Recombinant human interferon alpha 2b DNA fragment and encoding protein
A technology for recombinant human interferon and protein, applied in the field of recombinant human interferon DNA fragments and encoded proteins, can solve the problems of insufficient specific activity, low solubility, and failure to achieve maximum optimization, and achieve high anti-proliferative activity. Effect
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Embodiment 1
[0035] A recombinant human interferon α2b DNA fragment, which is the nucleotide sequence described in SEQ ID NO.1 in the sequence table. The design and synthesis of gene fragments are as follows:
[0036](1) The original sequence of human interferon α2b retrieved from the National Center for Biotechnology Information (NCBI) database, and based on the known amino acid sequence of interferon α2b on the market, after summarizing and analyzing a large number of documents about the active site of interferon, Directional design of a recombinant human interferon α2b DNA fragment (the nucleotide sequence described in SEQ ID NO.1 in the sequence table);
[0037] (2) Synthesis of recombinant human interferon α2b DNA fragment (nucleotide sequence described in SEQ ID NO.1 in the sequence table). Recombinant human interferon α2b contains 166 amino acids, and the length of its gene sequence is 498bp.
[0038] Primer design and synthesis:
[0039] Design and synthesize 8 pairs of primers ...
Embodiment 2
[0070] A recombinant human interferon α2b DNA fragment, which is the nucleotide sequence described in SEQ ID NO.2 in the sequence table. The steps are:
[0071] The 52nd, 53rd, and 55th amino acids were subjected to site-directed mutation using the large primer amplification method, and the downstream primer HX2 was designed to mutate the 161st amino acid, thereby obtaining the recombinant interferon sequence rh-mIFN-2 (described in the sequence table SEQ ID NO.2 nucleotide sequence);
[0072] Mutation primer design: (the wavy line is the mutation site)
[0073] Mut2(5'---3'): TTC ATG CAG CAC AAT CGC TTT CTG (SEQ ID NO.18)
[0074] Design downstream primers:
[0075] HX2 (the straight line is the restriction site): GTC AAG CTT TTC TTT GCT ACG CAG TTC (SEQ ID NO.19)
[0076] The specific process is:
[0077] The first round of reaction system (total volume is 100 μL): with upstream primer ES (SEQ ID NO.8) and mutation primer Mut2 (SEQ ID NO.18) as primers, with rh...
Embodiment 3
[0085] A recombinant human interferon α2b DNA fragment, which is the nucleotide sequence described in SEQ ID NO.4 in the sequence table. The steps are:
[0086] After obtaining the target fragment of rh-mIFN-2, use this sequence as a template and use the large primer method to perform site-directed mutation on amino acids 103 and 107, thereby obtaining the target sequence rh-mIFN-3 (SEQ ID NO.4 in the sequence table said nucleotide sequence);
[0087] Mutation primers are as follows:
[0088] Mut3(5'---3'): CAG CGG GGT TTC CAC GCC CAC CTGAAT CAC GCA (SEQ ID NO. 20);
[0089] Carry out PCR, obtain product, electrophoresis verification (see image 3 In the figure, swimming lane 1 is the first round of PCR product; swimming lane 2 is the second round of PCR product) using a PCR product purification kit to purify the product to obtain the number rh-mIFN-3 (the nucleoside described in the sequence table SEQ ID NO.4 acid sequence), stored at 4°C.
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