Preparation method and application of human heat shock protein gp96

A heat shock protein, insect cell technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of limited heat shock protein production, affecting the efficacy of vaccines, and easy loss of recombinant genes, achieving enhanced Immunological function, improving antiviral efficacy, and stabilizing expression system

Inactive Publication Date: 2015-02-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that gp96 plays a crucial role in the immunological function of Toll-like receptors (TLRs) in APCs, and the TLRs in gp96 gene-deficient DC cells lose the function of triggering natural immunity, resulting in transgenic mice Significantly reduced ability to fight infection
[0006] Due to limited tissue sources, the heat shock protein gp96 extracted from tissues has a limited yield and is difficult to apply industrially, and the gp96 protein extracted from tissues binds to antigenic polypeptides in tissue cells, affecting its combination with HBcAg and HBsAg, thereby affecting the efficacy of the vaccine
However, using Hansenula yeast to recombine gp96 protein in vitro, there are a series of problems such as easy loss of recombinant gene in engineering strains, low expression level, unstable protein and difficult purification.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application of human heat shock protein gp96
  • Preparation method and application of human heat shock protein gp96
  • Preparation method and application of human heat shock protein gp96

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1. Expression of human heat shock protein gp96 by insect baculovirus expression system

[0107] 1. Construction of Bacmid-gp96 plasmid

[0108] (1) Design and synthesis of gp96 primers

[0109] Design and synthesize the following primers:

[0110] Forward primer: 5'-G GAATTC ATGGACGATGAAGTTGATGT-3' (SEQ ID No. 1)

[0111] (The underlined sequence is the recognition site for EcoRI digestion)

[0112] Reverse primer: 5-GC TCTAGA TTACAATTCATCTTTTTCAGCTG-3' (SEQ ID No. 2)

[0113] (The underlined sequence is the recognition site for XbaI digestion)

[0114] (2) Extract the mRNA of human liver cancer cell HepG2, and synthesize cDNA by reverse transcription.

[0115] (3) With the cDNA of step (2) as a template, PCR amplification is carried out with the DNA molecules shown in SEQ ID No.1 and SEQ ID No.2 as primers to obtain a PCR amplification product, as shown in SEQ ID No.3 Show.

[0116] The 8th to 2356th position from the 5' end in SEQ ID No.3 is the gen...

Embodiment 2

[0142] Example 2, HBc 149 Antigen preparation

[0143] 1. pET21a-HBc 149 Plasmid construction

[0144] (1) HBc 149 Primer design and synthesis

[0145] Design and synthesize the following primers:

[0146] HBC149up primer: 5’-GGGAATTC catatg GACATTGACCCGTATAAAGAATTTG-3' (SEQ ID No.5)

[0147] (The underlined sequence is the recognition site for Nde I digestion)

[0148] HBC149down primer: 5'-CCG ctcgag TCAAACAACAGTAGTTTCCGGAAGT-3' (SEQ ID No. 6)

[0149] (The underlined sequence is the XhoI restriction recognition site)

[0150] (2) Using the pHBV plasmid as a template and using HBC149 up primer and HBC149 down primer as primers to perform PCR amplification to obtain a PCR amplification product, as shown in SEQ ID No.7.

[0151] From the 12th to the 458th position from the 5' end in SEQ ID No.7 is HBc 149 The coding gene of HBc 149 The amino acid sequence of is shown in SEQ ID No.8.

[0152] (3) Nde I and Xho I double-digest the DNA molecule shown in SEQ ID No.7...

Embodiment 3

[0167] Example 3, Preparation of Human Heat Shock Protein gp96 (Y-gp96) Expressed by Hansenula

[0168] Reference can be made to the literature "Li Y, Song H, Li J, Wang Y, Yan X, Zhao B, Zhang X, Wang S, Chen L, Qiu B, Meng S. Hansenula polymorpha expressed heat shock protein gp96exerts potent T cell activation activity as an adjuvant.J Biotechnol.20; 151(4):343-9." The method disclosed to prepare the human heat shock protein gp96 (Y-gp96) expressed by Hansenula yeast.

[0169] 1. Construction of pHFMDZ-R1L2GAmy-gp96 plasmid

[0170](1) The mRNA of human liver cancer cell HepG2 was extracted, and cDNA was synthesized by reverse transcription.

[0171] (2) Using the cDNA in step (1) as a template, and using the upstream primer 1 and the downstream primer 1 as primers, perform PCR amplification to obtain a PCR amplification product.

[0172] Upstream primer 1: 5'-CCGgaattcATGGACGATGAAGTTGATG-3'; (SEQ ID No.9)

[0173] (The underlined sequence is the recognition site for EcoR...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention discloses a preparation method and an application of a human heat shock protein gp96. The method is characterized in that the human heat shock protein gp96 is expressed in an insect cell. The human heat shock protein gp96 expressed by an insect cell expression system does not bind with an antigen polypeptide, so the human heat shock protein gp96 can be well bound with HBcAg and HBsAg, thereby the immune functions of the human heat shock protein gp96 are enhanced, and HBV therapeutic vaccines developed with the human heat shock protein gp96 secreted and expressed by the insect cell as an adjuvant can greatly improve the anti-HBV curative effect of the HBV therapeutic vaccines.

Description

technical field [0001] The invention relates to a preparation method and application of human heat shock protein gp96, belonging to the field of biomedicine. Background technique [0002] Although the occurrence of HBV can be effectively prevented by inoculation of hepatitis B virus (HBV) vaccine. However, there are still 350 million HBV carriers in the world, and 93 million people are HBV carriers in China alone. About 15-40% of chronic hepatitis B infection patients may develop fatal diseases, such as liver cirrhosis, liver failure and liver disease. Chronic hepatitis B infection seriously endangers public health. Existing treatments for patients with chronic hepatitis B include IFN-α and nucleosides or nucleoside analogs (such as lamivudine, adefovir, and entecavir), which are costly and only partially effective, and Can cause mutations that increase HBV drug resistance. Therefore, there is an urgent need to develop new therapeutic vaccines to complement or replace exi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C07K14/435C07K1/18C07K1/16A61K39/39A61P31/20
Inventor 孟颂东赵报刘炜炜邓蒙蒙陈立钊
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap