Man's serum albumin with man's parathormone (1-34) fusion protein and its application

A technology of human serum albumin and parathyroid hormone, applied in the field of genetic engineering, can solve the problems of restricting long-term application and high price

Inactive Publication Date: 2006-09-13
浙江优诺金生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing hPTH(1-34) preparations are expensive and require daily subcutaneous injections, which limits their long-term application

Method used

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  • Man's serum albumin with man's parathormone (1-34) fusion protein and its application
  • Man's serum albumin with man's parathormone (1-34) fusion protein and its application
  • Man's serum albumin with man's parathormone (1-34) fusion protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1 Production of HSA and hPTH (1-34) fusion protein construction of yeast engineering bacteria

[0159] 1. Materials and methods

[0160] (1) Experimental materials

[0161] Yeast Pichia Pastoris GS115 (his4 Mut + ), the expression plasmid pPIC9 is a product of Invitrogen Company. The cloning vector pGEM-T is a product of Promega Company.

[0162] TRIzol reagent, TaqDNA polymerase, restriction endonuclease, PCR product recovery kit, plasmid extraction kit, and agarose gel recovery kit were all purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. Engineering Technology Service Co., Ltd. synthesized it on its behalf, and the sequencing service was completed by Shanghai United Gene Biotechnology Service Company.

[0163] Yeast medium YPD, BMGY, BMMY, RDB, MM, MD components are the same as Invitrogen Pichia experiment manual. The goat anti-human serum albumin polyclonal antibody is the product of Beckman Company, and the goat anti-human hPT...

Embodiment 2

[0186] Example 2 Construction of pPIC9-HSA-hPTH(1-34) recombinant yeast expression plasmid

[0187] In order to secrete and express HSA and hPTH(1-34) from Pichia pastoris in the form of fusion protein, see Figure 2, select the pPIC9 plasmid as the vector, and insert it at the EcoRI and NotI sites downstream of the alcohol oxidase AOX promoter of the vector Genes for fusion proteins. The vector pPIC9 was double digested with EcoRI and NotI overnight, and the gel was recovered for use. The aforementioned pGEM-HSA plasmid was double-digested with EcoRI and BamHI, and a fragment of about 1.8 kb was recovered. Put the linearized pPIC9 vector after enzyme digestion and the cDNA fragments containing HSA cDNA and hPTH(1-34) into the ligation system according to the appropriate molar ratio, catalyze with T4 ligase, react overnight at 16°C, and transform the freshly prepared DH5α sensory State cells were spread on LB agar plates containing ampicillin to pick positive clones. After i...

Embodiment 3

[0189] Example 3 Construction of other types of yeast expression plasmids for HSA and hPTH(1-34) fusion genes

[0190]Enzymes based on other yeast expression vectors such as pPIC9, pPIC9K, pPICZα, pHIL-S1, pPIC6α, pGAPZα and other yeast secretion vectors and other yeast intracellular expression vectors such as pPIC3.5K, pPA0815, pPICZ, pHIL-D2, pPIC6, pGAPZ Cut the site, modify the 5' and (or) 3' ends of the fusion gene of HSA-hPTH (1-34), and use conventional methods of molecular biology such as PCR, blunt end filling, blunt end connection, etc., to insert expression The multiple cloning site region of the vector. Construction work is carried out according to ligation, transformation, enzyme digestion and identification, and the sequencing steps are the same as in Example 2.

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Abstract

The invention discloses a fusion protein of human sperm albumin and human parathyroid hormone (1-34), and DNA sequence coding it as well as bacteria, yeast, animal cells and plant cells that carry the DNA sequence. It contains a first region with at least 85% sequence isogeny with human sperm albumin and a second region with at least 85% sequence isogeny with hPTH (1-34), and can substitute, delete or add several aminophenol residues on the premise of not changing its own property; there is a peptide linkage between the two regions; it not only retains the functional actions of hPTH (1-34) to activate receptor and stimulate bon reconstruction but also remarkably prolongs in vivo half life, and it is a medicinal protein that can cure osteoporosis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to the synthesis of a fusion protein of human serum albumin and human parathyroid hormone (hPTH) (1-34) by DNA recombination technology, a recombinant vector containing the gene, and a The host transformed with this vector. Background technique [0002] Parathyroid hormone (PTH) is an important factor in regulating calcium and phosphorus metabolism in organisms. Human parathyroid hormone (hPTH) is a polypeptide hormone composed of 84 amino acids with a molecular weight of 9500. It is secreted by parathyroid chief cells. The initial synthesis product is 115 preproparathyroid hormones. preprohPTH is excised during the secretion process After 31 residues at the N-terminus, a mature straight-chain polypeptide hormone consisting of 84 amino acids is formed. As early as the late 1970s, Tregear et al. proved that only 34 amino acid residues at the N-terminus a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/14C12N15/16C07K14/435C07K14/635C07K14/765C12N15/81A61K38/16A61K38/29A61K38/38A61P19/10
Inventor 陈枢青陈静
Owner 浙江优诺金生物工程有限公司
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