrophoresis for nucleic acid, 120V, 32min, and observe the electrophoresis results under a UV gel imager. The PCR product CaIFN-α6 was recovered and purified with Cycle-Pure Kit.
[0044] Using the gene synthesized by the company as a template, the canine interferon-α7 gene fragment was amplified by polymerase chain reaction (PCR). The reaction system is as follows:
[0045]
[0046] PCR reaction progra...
Embodiment 3
[0048] Example 3 Preparation and linearization of pPICZαA-CaIFN-α6-α7 plasmid
[0049] After the pPICZαA-CaIFN-α6-α7 plasmid was sequenced correctly, it was transformed into Escherichia coli DH5a. The specific operation is as follows:
[0050] 1. Thaw a tube of 100 μl E.coli DH5a competent cells on ice, flick the tube wall to resuspend the cells. Add 10 μl of plasmid to the competent cells, flick a few times, and incubate on ice for 30 minutes.
[0051] Heat shock in a 2.42°C water bath for 90 seconds, then quickly place on ice for 5 minutes.
[0052] 3. Add 500 μl LB liquid medium and incubate at 37°C for 45-60 minutes.
[0053] 4. Centrifuge at 5000g for 3 minutes to collect the bacterial cells, spread a certain amount of bacterial cells evenly on low-salt LB plates containing 25 μg / ml Zeocine, and culture overnight.
[0054] Pick positive clones for expansion culture, and use the Plasmid Midi Preparation Kit (Plasmid Midi Preparation Kit) of Beyontian Biotechnology Co.,...
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