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Human serum amyloid A1 and preparation method and application thereof

A technology of amyloid protein and human serum, applied in the field of bioengineering, can solve problems such as difficult to obtain SAA1 and difficult to separate

Active Publication Date: 2012-07-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, SAA1 is expressed in Escherichia coli and combined with lipopolysaccharide (LPS) on the cell wall of Gram-negative bacteria. Since SAA1 has a strong binding effect with lipopolysaccharide and its lipids, it is difficult to separate once combined, and it is difficult to obtain different lipopolysaccharide-containing SAA1

Method used

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  • Human serum amyloid A1 and preparation method and application thereof
  • Human serum amyloid A1 and preparation method and application thereof
  • Human serum amyloid A1 and preparation method and application thereof

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preparation example Construction

[0098] The preparation method of the recombinant human serum amyloid A1 and its multimer of the present invention comprises the steps of:

[0099] (a) providing a yeast expression vector containing a gene encoding human serum amyloid A1;

[0100] (b) providing a yeast host cell containing said yeast expression vector;

[0101] (c) cultivating the yeast host cell under suitable expression conditions, thereby expressing the recombinant human serum amyloid A1 of the present invention, or the multimer of the present invention; and

[0102] (d) isolating the recombinant human serum amyloid A1 or its multimers from the culture product.

[0103] In another preferred example, the gene encoding human serum amyloid A1 is amplified, double-digested and then ligated to a commercially available vector (such as pPIC9K) to obtain the yeast expression vector described in step (a). pPIC9K is a secretory vector for high-efficiency expression of foreign proteins, with a signal peptide α-factor...

Embodiment 1

[0153] Preparation of recombinant hSAA1

[0154] Synthesize the following primers:

[0155] Upstream primer (SEQ ID NO: 3):

[0156] 5'GTCG CTC GAG AAA AGA GAG GCT CGAAGCTTCTTTTTCGTTC 3';

[0157] Downstream primer (SEQ ID NO: 4):

[0158] 5'GTCG GAA TTC TCA GTG GTG GTG GTG GTG GTG GTATTTCTCAGGCAG 3';

[0159]The human mRNA was extracted by conventional methods, and the cDNA obtained after reverse transcription was used as a template. The full-length human hSAA1 gene was amplified by conventional PCR using the above primers. The PCR amplified product was digested with XhoI / EcoRI, and inserted into the downstream of the α-factor signal peptide of the vector pPIC9k (purchased from Invitrogen) that had undergone the same digestion, to obtain the vector pPIC9k-hSAA1. When hSAA1 is secreted and expressed, the α-factor signal peptide will be cleaved, thereby releasing the N-terminus of hSAA1.

[0160] Using the electroporation method, the above-mentioned recombinant expression...

Embodiment 2

[0164] Preparation of hSAA1 polypeptide fragment

[0165] The his6-sumo-tev sequence was gene-synthesized by conventional methods, as shown in SEQ ID NO: 5, and the restriction site XhoI was used to insert the α-factor signal peptide downstream of the vector pPIC9k (Invitrogen Company) to obtain the his6-sumo-tev sequence with his6-sumo- The pPIC9k vector pPIC9k-his6-sumo-tev for tev.

[0166] Using the primers named P27-72 in Table 1, the human hSAA1 gene fragment encoding the polypeptide fragment P27-72 was respectively amplified by conventional PCR method. For the PCR amplification product, after double digestion with XhoI / EcoRI, insert it into the downstream of his6-sumo-tev in the vector pPIC9k-his6-sumo-tev that has undergone the same digestion, and obtain the vector pPIC9k-his6-sumo-tev-hSAA1P27 -72.

[0167] Using the electroporation method, the above-mentioned recombinant expression vector pPIC9k-his6-sumo-tev-hSAA1P27-72 was transferred into conventional Pichia pas...

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Abstract

The invention discloses a human serum amyloid A1 and a preparation method and an application thereof. The invention further discloses a polymer of the human serum amyloid A1 and a polypeptide fragment of the human serum amyloid A1. The human serum amyloid A1, the polymer and the polypeptide fragment disclosed by the invention have the capabilities of inhibiting the expression of inflammatory cytokines, and can be used for inhibiting inflammations and treatment inflammation-relevant diseases.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a human serum amyloid A1 and its preparation method and application. Background technique [0002] Serum Amyloid A (SAA) is a major acute phase protein secreted when the body is infected and injured. SAA is a general term for polymorphic proteins, consisting of related proteins (SAA1-SAA4) with independent genes. Among them, human serum amyloid A1 (SAA1) consists of 104 amino acids and is mainly synthesized by liver cells. Human SAA2 and human SAA1 have a difference of 7 amino acids. They are both acute phase proteins and have similar roles in inflammatory reactions. Human SAA3 is a pseudogene, and human SAA4 does not change much in the acute phase of the reaction, and is continuously produced in a small amount in the body. [0003] Studies have shown that the content of SAA1 in the serum of patients with acute and chronic inflammation changes, so it is an im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/81C12N1/19C12P21/02A61K38/17A61P29/00A61P19/02A61P9/10A61P9/00A61P1/18A61P1/16A61P3/10A61P13/12A61P3/04A61P21/00A61P19/06
Inventor 叶德全陈明杰周慧斌
Owner SHANGHAI JIAO TONG UNIV
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