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Receptor activator of nuclear factor kappa B ligand (RANKL)-HBaAg expression constructing object, yeast, manufacture method as well as application

A technology of constructs and expression cassettes, applied in the field of constructs for transforming yeast cells, can solve problems such as the imbalance of RANKL/OPG ratio

Active Publication Date: 2012-05-09
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Decreased hormone levels lead to up-regulation of RANKL expression and down-regulation of OPG expression, leading to an imbalance in the RANKL / OPG ratio

Method used

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  • Receptor activator of nuclear factor kappa B ligand (RANKL)-HBaAg expression constructing object, yeast, manufacture method as well as application
  • Receptor activator of nuclear factor kappa B ligand (RANKL)-HBaAg expression constructing object, yeast, manufacture method as well as application
  • Receptor activator of nuclear factor kappa B ligand (RANKL)-HBaAg expression constructing object, yeast, manufacture method as well as application

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preparation example Construction

[0068] The preparation method of the expression cassette of the present invention comprises the following steps: (1) inserting the coding sequence of the RANKL-HBsAg recombinant protein into a plasmid containing the start signal element AOX and the stop signal element AOX (TT) at a fixed point, so as to obtain a single-copy expression plasmid; (2) Transforming the single-copy expression plasmid to obtain a complete RANKL-HBsAg recombinant protein expression cassette.

[0069] In a preferred embodiment of the present invention, the plasmid used can be: pPIC3.5(K), pAO815 or pHIL-D2, etc., as long as it contains the start signal element AOX and the stop signal element AOX (TT); preferably pPIC3. 5(K) plasmid.

[0070] The coding sequence of the RANKL-HBsAg recombinant protein can be inserted between 5AOX1 and 3AOX(TT) of the plasmid, preferably between the EcoRI restriction site and the NotI restriction site. Conventional methods such as PCR can be used to amplify the expressio...

Embodiment 1

[0092] Example 1. Construction of single-copy expression plasmids

[0093] Human mesenchymal stem cells, BMP-2 recombinant adenovirus, modified plasmid pPIC3.5K (transformed on the basis of pPIC3.5K plasmid of Invitrogen Company) were purchased from Shanghai Institute of Biological Products; Escherichia coli TOP10 strain, Pichia pastoris GS115 Strains were purchased from Invitrogen Company.

[0094] 1. Preparation of RANKL cDNA

[0095] Human mesenchymal stem cells (purchased from Shanghai Institute of Biological Products) were cultured according to conventional methods. On the second day after subculture, 500 μl of BMP-2 recombinant adenovirus was added to the culture medium to induce human mesenchymal stem cells to differentiate into osteoblasts. After 3 days, Change the medium 1 / 3, digest and harvest after 7 days. The total RNA of cells was extracted, and RANKLcDNA was obtained by RT-PCR method. The obtained cDNA was identified by agarose electrophoresis as RANKL cDNA. ...

Embodiment 2

[0109] Example 2. Construction of high-copy expression plasmids

[0110] Using the principle of homologous enzymes, the complete expression cassette with 5′AOX, RANKL-HBsAg gene and transcription terminator was repeatedly inserted into the plasmid, and pPIC3.5Kgai-RH (single copy), pPIC3.5Kgai-RH2C ( 2 copies), pPIC3.5K-RH4C (4 copies) and pPIC3.5K-RH8C (8 copies) and other four plasmids with different copy numbers (such as Figure 4A shown). The specific process is as follows:

[0111] The plasmid pPIC3.5K-RH obtained in Example 1 was double digested with BglII, MluI and BamHI, MluI respectively. Recover large fragments from plasmids digested with BamHI and MluI, and small fragments from plasmids digested with BglII and MluI. The two sets of restriction fragments are connected, BamHI and BglII are homologous enzymes, and the restriction site disappears after connection, thus completing the connection of the two expression cassettes. The ligated product was transformed, th...

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Abstract

The invention provides a constructing object for expressing receptor activator of nuclear factor kappa B ligand (RANKL)-HBaAg recombinant protein and a preparation method thereof. The constructing object comprises 1 to 15 expression boxes, wherein each expression box comprises an initiating signal element alternative oxidase (AOX) (a), RANKL-HBsAg recombinant protein coding sequences (b) and a termination signal element AOX (TT). The invention also provides yeast cells, virus-like particles and a purpose of the particles, wherein the yeast cells comprise the constructing object, the virus-like particles are generated by the yeast cells and comprise the RANKL-HBsAg recombinant protein. The yeast cells constructed in the invention can be used for generating a large amount of RANKL-HBsAg recombinant protein, so the RANKL-HBsAg recombinant protein is used for treating or preventing diseases or symptoms such as osteoporosis and the like relative to RANK / RANKL / osteoprotegerin (OPG) systems.

Description

technical field [0001] The invention relates to the fields of bioengineering and immunity, in particular to a yeast expressing recombinant NF-κB receptor activator ligand (receptor activator of NF-κB ligand, RANKL)-hepatitis B surface antigen (HBsAg) Cells, constructs for transforming yeast cells, methods of their construction and uses. Background technique [0002] Bone is a dynamic tissue that is constantly being built, resorbed, and rebuilt throughout the lifespan of an organism. While osteoblasts continuously form new bone cells, they also induce osteoclasts to continuously dissolve and absorb bone. Osteoclasts attach to the bone surface and dissolve bone by releasing acidic media and proteases. On the one hand, they form resorption pits for osteoblasts to form new bone cells. On the other hand, they maintain the volume of the medullary cavity from being occupied by bone . The significance of the bone remodeling process is to prevent the accumulation of fatigue damage...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/19C12N7/01C12P21/02C07K19/00A61K48/00A61K39/00A61P19/10A61P19/02A61P19/04A61P29/00A61P35/00A61P9/00C12R1/84C12R1/865C12R1/78
Inventor 何成楼觉人
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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