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Recombinant equine serum albumin, and preparation method and application thereof

A technology of albumin and horse serum, which is applied in the field of recombinant expression protein and genetic engineering, can solve the problem of low expression of target protein and achieve high expression

Pending Publication Date: 2020-02-21
SHANGHAI SERUM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the secretion and expression of Pichia pastoris of equine serum albumin, the applicant found that the signal peptide of yeast α-mating factor (α-mating factor) was used as the signal peptide of albumin, and the expression level of the target protein was very low

Method used

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  • Recombinant equine serum albumin, and preparation method and application thereof
  • Recombinant equine serum albumin, and preparation method and application thereof
  • Recombinant equine serum albumin, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Equine Serum Albumin Amino Acid Sequence, Codon Optimization Based on Yeast Preference

[0031] In the process of transcribing amino acids from nucleic acids, there are 20 amino acids that make up proteins, 61 codons that encode amino acids, and there can be several codons corresponding to one amino acid. Yeast has a preference for codons, which leads to high translation efficiency of several codons for an amino acid in yeast cells, and low translation efficiency of some codons. The nucleotide sequence of horse serum albumin contains some codons with low translation efficiency in yeast cells (see SEQ ID No.1 for the nucleotide sequence of horse serum albumin, and SEQ ID No.4 for the amino acid sequence of horse serum albumin) . In order to improve the expression level of the target protein in cells, the present invention entrusted Shanghai Bioengineering Company (Sangong) to rewrite all the nucleotide sequences of horse serum albumin into sequences with the ...

Embodiment 2

[0032] Example 2. Construction of Pichia pastoris cell expression vector ESA / pHIL-D2

[0033] 20 microliters of reaction system containing 20 micrograms of ESA / pBluescript (prepared in Example 1), 20 units of EcoR I (purchased from Baobiology), digested at 37°C for 4 hours, followed by agarose electrophoresis, and the molecular weight was cut under UV light The horse serum albumin DNA (ESA cDNA) was about 1.6Kb, and the DNA was recovered with Biotech DNA Recovery Kit.

[0034] The pHIL-D2 plasmid linearization reaction conditions are as follows: 20 microliters of reaction system containing 10 micrograms of pHIL-D2 plasmid, 20 units of EcoR I (purchased from Baobiology), digested at 37°C for 4 hours, and then digested with an equal volume of phenol:chloroform ( 1:1, v / v) for extraction once, and then ethanol precipitation to recover DNA. Place the ethanol solution on ice for 15 minutes and then centrifuge at 14,000 rpm for 10 minutes to recover the precipitated DNA, and disso...

Embodiment 3

[0037] Example 3. Construction of a Pichia cell line expressing recombinant equine serum albumin (ESA / GS115)

[0038] In a 40 microliter reaction system, 40 micrograms of Pichia pastoris cell expression vector ESA / pHIL-D2 plasmid DNA, 40 units of Sal I (purchased from Baobio) were contained, and digested at 37°C for 4 hours to linearize it. The final reaction was extracted once with an equal volume of phenol:chloroform (1:1, v / v), and then precipitated with ethanol to recover DNA. Place the ethanol solution on ice for 15 minutes and centrifuge at 14,000 rpm for 10 minutes to recover the precipitated DNA, and dissolve the DNA with 20 microliters of 10 mmol / L Tris-HCl (pH 8.0).

[0039] Pick GS115 single clones and add them to a 50ml conical tube containing 5ml YPD medium, and grow overnight at 30°C and 200rpm. Add 0.5 ml cultured GS115 to a 2 liter flask containing 500 ml fresh YPD (1% yeast extract, 2% peptone, 2% glucose) medium, grow overnight at 30°C, 200rpm until OD 600 ...

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Abstract

The invention discloses recombinant equine serum albumin and a preparation method thereof. The nucleotide sequence (shown as SEQ ID NO : 2) of coding equine serum albumin optimized by codon is constructed into an eukaryotic cell inducible expression vector to transfer into eukaryotic cells to perform inducible expression after culturing, and the recombinant equine serum albumin can be obtained through purification. An application of the recombinant equine serum albumin in the preparation of rabies vaccines for immunizing horses is also disclosed. A high anti-rabies antibody is obtained after arabies antigen using the recombinant equine serum albumin as a stabilizing agent immunizes the horses, and antibodies against human serum albumin are not detected. The obtained recombinant equine serum albumin can be used as the stabilizing agent of the rabies vaccines for immunizing the horses by replacing the human serum albumin, so that the recombinant equine serum albumin can be used for producing high-quality equine anti-rabies virus serum.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to the field of recombinantly expressed proteins, specifically to the expression of a mammalian plasma protein, especially to a recombinant equine serum albumin (Equine serum albumin, ESA) and its preparation method; in addition, the present invention The invention also relates to the application of the recombinant horse serum albumin obtained by the above method. Background technique [0002] Serum albumin is the most important protein in mammalian plasma, and it is widely used as an excipient or protective agent in the pharmaceutical industry to protect the stability of active ingredients of drugs. Many viral vaccines use albumin as a protective agent. Human rabies vaccine is one of them. [0003] Rabies is an acute fatal central nervous system (CNS) natural foci disease caused by Rabies virus (RABV) in humans and all warm-blooded animals. The disease is dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/12C07K14/765A61K47/42A61K39/205A61P31/14
CPCA61K39/12A61K47/42A61K2039/552A61P31/14C07K14/765C12N15/815C12N2760/20134C12N2800/22
Inventor 范铁炯胡金贵胡双锋孙九如梁光军史小月柏伟李鑫
Owner SHANGHAI SERUM BIOTECH
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