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Fusion protein for treating alzheimer's disease and preparation method thereof

A technology for Alzheimer's disease and fusion protein, which is applied in the field of fusion protein for treating Alzheimer's disease and its preparation

Inactive Publication Date: 2012-04-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the trial was later stopped because several patients involved in the trial developed severe brain inflammation

Method used

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  • Fusion protein for treating alzheimer's disease and preparation method thereof
  • Fusion protein for treating alzheimer's disease and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Construction of the recombinant expression vector of CTB and human Aβ42 fusion gene

[0036] The human Aβ42 gene was obtained by one chain extension reaction and two PCR methods: (1) chain extension: the reaction system contained primers F2 and R2, 1 unit of Taq enzyme and other conventional PCR reagents; the reaction conditions were denaturation at 94°C for 10 minutes, After annealing at 55°C for 5 minutes, let F2 and R2 serve as primers and templates for each other and extend at 72°C for 5 minutes, and the product obtained is denoted as SegI; (2) The first PCR: SegI is used as a template, F1 and R1 are used as primers, and the reaction conditions are: Pre-denaturation at 94°C for 5 minutes, amplification at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 30 cycles of reaction, and finally 72°C for 5 minutes. At this time, the product was recorded as Seg II, and the DNA cleaning kit was used Purify the product; use BamHI and XhoI double enzyme...

Embodiment 2

[0038] Example 2: Obtaining of CTB and human Aβ42 fusion gene recombinant baculovirus

[0039] Take 5ul insect baculovirus transfer plasmid pBac-TNS containing CTB and human Aβ42 fusion gene and 6ul wild silkworm nuclear polyhedrosis virus DNA for co-transfection. Take 6ul Lipofectin (Invitrogen Company) and add 100ul serum-free TC-100 medium and mix well. The BmN cells previously cultured in a 35mm Dish were washed twice with serum-free TC-100 (Invitrogen Company) medium, and the transfer plasmid and Lipofectin mixture was added dropwise, cultured at 27°C for 4-5 days, and the supernatant was collected for the second stage. One round of plaque screening. Take 5ul of the supernatant to infect the BmN cells in a 35mm Dish, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting point agarose. Pick plaques after 4-5 days, infect BmN cells for 3-4 days, save the supernatant, lyse the cells with NaOH for Southern hybridization, and tak...

Embodiment 3

[0040] Example 3: Expression of fusion gene of CTB and human Aβ42 in silkworm larvae and pupae

[0041] Take the amplified recombinant baculovirus containing fusion gene of CTB and human Aβ42 and inject it into fifth instar silkworm (Bombyx mori) larvae and pupae, inject about 2ul per head (titer is 1×107 / ml), take 24, The silkworm lymph and pupal blood expressed at 48, 72, 96, 120 and 144 hours were centrifuged at 6000rpm for 10min to get the supernatant, diluted 10 times with PBS pH7.4, and added an equal volume of 2× protein loading buffer (100Mm Tris. HCl, 4% SDS, 0.1% bromophenol blue, 10% glycerol), take 10ul for SDS-PAGE analysis. The results showed that the fusion protein of CTB and human Aβ42 was highly expressed in silkworm and pupae, and the molecular weight of the expressed product was about 20kD by Western hybridization. The hemolymph of silkworm larvae and pupal bodies containing the expressed fusion protein was subjected to high-speed freezing and centrifugatio...

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Abstract

The invention relates to a fusion protein for treating alzheimer disease, and a preparation method thereof. The invention provides the fusion protein for treating the alzheimer disease and also provides a nucleotide sequence expressing the fusion protein. The invention further provides an expression vector which contains the nucleotide sequence and a recombinant virus containing the expression vector. The recombinant virus refers to the recombinant virus which is stored in the General Microbiological Center of China Committee for Culture Collection of Microorganisms, with the storage code of CGMCC No.1795. The animal oral experiment shows that the provided fusion protein can generate a high-level antibody and becomes a novel method for preparing an oral medicine for treating the alzheimerdisease.

Description

technical field [0001] The invention relates to a fusion protein for treating Alzheimer's disease and a preparation method thereof. Background technique [0002] Alzheimer's disease (AD), also known as senile dementia, is the most common and severe central nervous system degenerative disease among the elderly. More than a quarter of the world's total cases, and an average of 300,000 elderly people join the ranks every year. With the prominence of the population aging problem in our country, the prevention and treatment of senile dementia have become more and more important. Clinically, it mainly manifests as the overall decline of the central nervous system, and the gradual loss of cognitive function and memory, resulting in obstacles to self-care ability and abnormal mental behavior, and eventually leads to death. The most prominent neuropathological features of AD are the formation of senile plaques, neurofibrillary tangles and massive death of nerve cells, and the late ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/866C12N7/01A61P25/28
Inventor 金勇丰张耀洲李霁
Owner ZHEJIANG UNIV
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