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Fusion protein and method for preparing same

A technology of fusion protein and phycocyanin, which is applied in the direction of chemical instruments and methods, hybrid peptides, peptides, etc., can solve the problem of distinguishing exogenous proteins from other endogenous homologues, affecting the yield of target proteins, and large Large-scale production is unrealistic and other problems, to achieve the effect of saving post-processing steps, increasing biological activity, and increasing stability

Inactive Publication Date: 2006-01-18
OCEAN UNIV OF CHINA
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  • Claims
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Problems solved by technology

Therefore, new cells need to be constantly prepared for infection, which is very impractical for large-scale production
And in eukaryotic cells, it is difficult to distinguish exogenous proteins from other endogenous homologues
[0005] In short, regardless of prokaryotic or eukaryotic expression, tedious follow-up work such as protein extraction, cleavage, and purification cannot be avoided. During the purification process, because the calcitonin molecule is too small, it is extremely unstable in solution, which seriously affects the yield of the target protein. Rate

Method used

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  • Fusion protein and method for preparing same
  • Fusion protein and method for preparing same
  • Fusion protein and method for preparing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1. Chimeric gene preparation:

[0086] Based on human and salmon calcitonin as a guide, computer-aided software is used to design a series of chimeras, and to screen calcitonin analogs with high activity and low antigenicity, that is, they can reduce blood calcium and have similar antigenicity to human CT CT analogues. The chimeric calcitonin gene was obtained by combining chemical synthesis and enzymes, and introduced into the cloning vector pUC19 to construct the recombinant plasmid pUC19-cCT. After sequencing, it was consistent with the expected sequence, as shown in Figure 1.

Embodiment 2

[0087] Embodiment 2. Fusion gene preparation:

[0088] The phycocyanin β subunit gene of Arthrospira (Spirulina) / Arthrospira (Spirulina) platensis FACHB341 was fused upstream of the above-mentioned chimeric calcitonin gene, and a connecting sequence was designed between the two genes so that the two peptides were folded Relatively independently, construct a cloning vector containing a fusion gene, the steps are as follows;

[0089] ①Design a pair of primers: Design primers cpcB1 and cpcB2 using Arthrospira (Spirulina) phycocyanin β subunit as a template, including EcoR I and Nde I sites in primer cpcB1, and BamH I sites in primer cpcB2 , which is also the reverse encoding of the two amino acids of the linker (GlySer), followed by the reverse complementary coding sequence encoding the 5 amino acids of the N-terminal of the linker peptide (GlyGlyGlySerGly) and the 8 amino acids of the C-terminal of cpcB, and at the 3' of primer cpcB2 Design degenerate primers at the end, elimin...

Embodiment 3

[0099] Embodiment 3. Expression of fusion gene:

[0100] ① Construction of an expression vector containing a fusion gene: cut the recombinant plasmid pUC-BCT in step 5 of the above (2) with NdeI and SphI, then connect it into the expression vector pLEX cut with the same endonuclease, use pLEX sequencing primers and Double PCR screening with cpcB1-CT2 specific primers, combined with enzyme digestion identification to screen the recombinant expression plasmid, named pBCT; see Figure 2 for the construction flow chart of the prokaryotic expression vector;

[0101] ② Transform Escherichia coli GI724:

[0102] Transform Escherichia coli GI724 with calcium chloride and manganese chloride transformation method, pick out the colony of the target clone on the RMG-Amp plate, and incubate obliquely at 30°C for 12-16 hours;

[0103] ③ Select a single clone from the plate on the first day and place it in 1ml RM medium (100μg / ml Amp), culture at 30°C with shaking at 200-225rpm / min, overnigh...

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Abstract

The present invention is fusion protein constituted through fusing phycocyanin beta-subunit gene in the upstream of human and salmon calcitonin chimera gene. The fusion protein has length 648 bp, molecular weight 23 KDa, amino acid sequence with 100 % homology with the sequence shown in SEQ No. 2 and polypeptide with 213 residues; and has prokaryotic expression vector with preservation number of CCTCC1261. The calcitonin chimera gene has length 102 bp; and the phycocyanin beta-subunit gene is cpcB gene of length 519 bp and polypeptide coding 171 amino acid residues. The constitution of the fusion protein needs no processing and purification, and this ensures the high expression amount of the calcitonin, increases the stability in clinical application, lowers the antigenicity of calcitonin and raises its bioactivity.

Description

technical field [0001] The present invention relates to the transformation of calcitonin gene. Specifically, it is a fusion protein and its preparation method. The protein is obtained by expressing the upstream of human and salmon chimeric calcitonin genes by fusing the phycocyanin β subunit gene, which belongs to molecular biology technology and biological Engineering technology intersects technical fields. Background technique [0002] In the prior art, calcitonin (calcitonin, referred to as: CT) is a polypeptide that regulates calcium and phosphorus metabolism secreted by thyroid parafollicular cells (C cells) of mammals or posterior parotid glands of other vertebrates such as fish and birds. hormone. Calcitonin is a polypeptide composed of 32 amino acids. Its main function in the body is to inhibit the activity of osteoclasts, reduce bone resorption, and promote calcium deposition in bone, thereby reducing blood calcium. Clinically, calcitonin is widely used in the tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62
Inventor 张学成王玉梅周一江臧晓南
Owner OCEAN UNIV OF CHINA
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