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Method for preparing recombined human amyloid A beta 42 and application thereof

An amyloid and protein technology, applied in the field of fusion proteins, can solve the problems of difficulty in synthesis, increase in cost, application limitations, etc., and achieve the effects of low cost and high protein yield

Inactive Publication Date: 2008-09-24
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] due to Aβ 42 The 33-42 amino acid residues at the C-terminus of the peptide are highly hydrophobic, and the 28-42 residues form a β-sheet conformation, directly soluble in Aβ in aqueous solution 42 Peptides aggregate immediately after dissolution to form insoluble precipitates. Aβ currently used in research and clinical applications is usually chemically synthesized high-purity peptides. With the growth of synthetic peptide chains, the difficulty and cost of synthesis increase, and the application is limited.

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  • Method for preparing recombined human amyloid A beta 42 and application thereof

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Embodiment 1

[0029] Example 1: pGEX-4T-1 / Aβ 42 Plasmid construction

[0030] (1) Obtaining cDNA: The total RNA of SH-SY5Y cells from human neuroma blasts was extracted, and the cDNA of SH-SY5Y cells was obtained by reverse synthesis using oligo(dT) as a primer.

[0031] (2) Primer design and PCR: search genbank, based on human Aβ 42 The sequence and the multiple cloning site primers of the vector pGEX-4T-1 were designed.

[0032] upstream primer P 1 : 5'-CG GGATCC GATGCAGAATTCCGACATGACTCAG-3', introduce BamHI restriction site,

[0033] downstream primer P 2 : 5'ACGC GTC GAC CTACGCTATGACAACACCGCCCA-3', introducing a SalI restriction site and a stop codon TAG.

[0034] Using human SH-SY5Y cell cDNA as template for PCR amplification, the product fragment length is 147bp. Amplification conditions: Pfu DNA polymerase, 30 cycles of amplification at 94°C for 30s, 60°C for 30s, and 72°C for 1min.

[0035] (3) Double enzyme digestion and ligation of the PCR product and the vector: BamHI a...

Embodiment 2

[0043] Example 2: GST-Aβ 42 Prokaryotic expression and purification of fusion proteins

[0044] (5) Pre-expression. Transform the expression strain Escherichia coli BL21 with the recombinant plasmid with the identified sequence and correct insertion direction. The molecular weight of the GST protein expressed by the empty vector pGEX-4T-1 is 27KD, GST-Aβ 42 The molecular weight of the fusion protein is about 32KD. Induce with 1mM IPTG at 37°C for 0, 1, 3, 5 hours, thaw the centrifuged cells with 1×SDS lysate, boil at 95°C for 5min, analyze by SDS-PAGE electrophoresis, and make corresponding antibodies (GST and Aβ 42 ) western blot analysis. Both SDS-PAGE and Western blot showed positive bands, indicating GST-Aβ 42 The fusion protein has been expressed in expression bacteria.

[0045] Induced condition optimization. Constructed pGEX-4T-1 / Aβ 42 / BL 21 expression strains were cultured at 37°C or 25°C, inoculated with fresh bacteria at a ratio of 1:100, and cultured with s...

Embodiment 3

[0049] Example 3: Aβ 42 Protein digestion and identification

[0050] (8) Aβ 42 Protease digestion and purification: purified GST-Aβ 42 The fusion protein was treated with thrombin, 10U / mg fusion protein, reacted in pH7.4, 50mM PBS phosphate buffer system at 23°C for 16 hours, and then passed through Glutathione Sepharose 4B affinity column to remove enzyme-cleaved GST protein, benzene Formamidine column removes thrombin to obtain pure Aβ 42 protein;

[0051] (9) Aβ 42 Western blotting identification of protein: the above-mentioned Aβ obtained by enzyme digestion and double affinity purification 42 Anti-Aβ 42 Antibody and anti-GST antibody were identified by Western blotting, and the results showed that the obtained protein could only interact with anti-Aβ 42 Antibody reaction, but not with anti-GST antibody, confirms that the protein is Aβ 42 protein;

[0052] (10) Aβ 42 Protein aggregation analysis: using the fluorescent substance Thioflavin-T that can specifically...

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Abstract

The invention provides a method for preparing recombinant human amyloid protein A Beta42 and application of the recombinant human amyloid protein, which belongs to the technical field of fusion protein. In the method, PCR sense primer P1 of the human amyloid A Beta42 is designed; a BamHI restriction enzyme cutting site, an anti-sense primers P2, a Sall restriction enzyme cutting site and a terminator codon TAG are introduced according to the sequence of the precusor protein APP of the human amyloid and multiple cloning sites of the cloning vector pGEX-4T-1. cDNA of human SH-SY5Y cells is taken as a templet for amplifing PCR; the length of the fragment of the product is 147bp. The Beta42 fragment of human APP from 672 to 713 is encoded. The A Beta42 gene fragment sequence is combined into a prokaryotic expression vector pGEX-4T-1 to form the prokaryotic expression plasmids pGEX-4T-1 / A Beta42 of human A Beta42. PGEX-4T-1 / A Beta42 is transformed into colibacillus BL21 (DE3), and purified activated recombinant human amyloid A Beta42 is got through induced expression, separation and enzyme cutting.

Description

technical field [0001] A kind of recombinant human amyloid Aβ 42 The preparation method and application of the method, the invention relates to a method for constructing, expressing and purifying recombinant human amyloid prokaryotic expression plasmid, which belongs to the technical field of fusion proteins. Background technique [0002] Alzheimer's disease (Alzheimer's disease, AD) is a kind of neurological chronic disabling and fatal disease that often occurs in the elderly. Experts predict that it will become the first killer that endangers human health in this century. There are 6 million dementia patients in my country, accounting for 1 / 4 of the world's total. With the increase of the aging population, there will be 1 million dementia patients every year, that is, 2 new cases per minute. Therefore, it is of great social and practical significance to carry out research on AD. [0003] In 1984, Glenner first successfully isolated an amyloid peptide with a relative mole...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/47A61K38/17A61P25/28G01N33/68
Inventor 俞惠新张莉谭成陆春雄林秀峰陈波宋翠翠曹国宪张荣军黄群徐希杰
Owner JIANGSU INST OF NUCLEAR MEDICINE
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