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Recombinant protein as well as preparation method and application thereof

A recombinant protein and protein technology, applied in biochemical equipment and methods, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of connection ratio, difficult control of the molecular size of the connection site, and obstacles to drug quality control, etc., to improve Memory and cognition levels, reduced activation levels, simple effects of preparation

Active Publication Date: 2018-07-24
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in vaccines prepared with KLH as a carrier, the connection ratio, connection site, and molecular size between KLH and antigenic epitopes are not easy to control, which has caused obstacles to the quality control of drug development

Method used

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  • Recombinant protein as well as preparation method and application thereof
  • Recombinant protein as well as preparation method and application thereof
  • Recombinant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation and Characterization of Recombinant Proteins

[0068] Will Tau 294-305 The gene is inserted between the codon sites of Asp78 and Pro79 in the immunodominant region of the truncated hepatitis B virus core protein (HBc) gene (coding amino acids 1 to 149 on HBc) to obtain T294-HBc recombinant protein; T294 will be expressed -The nucleic acid sequence of the HBc recombinant protein is cloned into the PBR327 vector to obtain the T294-HBc-PBR recombinant vector; the T294-HBc-PBR recombinant vector is transformed into Escherichia coli BL21 competent cells, and the Escherichia coli cells are supplemented with 1% casamino acids, Culture in M9 salt medium with 0.2% glucose and 50 μg / mL ampicillin at 37° C. for 20-24 hours.

[0069]Collect the cells by centrifugation, resuspend 2 g of cells in 40 mL of lysis buffer, and sonicate in an ice bath for 30 minutes; then, centrifuge at 12,000 rpm for 15 minutes, add ammonium sulfate to the supernatant to 33% satura...

Embodiment 2

[0073] Embodiment 2 Vaccine immunity and antibody detection

[0074] (1) Animal immunity

[0075] The 5-month-old Tau.P301S transgenic mice were randomly divided into PBS group, HBc control group, R1 group, R2 group, R3 group, R4 group and T294-HBc group, with 7 mice in each group. The same wild-type C57BL / 6J mice served as the WT control group.

[0076] The above 8 groups of mice were treated as follows:

[0077] Inject 100 μg of recombinant protein + aluminum adjuvant to mice in R1 group, R2 group, R3 group, R4 group and T294-HBc group, inject the same dose of PBS + aluminum adjuvant to mice in PBS group, and inject 100 μg to mice in HBc control group Equal doses of HBc carrier + aluminum adjuvant were injected into WT control mice with equal doses of aluminum adjuvant, and the mice were subcutaneously injected on days 0, 14, 28 and 42, with an interval of two weeks between each injection.

[0078] (2) Antibody titer and EC 50 detection

[0079] On the 10th day after ea...

Embodiment 3

[0092] Example 3 T294-HBc vaccine improves the cognitive level of Tau.P301S mice

[0093] In order to evaluate the therapeutic effect of T294-HBc vaccine on Tau.P301S mice, the mice after immunotherapy were subjected to the following behavioral experiments: forced Y maze, novel object recognition, water maze and spontaneous Y maze.

[0094] (1) Forced Y maze

[0095] The Y maze used in this experiment is composed of three arms with an angle of 120 degrees to each other. The end of each arm is marked with a different pattern. The three arms are randomly designated as the start arm, the new detection arm and the exploration arm.

[0096] In the training phase, put the mouse into the starting arm and let it explore freely in the starting arm and the exploration arm for 5 minutes. At this time, the new arm is closed; after 30 minutes, open the new arm and put the mouse in the Initiate arm and allow mice to explore freely for 5 min. The time each mouse stayed in the new arm and t...

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Abstract

The invention provides recombinant protein as well as a preparation method and application thereof. The recombinant protein comprises Tau protein and hepatitis B virus core protein. The recombinant protein adopts Tau294-305 and the hepatitis B virus core protein to perform recombination, T294-HBc for fusion expression is self-assembled into HBc chimeric virus-like particles, with surface displaying Tau294-305, and vaccine is good in homogeneity, simple to prepare, high in immunogenicity and small in side effect, and has a good application prospect in terms of the treatment of dementia, such asalzheimer disease, frontotemporal dementia, corticobasal degeneration, Pick disease and progressive supranuclear palsy, caused by Tau lesion.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a recombinant protein and its preparation method and application, in particular to a recombinant protein of Tau protein and hepatitis B virus core protein and its preparation method and application. Background technique [0002] Alzheimer's disease, frontotemporal dementia, corticobasal degeneration, Pick's disease, and progressive supranuclear palsy are major neurodegenerative diseases that can cause symptoms such as dementia. In 2015, there were approximately 46.8 million dementia patients worldwide, and the number of patients is expected to reach 113.5 million by 2050 (World Alzheimer Report 2016: Improving healthcare for people living with dementia.). Among them, the pathological features of Alzheimer's disease are mainly neuron loss, hyperphosphorylated microtubule-associated protein Tau protein aggregation in neurons to form neurofibrillary tangles, and accumulation and deposition...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K39/00A61P25/28A61P25/00
CPCA61K39/0007C07K14/005C07K14/47C07K2319/00C12N2730/10122C12N2730/10123
Inventor 刘瑞田季梅谢喜秀
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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