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Fusion protein containing haman parathyroxin 1-34 and its expression vector

A parathyroid hormone and fusion protein technology, applied in the field of genetic engineering, can solve problems such as complex preparation process

Active Publication Date: 2007-01-24
BEIJING GENETECH PHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the need to use double enzyme digestion, the preparation process disclosed in this patent application is also relatively complicated
[0005] It has long been known that thioredoxin (Thiroedoxin, sometimes referred to as "Trx") is a protein commonly found in yeast, bacteria, animals, and plants. This protein is also a commonly used PTH (1-34) expression host, such as Endogenous protein of yeast or E. coli, but so far no one has used this protein to fuse with PTH(1-34) to easily produce hPTH(1-34)

Method used

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  • Fusion protein containing haman parathyroxin 1-34 and its expression vector

Examples

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Embodiment 1

[0029] Example 1 Design and artificial synthesis of DNA sequences expressing hPTH(1-34)

[0030] According to the amino acid sequence of hPTH (1-34) (see Table 1) and the codon preference of E. coli, an optimized DNA sequence suitable for expression in E. coli was synthesized artificially. When synthesizing hPTH(1-34) gene, KpnI restriction site GGTACC was introduced at the 5' end of the gene, stop codon TAA and SalI restriction site GTCGAC were introduced at the 3' end, and KpnI enzyme was introduced at the 5' end After the cleavage site, add the coding sequence GACGACGACGACAAG of the enterokinase cleavage recognition site to obtain sequence 1 (see Figure 10 ). To facilitate subsequent subcloning, the synthetic gene was cloned into pUC18 for preservation of the synthetic DNA sequence. Plasmid pUC18 contains the same KpnI and SalI restriction sites as plasmid pET32a(+).

[0031] serial number

[0032] Note: The amino acid sequence is marked from the N-terminus ...

Embodiment 2

[0033] Example 2 Construction process of recombinant plasmid

[0034] The following molecular cloning techniques, unless otherwise specified, refer to the literature: Molecular Cloning Experiment Guide (translated by Huang Peitang et al., [US] Sambrook et al., Science Press, 2002). The DNA extraction kit (UNIQ-10), DNA gel recovery kit (UNIQ-10) and ligation kit used in DNA manipulation were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. The cloning vector pUC18 and restriction enzymes were purchased from Fermentas LifeScience Company. Expression vector pET32a(+), E. coli TOP10 and BL21(DE3) were purchased from Novagen Company. The E. coli host for cloning is TOP10, and the E. coli host for expression is BL21 (DE3). The BL21(DE3) genotype was: hsdS gal (λcIts857ind1 Sam7 nin5 lacUV5-T7 geneI). BL21(DE3) carries the T7RNA polymerase gene. Under the induction of IPTG, a large amount of T7RNA polymerase is produced, thus enabling the expression of e...

Embodiment 3

[0057] Example 3 Induced expression of engineering bacteria

[0058] Escherichia coli BL21(DE3) is transformed with the recombinant plasmid pET-PTH(1-34) DNA, and the obtained is the genetically engineered bacteria for expressing the rhPTH(1-34) fusion protein. Pick 8 single colonies, culture overnight at 37°C in 5ml LB medium (1% peptone, 0.5% yeast extract, 1% NaCl) containing 100μg / ml Amp, transfer to 50ml containing 100μg / ml Amp cultured in LB medium at 37°C, and the remaining bacterial solution was subpackaged with 15% glycerol and frozen. OD 600 When it reached 0.5, IPTG was added to a final concentration of 0.5mM to induce expression, and samples were taken for SDS-PAGE electrophoresis after 4 hours. Compared with the uninduced control, the induced recombinants all had an expression band with an expected molecular weight of about 20 kDa, and the expression amount was more than 25% of the total bacterial protein. Bacteria No. 6 with the highest yield was taken as the...

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Abstract

The present invention provides a kind of fusion protein, which possesses thioredoxin sequence and parathormone peptide 1-34 located in the downstream of the thioredoxin. Preferably, the fusion protein possesses also connecting peptide containing proteolytic enzyme recognizing site and located between the thioredoxin and the parathormone peptide 1-34. From the fusion protein, human parathormone peptide 1-34 may be prepared.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a fusion protein containing human parathyroid hormone 1-34 and an expression vector thereof. Background technique [0002] Parathyroid hormone (Parathyroid Hormone, PTH) is synthesized by the parathyroid hormone chief cell and consists of 84 amino acids. It is increasingly recognized that PTH can increase the number of osteoblasts, which are very active in synthesizing new bone matrix, and can alter gene expression in bone in vivo. PTH also has the functions of lowering blood pressure, regulating the expression of vitamin D receptor (VDR), and regulating the activity of alkaline phosphatase. Tregear et al. (Tregear GW et al., Endocrinology, 1973, 93:1349) proved by experiments that only 1-34 amino acid residues at the amino terminal are required for PTH to play the role of calcium and phosphorus regulating molecules. However, PTH(1-34) does not contain cysteine ​​an...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63
CPCC07K14/635C07K2319/00C07K2319/35C12N9/0036
Inventor 张仁怀杨立明阳勇刘社际黄碧莲何凯刘德林
Owner BEIJING GENETECH PHARML
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