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A long-acting recombinant human filaggrin expressed by Saccharomyces cerevisiae and its application

A technology of Saccharomyces cerevisiae and filaggrin, applied in the emerging biomedical field, can solve the problems of low biological activity of human filaggrin, easy formation of inclusion bodies, and restrictions on popularization and application, and achieve low cost, reduced production cost, and improved production process. simple effect

Active Publication Date: 2022-04-15
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the disadvantages of complex process, high cost, and limited popularization and application of natural purified human filaggrin; existing prokaryotic expression systems in the prior art have defects such as easy formation of inclusion bodies and low biological activity of obtained human filaggrin. Technical problem, the present invention provides a Saccharomyces cerevisiae expressing long-acting recombinant human filaggrin, a Saccharomyces cerevisiae signal peptide recombinant plasmid, a Saccharomyces cerevisiae secretion expression vector, an Saccharomyces cerevisiae auxotrophic strain, and a Saccharomyces cerevisiae Application of Yeast Expressed Long-acting Recombinant Human Filaggrin in Moisturizing Cosmetics

Method used

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  • A long-acting recombinant human filaggrin expressed by Saccharomyces cerevisiae and its application
  • A long-acting recombinant human filaggrin expressed by Saccharomyces cerevisiae and its application
  • A long-acting recombinant human filaggrin expressed by Saccharomyces cerevisiae and its application

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Effect test

Embodiment 1

[0039] This example discloses a long-acting recombinant human filaggrin expressed by Saccharomyces cerevisiae. The long-acting recombinant human filaggrin is a recombinant protein formed by fusing a monomer structure at the C-terminal of the natural human FLG repeat unit with HSA, and the recombinant protein can prevent the loss of epidermal water. And the recombinant protein should fuse hFLG truncated protein with HSA protein, which effectively improves the stability of the recombinant protein, and has simple production process, low cost, uniform product and no immunogenicity.

[0040] The nucleotide sequence of the long-acting recombinant human filaggrin is shown in Seq ID NO.1, and the amino acid sequence corresponding to the nucleotide sequence of the long-acting recombinant human filaggrin is shown in Seq ID NO.2.

[0041] The amino acid sequence of long-acting recombinant human filaggrin includes the amino acid sequence of filaggrin and the amino acid sequence of human s...

Embodiment 2

[0052] This embodiment discloses a signal peptide recombinant plasmid of Saccharomyces cerevisiae. The Saccharomyces cerevisiae signal peptide recombinant plasmid uses the Saccharomyces cerevisiae as described in Example 1 to express the long-acting recombinant human filaggrin gene.

[0053] The construction of the Saccharomyces cerevisiae signal peptide recombinant plasmid comprises the following steps:

[0054] Firstly, synthetic primers were designed according to the sequence of Saccharomyces cerevisiae Mfα gene, and the synthetic primers included forward primer Mfα-F and reverse primer Mfα-R. In this example, the forward primer Mfα-F is shown in Seq ID NO.3, and the reverse primer Mfα-R is shown in Seq ID NO.4.

[0055] Secondly, the signal peptide Mfα gene in the yeast is amplified by RT-PCR to obtain the RT-PCR amplification product, and the RT-PCR amplification product is recovered by gel to obtain the gel recovery product.

[0056] Then the product recovered from the...

Embodiment 3

[0070] This embodiment provides a secretory expression vector of Saccharomyces cerevisiae, which contains the gene for expressing long-acting recombinant human filaggrin in Saccharomyces cerevisiae as described in Example 1. The construction of the Saccharomyces cerevisiae secretion expression vector pYES2 / CT-Mfα-hFLG-HSA specifically includes the following steps:

[0071] The plasmid pYES2 / CT-Mfα and plasmid pMD19-T-hFLG-HSA were double-digested with endonuclease BamH I and endonuclease Xba I respectively. The enzyme digestion reaction system is:

[0072]

[0073] The enzyme digestion reaction was carried out in a metal bath at 37°C for 3 hours, and the digestion products were detected by 2% agarose gel electrophoresis, and the hFLG-HSA gene fragment and the pYES2 / CT-Mfα vector were recovered by gel cutting, and then ligated with T4 DNA ligase. The connection reaction system is:

[0074]

[0075] The reaction conditions were 14 hours at 16°C, and the recombinant plasmi...

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Abstract

The invention discloses a long-acting recombinant human filaggrin expressed by Saccharomyces cerevisiae and its application. The nucleotide sequence of the long-acting recombinant human filaggrin is shown in Seq ID NO.1. The long-acting recombinant human filaggrin The amino acid sequence corresponding to the nucleotide sequence is shown in Seq ID NO.2. The amino acid sequence of long-acting recombinant human filaggrin includes the amino acid sequence of filaggrin and the amino acid sequence of human serum albumin. The amino acid sequence of filaggrin includes a monomer structure corresponding to the C-terminus of the filaggrin nucleotide sequence repeat amino acid sequence. The long-acting recombinant human filaggrin provided by the invention can promote the aggregation of keratin intermediate filaments and the formation of disulfide bonds between intermediate filaments, prevent the loss of epidermal water and the invasion of external allergic substances, and reduce the apoptosis of epidermal cells induced by ultraviolet rays sensitivity. The fusion of HAS effectively improves the stability of the recombinant protein.

Description

technical field [0001] The present invention relates to the emerging field of biomedicine, in particular to a Saccharomyces cerevisiae expressing long-acting recombinant human filaggrin, a Saccharomyces cerevisiae signal peptide recombinant plasmid, a Saccharomyces cerevisiae secretion expression vector, an Saccharomyces cerevisiae auxotrophic strain, and An application of Saccharomyces cerevisiae expressing long-acting recombinant human filaggrin in moisturizing cosmetics. Background technique [0002] Filaggrin (flaggrin, FLG), also known as intermediate filaggrin, has a relative molecular mass of about 35,000 and mainly exists in the granular layer and transparent layer of the epidermis. Its precursor filaggrin is a highly phosphorylated, histidine-rich, water-insoluble and inactive polypeptide with a relative molecular mass of about 435,000. The coding gene FLG of profilaggrin is located in the epidermal differentiation complex, and the molecule starts with an N-termina...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/81C12N1/19A61K8/64A61Q19/00C12R1/865
CPCC07K14/47C12N15/81A61K8/64A61Q19/00
Inventor 赵俊刘洁刘家炉凡玉芳孔国庆周炜何志远
Owner WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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