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Serum amyloid protein A mutant as well as application and preparation method thereof

A protein mutant, serum starch technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the limitation of SAA application, it is not easy to obtain SAA protein with natural structure, and stability is not stable. Good in vitro stability, improve in vitro stability, and retain the effect of antigenicity

Inactive Publication Date: 2020-02-04
宁波赛珀生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the recombinantly expressed SAA protein is very easy to form aggregation and degradation, the stability in vitro is very poor, easy to degrade, and it is not easy to obtain SAA protein with natural structure, these shortcomings greatly limit the application of SAA

Method used

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  • Serum amyloid protein A mutant as well as application and preparation method thereof
  • Serum amyloid protein A mutant as well as application and preparation method thereof
  • Serum amyloid protein A mutant as well as application and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Design of the amino acid sequence of the SAA mutant:

[0025] According to the amino acid sequence of the SAA protein published on the NCBI website and the characteristics of the sequence, the 52nd amino acid was replaced by Ala with Val, the 57th amino acid was replaced by Val with Ala, the 60th amino acid was replaced by Asp with Asn, and the 71st amino acid was replaced by Asp. The amino acid was replaced by Arg by His. Under the premise of retaining the antigenicity of natural SAA, the amino acid sequence of the obtained SAA mutant is shown in SEQ ID NO.1. The stability of this mutant is significantly better than that of native serum amyloid A.

Embodiment 2

[0027] Optimization and acquisition of SAA mutant gene sequence:

[0028] The nucleotide sequence of the SAA mutant was artificially optimized using the dominant codons of Escherichia coli, and finally the nucleotide sequence shown in SEQ ID NO.2 was obtained. The SAA mutant gene composed of dominant codons of Escherichia coli is obtained by annealing extension PCR technology, and the specific operation steps can refer to the Chinese invention patent with the authorized announcement number CN 104610443B.

Embodiment 3

[0030] Construction of SAA mutant expression vector:

[0031] The SAA mutant gene sequence obtained in Example 2 was double-digested with NdeI and XhoI and replaced between XhoI / NdeI of the prokaryotic expression vector pET-32a (+) (according to Merck Millipore, catalog number 69015) The fragment of pET-32a(+)-SAA (mutant type) was obtained.

[0032] Recombinant plasmid pET-32a(+)-SAA mutant), pET-32a(+) and SAA mutant gene were identified by XhoI and NdeI double enzyme digestion, the results are as follows figure 1 As shown, where M: DL5000Marker; 1: pET-32a(+)-SAA (mutant) recombinant plasmid double digestion; 2: pET-32a(+) plasmid double digestion; 3; SAA mutant gene double digestion . Depend on figure 1 It can be seen that after the recombinant plasmid is digested with Xho I and Nhe I, two bands with the expected molecular weight can be seen in electrophoresis, which is consistent with the size of pET-32a(+) and the SAA mutant gene.

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Abstract

The invention discloses a serum amyloid protein A mutant which has an amino acid sequence as shown in SEQ ID NO.1 and still has antigenicity of serum amyloid protein after several amino acids are replaced, so that a unique mutant is obtained. The SAA mutant is obtained by replacing four amino acids in natural SAA protein, so that the in-vitro stability of the SAA mutant is improved. Experiments prove that obtained SAA protein is soluble mature protein, the activity of the SAA protein is kept to the maximum extent, and the antigenicity of natural SAA protein is reserved. The invention also discloses application and a preparation method of the serum amyloid protein A mutant.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a serum amyloid A mutant. The invention also relates to the application and preparation method of the mutant. Background technique [0002] Serum Amyloid Protein A (SAA) is an acute phase response protein, which belongs to the heterogeneous protein in the apolipoprotein family, with a relative molecular weight of about 12 000. After the SAA synthesized in the liver is released into the blood, it quickly combines with HDL. The metabolism in the body is mainly degraded by proteases in serum, cell surface and intracellular, and the liver is the main degradation site. SAA produced by extrahepatic cells is mainly degraded by proteases on the cell surface or in cells through intercellular adhesion and endocytosis. SAA is the most acutely reflected among the acute phase proteins. When the disease improves and recovers, SAA first declines and returns to normal. In the past 20 y...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/70C12N1/21C12R1/19
CPCC07K14/47C12N15/70
Inventor 王民燕刘东海褚旭明夏杰王益栋李雪梅
Owner 宁波赛珀生物技术有限公司
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