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A nucleic acid molecule encoding parathyroid hormone protein or a fragment thereof

A technology of parathyroid hormone and nucleic acid molecules, applied in the direction of parathyroid hormone, peptide/protein components, nucleic acid carriers, etc., can solve the problems of unbearable patient, poor patient compliance, small molecular weight, etc., to reduce treatment costs, improve compliance effect

Active Publication Date: 2022-02-18
BEIJING NORTHLAND BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because PTH does not contain cysteine, PTH (1-34aa) and PTH (1-84aa) are very unstable in the body, and their molecular weight is small, so they are easily filtered by the glomerulus, so the half-life in the body is relatively short
In order to achieve the therapeutic effect, frequent high-dose medication is generally required (usually subcutaneous injection once a day for several months), but such frequent medication combined with a long treatment cycle makes it difficult for patients to bear, and it will cause serious clinical problems. Headache, vomiting, fever and other adverse reactions, poor patient compliance

Method used

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  • A nucleic acid molecule encoding parathyroid hormone protein or a fragment thereof
  • A nucleic acid molecule encoding parathyroid hormone protein or a fragment thereof
  • A nucleic acid molecule encoding parathyroid hormone protein or a fragment thereof

Examples

Experimental program
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Embodiment 1

[0070] Embodiment 1: Construction of recombinant vector

[0071] (1) Synthesis of the target gene

[0072] Using the nucleotide sequence encoding natural human PTH protein as a template, synthesize the nucleotide sequence shown in SEQ ID NO: 25 or SEQ ID NO: 27, wherein the sequence SEQ ID NO: 25 includes from 5' to 3' : the nucleotide sequence (SEQ ID NO:23) of encoding signal peptide, the nucleotide sequence (SEQ ID NO:3) and stop codon TGATAG (SEQID NO:18) of encoding PTH (1-34aa); Sequence SEQ ID NO: 27 includes from 5' to 3': nucleotide sequence (SEQ ID NO:23) encoding signal peptide, nucleotide sequence (SEQ ID NO:5) encoding PTH (1-84aa) and stop codon Sub-TGATAG (SEQ ID NO: 18).

[0073] (2) Construction of recombinant vector

[0074] The above synthesized sequences of SEQ ID NO: 25 and SEQ ID NO: 27 were respectively cloned into pCK vectors to obtain pCK-PTH(1-34) and pCK-PTH(1-84) recombinant vectors. The pCK vector sequence is shown in SEQ ID NO: 17 (see Chine...

Embodiment 2

[0077] Embodiment 2: the fermentation of engineering bacterium

[0078] (1) Activation of bacteria

[0079] Take out the engineering bacteria from the working seed bank, use an inoculation loop to dip the bacterial liquid from the cryotube, streak on the LB solid plate containing kanamycin sulfate solution, and culture overnight at 37°C.

[0080] (2) Preparation and fermentation of bacterial liquid

[0081] Pick colonies from the plate, transfer the colonies to LB medium containing kanamycin sulfate solution, place them in a shaker, and culture them at 37°C for about 4-8 hours, and the obtained liquid culture is the primary seed liquid. The primary seed liquid is transferred to the LB medium containing kanamycin sulfate solution, placed on a shaker, and cultivated at 37°C for about 15-20h, and the obtained liquid culture is the secondary seed liquid. Inoculate the secondary seed liquid into a 30L fermenter, and when the cell concentration OD600 reaches 5-8, transfer the fe...

Embodiment 3

[0084] Embodiment 3: Purification of recombinant vector

[0085] (1) Pre-treatment and concentration: Weigh 1200 g of frozen bacteria, after alkaline lysis and neutralization, filter, collect the supernatant, and concentrate to 1-2 L, centrifuge at 6000 rpm for 20 min, and collect the supernatant after centrifugation.

[0086] (2) Chromatography: Use molecular sieve chromatography, affinity chromatography, and ion exchange chromatography to further separate pCK-PTH (1-34) and pCK-PTH (1-84) recombinant vectors, and change the liquid through ultrafiltration, Store the pCK-PTH(1-34) and pCK-PTH(1-84) recombinant vectors in stable preparation solutions, respectively.

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Abstract

The present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a parathyroid hormone (PTH) protein or a fragment thereof and an encoding termination region. The present application also relates to vectors and host cells comprising said nucleic acid molecules. The present application also relates to a pharmaceutical composition containing the nucleic acid molecule, and uses of the pharmaceutical composition.

Description

technical field [0001] The present application relates to a nucleic acid molecule comprising a nucleotide sequence encoding a parathyroid hormone (PTH) protein or a fragment thereof and an encoding termination region. The present application also relates to vectors and host cells comprising said nucleic acid molecules. The present application also relates to a pharmaceutical composition containing the nucleic acid molecule, and uses of the pharmaceutical composition. Background technique [0002] Osteoporosis (osteoporosis, OP) is common in the elderly and postmenopausal women. It is manifested by increased bone fragility and susceptibility to fractures. The reason is a decrease in bone mass in the body and a breakdown in the microarchitecture of bone tissue. Osteoporosis can occur at any age and can be divided into primary, secondary and idiopathic osteoporosis. At present, the treatment drugs for osteoporosis mainly start from two aspects, one is to promote the formati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/635C07K19/00C12N15/16C12N15/62C12N15/70C12N1/21A61K48/00A61K38/29A61K9/08A61K9/19A61P19/10A61P5/22A61P3/12C12R1/19
CPCC07K14/635C12N15/70A61K48/005A61K48/0008A61K9/0019A61K9/08A61K9/19A61P19/10A61P5/22A61P3/12C07K2319/02C12N2800/22A61K38/00
Inventor 聂李亚许松山马素永汤晓闯
Owner BEIJING NORTHLAND BIOTECH